WorldWideScience

Sample records for bioluminescence

  1. Bioluminescence.

    Science.gov (United States)

    Jones, M. Gail

    1993-01-01

    Describes bioluminescence and the chemistry of how it occurs. Presents information for conducting the following classroom activities: (1) firefly mimic; (2) modeling deep-sea fish; (3) sea fireflies; and (4) the chemistry of light. (PR)

  2. Destabilized bioluminescent proteins

    Science.gov (United States)

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  3. Cellular bioluminescence imaging.

    Science.gov (United States)

    Welsh, David K; Noguchi, Takako

    2012-08-01

    Bioluminescence imaging of live cells has recently been recognized as an important alternative to fluorescence imaging. Fluorescent probes are much brighter than bioluminescent probes (luciferase enzymes) and, therefore, provide much better spatial and temporal resolution and much better contrast for delineating cell structure. However, with bioluminescence imaging there is virtually no background or toxicity. As a result, bioluminescence can be superior to fluorescence for detecting and quantifying molecules and their interactions in living cells, particularly in long-term studies. Structurally diverse luciferases from beetle and marine species have been used for a wide variety of applications, including tracking cells in vivo, detecting protein-protein interactions, measuring levels of calcium and other signaling molecules, detecting protease activity, and reporting circadian clock gene expression. Such applications can be optimized by the use of brighter and variously colored luciferases, brighter microscope optics, and ultrasensitive, low-noise cameras. This article presents a review of how bioluminescence differs from fluorescence, its applications to cellular imaging, and available probes, optics, and detectors. It also gives practical suggestions for optimal bioluminescence imaging of single cells.

  4. Bioluminescent bioreporter integrated circuit

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Michael L. (Knoxville, TN); Sayler, Gary S. (Blaine, TN); Paulus, Michael J. (Knoxville, TN)

    2000-01-01

    Disclosed are monolithic bioelectronic devices comprising a bioreporter and an OASIC. These bioluminescent bioreporter integrated circuit are useful in detecting substances such as pollutants, explosives, and heavy-metals residing in inhospitable areas such as groundwater, industrial process vessels, and battlefields. Also disclosed are methods and apparatus for environmental pollutant detection, oil exploration, drug discovery, industrial process control, and hazardous chemical monitoring.

  5. Theoretical Study of Dinoflagellate Bioluminescence.

    Science.gov (United States)

    Wang, Ming-Yu; Liu, Ya-Jun

    2017-03-01

    Dinoflagellates are the most ubiquitous luminescent protists in the marine environment and have drawn much attention for their crucial roles in marine ecosystems. Dinoflagellate bioluminescence has been applied in underwater target detection. The luminescent system of dinoflagellates is a typical luciferin-luciferase one. However, the excited-state oxyluciferin is not the light emitter of dinoflagellate bioluminescence as in most luciferin-luciferase bioluminescent organisms. The oxyluciferin of bioluminescent dinoflagellates is not fluorescent, whereas its luciferin emits bright fluorescence with similar wavelength of the bioluminescence. What is the light emitter of dinoflagellate bioluminescence and what is the chemical process of the light emission like? These questions have not been answered by the limited experimental evidence so far. In this study, for the first time, the density functional calculation is employed to investigate the geometries and properties of luciferin and oxyluciferin of bioluminescent dinoflagellate. The calculated results agree with the experimental observations and indicate the luciferin or its analogue, rather than oxyluciferin, is the bioluminophore of dinoflagellate bioluminescence. A rough mechanism involving energy transfer is proposed for dinoflagellate bioluminescence.

  6. BIOLUMINESCENCE IMAGING: PROGRESS AND APPLICATIONS

    OpenAIRE

    Badr, Christian E.; Tannous, Bakhos A

    2011-01-01

    Application of bioluminescence imaging has grown tremendously in the past decade and has significantly contributed to the core conceptual advances in biomedical research. This technology provides valuable means for monitoring of different biological processes for immunology, oncology, virology and neuroscience. In this review, we will discuss current trends in bioluminescence and its application in different fields with emphasis on cancer research.

  7. Chemiluminescence and bioluminescence microbe detection

    Science.gov (United States)

    Taylor, R. E.; Chappelle, E.; Picciolo, G. L.; Jeffers, E. L.; Thomas, R. R.

    1978-01-01

    Automated biosensors for online use with NASA Water Monitoring System employs bioluminescence and chemiluminescence techniques to rapidly measure microbe contamination of water samples. System eliminates standard laboratory procedures requiring time duration of 24 hours or longer.

  8. Bioluminescence Potential Modeling and Forecasting

    Science.gov (United States)

    2013-05-22

    bioluminescence in the wakes of ships, breaking waves, around the bodies of rapidly moving fish and mammals , and from simple agitation of the water with one’s hand...history of brilliant displays of bioluminescence in the wakes of ships, breaking waves, around the bodies of rapidly moving fish and mammals , and from...during the earlier stages of upwelling development. Later, the observed deep offshore BL potential maximum disappeared and became a shallower and much

  9. Circular polarization observed in bioluminescence

    NARCIS (Netherlands)

    Wijnberg, Hans; Meijer, E.W.; Hummelen, J.C.; Dekkers, H.P.J.M.; Schippers, P.H.; Carlson, A.D.

    1980-01-01

    While investigating circular polarization in luminescence, and having found it in chemiluminescence, we have studied bioluminescence because it is such a widespread and dramatic natural phenomenon. We report here that left and right lanterns of live larvae of the fireflies, Photuris lucicrescens and

  10. Combining fluorescence and bioluminescence microscopy.

    Science.gov (United States)

    Goda, Kazuhito; Hatta-Ohashi, Yoko; Akiyoshi, Ryutaro; Sugiyama, Takashi; Sakai, Ikuko; Takahashi, Takeo; Suzuki, Hirobumi

    2015-08-01

    Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.

  11. Bioluminescence imaging in live cells and animals.

    Science.gov (United States)

    Tung, Jack K; Berglund, Ken; Gutekunst, Claire-Anne; Hochgeschwender, Ute; Gross, Robert E

    2016-04-01

    The use of bioluminescent reporters in neuroscience research continues to grow at a rapid pace as their applications and unique advantages over conventional fluorescent reporters become more appreciated. Here, we describe practical methods and principles for detecting and imaging bioluminescence from live cells and animals. We systematically tested various components of our conventional fluorescence microscope to optimize it for long-term bioluminescence imaging. High-resolution bioluminescence images from live neurons were obtained with our microscope setup, which could be continuously captured for several hours with no signs of phototoxicity. Bioluminescence from the mouse brain was also imaged noninvasively through the intact skull with a conventional luminescence imager. These methods demonstrate how bioluminescence can be routinely detected and measured from live cells and animals in a cost-effective way with common reagents and equipment.

  12. Bioluminescence assay for cell viability.

    Science.gov (United States)

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N

    2015-06-01

    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  13. Bioluminescence tomography based on the phase approximation model

    OpenAIRE

    Cong, W; Wang, G.

    2010-01-01

    A reconstruction method of bioluminescence sources is proposed based on a phase approximation model. Compared with the diffuse approximation, this phase approximation model more correctly predicts bioluminescence photon propagation in biological tissues, so that bioluminescence tomography can accurately locate and quantify the distribution of bioluminescence sources. The compressive sensing (CS) technique is applied to regularize the inverse source reconstruction to enhance numerical stabilit...

  14. The Chemical Basis of Fungal Bioluminescence.

    Science.gov (United States)

    Purtov, Konstantin V; Petushkov, Valentin N; Baranov, Mikhail S; Mineev, Konstantin S; Rodionova, Natalja S; Kaskova, Zinaida M; Tsarkova, Aleksandra S; Petunin, Alexei I; Bondar, Vladimir S; Rodicheva, Emma K; Medvedeva, Svetlana E; Oba, Yuichi; Oba, Yumiko; Arseniev, Alexander S; Lukyanov, Sergey; Gitelson, Josef I; Yampolsky, Ilia V

    2015-07-06

    Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

  15. Repeated and Widespread Evolution of Bioluminescence in Marine Fishes.

    Science.gov (United States)

    Davis, Matthew P; Sparks, John S; Smith, W Leo

    2016-01-01

    Bioluminescence is primarily a marine phenomenon with 80% of metazoan bioluminescent genera occurring in the world's oceans. Here we show that bioluminescence has evolved repeatedly and is phylogenetically widespread across ray-finned fishes. We recover 27 independent evolutionary events of bioluminescence, all among marine fish lineages. This finding indicates that bioluminescence has evolved many more times than previously hypothesized across fishes and the tree of life. Our exploration of the macroevolutionary patterns of bioluminescent lineages indicates that the present day diversity of some inshore and deep-sea bioluminescent fish lineages that use bioluminescence for communication, feeding, and reproduction exhibit exceptional species richness given clade age. We show that exceptional species richness occurs particularly in deep-sea fishes with intrinsic bioluminescent systems and both shallow water and deep-sea lineages with luminescent systems used for communication.

  16. Analytical Applications of Bioluminescence and Chemiluminescence

    Science.gov (United States)

    Chappelle, E. W. (Editor); Picciolo, G. L. (Editor)

    1975-01-01

    Bioluminescence and chemiluminescence studies were used to measure the amount of adenosine triphosphate and therefore the amount of energy available. Firefly luciferase - luciferin enzyme system was emphasized. Photometer designs are also considered.

  17. Quantitative bioluminescence imaging of mouse tumor models.

    Science.gov (United States)

    Tseng, Jen-Chieh; Kung, Andrew L

    2015-01-05

    Bioluminescence imaging (BLI) has become an essential technique for preclinical evaluation of anticancer therapeutics and provides sensitive and quantitative measurements of tumor burden in experimental cancer models. For light generation, a vector encoding firefly luciferase is introduced into human cancer cells that are grown as tumor xenografts in immunocompromised hosts, and the enzyme substrate luciferin is injected into the host. Alternatively, the reporter gene can be expressed in genetically engineered mouse models to determine the onset and progression of disease. In addition to expression of an ectopic luciferase enzyme, bioluminescence requires oxygen and ATP, thus only viable luciferase-expressing cells or tissues are capable of producing bioluminescence signals. Here, we summarize a BLI protocol that takes advantage of advances in hardware, especially the cooled charge-coupled device camera, to enable detection of bioluminescence in living animals with high sensitivity and a large dynamic range.

  18. Circadian control sheds light on fungal bioluminescence.

    Science.gov (United States)

    Oliveira, Anderson G; Stevani, Cassius V; Waldenmaier, Hans E; Viviani, Vadim; Emerson, Jillian M; Loros, Jennifer J; Dunlap, Jay C

    2015-03-30

    Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi-only 71 species, all within the ∼ 9,000 fungi of the temperate and tropical Agaricales order-are reported from among ∼ 100,000 described fungal species [6, 7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a byproduct of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature-compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millennia [8-15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin "mushrooms," internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy, where wind flow is greatly reduced.

  19. Bioluminescence in Dinoflagellates: Evidence that the Adaptive Value of Bioluminescence in Dinoflagellates is Concentration Dependent.

    Science.gov (United States)

    Hanley, Karen A; Widder, Edith A

    2017-03-01

    Three major hypotheses have been proposed to explain why dinoflagellate bioluminescence deters copepod grazing: startle response, aposematic warning, and burglar alarm. These hypotheses propose dinoflagellate bioluminescence (A) startles predatory copepods, (B) warns potential predators of toxicity, and (C) draws the attention of higher order visual predators to the copepod's location. While the burglar alarm is the most commonly accepted hypothesis, it requires a high concentration of bioluminescent dinoflagellates to be effective, meaning the bioluminescence selective advantage at lower, more commonly observed, dinoflagellate concentrations may result from another function (e.g. startle response or aposematic warning). Therefore, a series of experiments was conducted to evaluate copepod grazing (Acartia tonsa) on bioluminescent dinoflagellates (during bioluminescent and nonbioluminescent phases, corresponding to night and day, respectively) at different concentrations (10, 1000, and 3000 cells mL(-1) ), on toxic (Pyrodinium bahamense var. bahamense) and nontoxic (Lingulodinium polyedrum) bioluminescent dinoflagellates, and in the presence of nonluminescent diatoms (Thalassiosira eccentrica). Changes in copepod ingestion rates, clearance rates, and feeding preferences as a result of these experimental factors, particularly during the mixed trails with nonluminescent diatoms, indicate there is a concentration threshold at which the burglar alarm becomes effective and below which dinoflagellate bioluminescence functions as an aposematic warning.

  20. Noninvasive bioluminescence imaging in small animals.

    Science.gov (United States)

    Zinn, Kurt R; Chaudhuri, Tandra R; Szafran, April Adams; O'Quinn, Darrell; Weaver, Casey; Dugger, Kari; Lamar, Dale; Kesterson, Robert A; Wang, Xiangdong; Frank, Stuart J

    2008-01-01

    There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling. A 3-dimensional (3D) capability for bioluminescence imaging is now available, but is more expensive and less efficient; other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. All of these problems make it unlikely that the method will be extended to human studies. However, in small animal models, bioluminescence imaging is now routinely applied to serially detect the location and burden of xenografted tumors, or identify and measure the number of immune or stem cells after an adoptive transfer. Bioluminescence imaging also makes it possible to track the relative amounts and locations of bacteria, viruses, and other pathogens over time. Specialized applications of bioluminescence also follow tissue-specific luciferase expression in transgenic mice, and monitor biological processes such as signaling or protein interactions in real time. In summary, bioluminescence imaging has become an important component of biomedical research that will continue in the future.

  1. Bioluminescence microscopy using a short focal-length imaging lens

    OpenAIRE

    Ogoh, K; Akiyoshi, R; May-Maw-Thet,; Sugiyama, T; Dosaka, S; Hatta-Ohashi, Y; Suzuki, H.

    2014-01-01

    Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera....

  2. Discovery of New Substrates for LuxAB Bacterial Bioluminescence.

    Science.gov (United States)

    Jiang, Tianyu; Wang, Weishan; Wu, Xingkang; Wu, Wenxiao; Bai, Haixiu; Ma, Zhao; Shen, Yuemao; Yang, Keqian; Li, Minyong

    2016-08-01

    In this article, four novel substrates with long halftime have been designed and synthesized successfully for luxAB bacterial bioluminescence. After in vitro and in vivo biological evaluation, these molecules can emit obvious bioluminescence emission with known bacterial luciferase, thus indicating a new promising approach to developing the bacterial bioluminescent system.

  3. Bioluminescence patterns among North American Armillaria species.

    Science.gov (United States)

    Mihail, Jeanne D

    2015-06-01

    Bioluminescence is widely recognized among white-spored species of Basidiomycota. Most reports of fungal bioluminescence are based upon visual light perception. When instruments such as photomultipliers have been used to measure fungal luminescence, more taxa have been discovered to produce light, albeit at a range of magnitudes. The present studies were undertaken to determine the prevalence of bioluminescence among North American Armillaria species. Consistent, constitutive bioluminescence was detected for the first time for mycelia of Armillaria calvescens, Armillaria cepistipes, Armillaria gemina, Armillaria nabsnona, and Armillaria sinapina and confirmed for mycelia of Armillaria gallica, Armillaria mellea, Armillaria ostoyae, and Armillaria tabescens. Emission spectra of mycelia representing all species had maximum intensity in the range 515-525 nm confirming that emitted light was the result of bioluminescence rather than chemiluminescence. Time series analysis of 1000 consecutive luminescence measurements revealed a highly significant departure from random variation. Mycelial luminescence of eight species exhibited significant, stable shifts in magnitude in response to a series of mechanical disturbance treatments, providing one mechanism for generating observed luminescence variation.

  4. Immobilized Bioluminescent Reagents in Flow Injection Analysis.

    Science.gov (United States)

    Nabi, Abdul

    Available from UMI in association with The British Library. Bioluminescent reactions exhibits two important characteristics from an analytical viewpoint; they are selective and highly sensitive. Furthermore, bioluminescent emissions are easily measured with a simple flow-through detector based on a photomultiplier tube and the rapid and reproducible mixing of sample and expensive reagent is best achieved by a flow injection manifold. The two most important bioluminescent systems are the enzyme (luciferase)/substrate (luciferin) combinations extracted from fireflies (Photinus pyralis) and marine bacteria (Virio harveyi) which requires ATP and NAD(P)H respectively as cofactors. Reactions that generate or consume these cofactors can also be coupled to the bioluminescent reaction to provide assays for a wide range of clinically important species. A flow injection manifold for the study of bioluminescent reactions is described, as are procedures for the extraction, purification and immobilization of firefly and bacterial luciferase and oxidoreductase. Results are presented for the determination of ATP using firefly system and the determination of other enzymes and substrates participating in ATP-converting reactions e.g. creatine kinase, ATP-sulphurylase, pyruvate kinase, creatine phosphate, pyrophosphate and phophoenolypyruvate. Similarly results are presented for the determination of NAD(P)H, FMN, FMNH_2 and several dehydrogenases which produce NAD(P)H and their substrates, e.g. alcohol, L-lactate, L-malate, L-glutamate, Glucose-6-phosphate and primary bile acid.

  5. In vivo cell tracking with bioluminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Eun; Kalimuthu, Senthilkumar; Ahn, Byeong Cheol [Dept. of Nuclear Medicine, Kyungpook National University School of Medicine and Hospital, Daegu (Korea, Republic of)

    2015-03-15

    Molecular imaging is a fast growing biomedical research that allows the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In vivo tracking of cells is an indispensable technology for development and optimization of cell therapy for replacement or renewal of damaged or diseased tissue using transplanted cells, often autologous cells. With outstanding advantages of bioluminescence imaging, the imaging approach is most commonly applied for in vivo monitoring of transplanted stem cells or immune cells in order to assess viability of administered cells with therapeutic efficacy in preclinical small animal models. In this review, a general overview of bioluminescence is provided and recent updates of in vivo cell tracking using the bioluminescence signal are discussed.

  6. Monitoring of environmental pollutants by bioluminescent bacteria.

    Science.gov (United States)

    Girotti, Stefano; Ferri, Elida Nora; Fumo, Maria Grazia; Maiolini, Elisabetta

    2008-02-04

    This review deals with the applications of bioluminescent bacteria to the environmental analyses, published during the years 2000-2007. The ecotoxicological assessment, by bioassays, of the environmental risks and the luminescent approaches are reported. The review includes a brief introduction to the characteristics and applications of bioassays, a description of the characteristics and applications of natural bioluminescent bacteria (BLB), and a collection of the main applications to organic and inorganic pollutants. The light-emitting genetically modified bacteria applications, as well as the bioluminescent immobilized systems and biosensors are outlined. Considerations about commercially available BLB and BLB catalogues are also reported. Most of the environmental applications, here mentioned, of luminescent organisms are on wastewater, seawater, surface and ground water, tap water, soil and sediments, air. Comparison to other bioindicators and bioassay has been also made. Various tables have been inserted, to make easier to take a rapid glance at all possible references concerning the topic of specific interest.

  7. Detection of bacteria with bioluminescent reporter bacteriophage.

    Science.gov (United States)

    Klumpp, Jochen; Loessner, Martin J

    2014-01-01

    Bacteriophages are viruses that exclusively infect bacteria. They are ideally suited for the development of highly specific diagnostic assay systems. Bioluminescent reporter bacteriophages are designed and constructed by integration of a luciferase gene in the virus genome. Relying on the host specificity of the phage, the system enables rapid, sensitive, and specific detection of bacterial pathogens. A bioluminescent reporter phage assay is superior to any other molecular detection method, because gene expression and light emission are dependent on an active metabolism of the bacterial cell, and only viable cells will yield a signal. In this chapter we introduce the concept of creating reporter phages, discuss their advantages and disadvantages, and illustrate the advances made in developing such systems for different Gram-negative and Gram-positive pathogens. The application of bioluminescent reporter phages for the detection of foodborne pathogens is emphasized.

  8. Optimisation of acquisition time in bioluminescence imaging

    Science.gov (United States)

    Taylor, Shelley L.; Mason, Suzannah K. G.; Glinton, Sophie; Cobbold, Mark; Styles, Iain B.; Dehghani, Hamid

    2015-03-01

    Decreasing the acquisition time in bioluminescence imaging (BLI) and bioluminescence tomography (BLT) will enable animals to be imaged within the window of stable emission of the bioluminescent source, a higher imaging throughput and minimisation of the time which an animal is anaesthetised. This work investigates, through simulation using a heterogeneous mouse model, two methods of decreasing acquisition time: 1. Imaging at fewer wavelengths (a reduction from five to three); and 2. Increasing the bandwidth of filters used for imaging. The results indicate that both methods are viable ways of decreasing the acquisition time without a loss in quantitative accuracy. Importantly, when choosing imaging wavelengths, the spectral attenuation of tissue and emission spectrum of the source must be considered, in order to choose wavelengths at which a high signal can be achieved. Additionally, when increasing the bandwidth of the filters used for imaging, the bandwidth must be accounted for in the reconstruction algorithm.

  9. In vivo cell tracking with bioluminescence imaging.

    Science.gov (United States)

    Kim, Jung Eun; Kalimuthu, Senthilkumar; Ahn, Byeong-Cheol

    2015-03-01

    Molecular imaging is a fast growing biomedical research that allows the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In vivo tracking of cells is an indispensable technology for development and optimization of cell therapy for replacement or renewal of damaged or diseased tissue using transplanted cells, often autologous cells. With outstanding advantages of bioluminescence imaging, the imaging approach is most commonly applied for in vivo monitoring of transplanted stem cells or immune cells in order to assess viability of administered cells with therapeutic efficacy in preclinical small animal models. In this review, a general overview of bioluminescence is provided and recent updates of in vivo cell tracking using the bioluminescence signal are discussed.

  10. Lighting up bioluminescence with coelenterazine: strategies and applications.

    Science.gov (United States)

    Jiang, Tianyu; Du, Lupei; Li, Minyong

    2016-04-01

    Bioluminescence-based techniques, such as bioluminescence imaging, BRET and dual-luciferase reporter assay systems, have been widely used to examine a myriad of biological processes. Coelenterazine (CTZ), a luciferin or light-producing compound found in bioluminescent organisms, has sparked great curiosity and interest in searching for analogues with improved photochemical properties. This review summarizes the current development of coelenterazine analogues, their bioluminescence properties, and the rational design of caged coelenterazine towards biotargets, as well as their applications in bioassays. It should be emphasized that the design of caged luciferins can provide valuable insight into detailed molecular processes in organisms and will be a trend in the development of bioluminescent molecules.

  11. Using bioluminescence imaging in glioma research.

    Science.gov (United States)

    Luwor, Rodney B; Stylli, Stanley S; Kaye, Andrew H

    2015-05-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumour and has the worst prognosis. Over the last decade, the use of bioluminescence imaging technology has rapidly become widespread to further understand the mechanisms that drive GBM development and progression. Pre-clinical evaluation and optimisation of therapeutic efficacy in GBM research has also utilised this simple non-invasive technology. Here we summarise recent advances made in glioma biology and therapeutic intervention using bioluminescence imaging. This review also describes the current knowledge regarding the use of luciferase-based reporters in examining the role of specific cancer signalling cascades that promote glioma progression.

  12. A Multichannel Bioluminescence Determination Platform for Bioassays.

    Science.gov (United States)

    Kim, Sung-Bae; Naganawa, Ryuichi

    2016-01-01

    The present protocol introduces a multichannel bioluminescence determination platform allowing a high sample throughput determination of weak bioluminescence with reduced standard deviations. The platform is designed to carry a multichannel conveyer, an optical filter, and a mirror cap. The platform enables us to near-simultaneously determine ligands in multiple samples without the replacement of the sample tubes. Furthermore, the optical filters beneath the multichannel conveyer are designed to easily discriminate colors during assays. This optical system provides excellent time- and labor-efficiency to users during bioassays.

  13. Bioluminescent bioreporter sensing of foodborne toxins

    Science.gov (United States)

    Fraley, Amanda C.; Ripp, Steven; Sayler, Gary S.

    2004-06-01

    Histamine is the primary etiological agent in the foodborne disease scombrotoxicosis, one of the most common food toxicities related to fish consumption. Procedures for detecting histamine in fish products are available, but are often too expensive or too complex for routine use. As an alternative, a bacterial bioluminescent bioreporter has been constructed to develop a biosensor system that autonomously responds to low levels of histamine. The bioreporter contains a promoterless Photorhabdus luminescens lux operon (luxCDABE) fused with the Vibrio anguillarum angR regulatory gene promoter of the anguibactin biosynthetic operon. The bioreporter emitted 1.46 times more bioluminescence than background, 30 minutes after the addition of 100mM histamine. However, specificity was not optimal, as this biosensor generated significant bioluminescence in the presence of L-proline and L-histidine. As a means towards improving histamine specificity, the promoter region of a histamine oxidase gene from Arthrobacter globiformis was cloned upstream of the promotorless lux operon from Photorhabdus luminescens. This recently constructed whole-cell, lux-based bioluminescent bioreporter is currently being tested for optimal performance in the presence of histamine in order to provide a rapid, simple, and inexpensive model sensor for the detection of foodborne toxins.

  14. Bioluminescent bioreporter integrated circuit detection methods

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Michael L.; Paulus, Michael J.; Sayler, Gary S.; Applegate, Bruce M.; Ripp, Steven A.

    2005-06-14

    Disclosed are monolithic bioelectronic devices comprising a bioreporter and an OASIC. These bioluminescent bioreporter integrated circuit are useful in detecting substances such as pollutants, explosives, and heavy-metals residing in inhospitable areas such as groundwater, industrial process vessels, and battlefields. Also disclosed are methods and apparatus for detection of particular analytes, including ammonia and estrogen compounds.

  15. Multicolor Bioluminescence Obtained Using Firefly Luciferin.

    Science.gov (United States)

    Kiyama, Masahiro; Saito, Ryohei; Iwano, Satoshi; Obata, Rika; Niwa, Haruki; Maki, Shojiro A

    2016-01-01

    Firefly bioluminescence is widely used in life science research as a useful analysis tool. For example, the adenosine-5`-triphosphate (ATP)-dependent enzymatic firefly bioluminescence reaction has long been utilized as a microbial monitoring tool. Rapid and sensitive firefly luciferin-luciferase combinations are used not only to measure cell viability but also for reporter-gene assays. Recently, bioluminescence was utilized as a noninvasive, real-time imaging tool for living subjects to monitor cells and biological events. However, the number of commercialized luciferase genes is limited and tissue-permeable near-infrared (NIR) region emitting light is required for in vivo imaging. In this review, recent studies describing synthetic luciferin analogues predicted to have red-shifted bioluminescence are summarized. Luciferase substrates emitting red, green, and blue light that were designed and developed in our laboratory are presented. The longest emission wavelength of the synthesized luciferin analogues was recorded at 675 nm, which is within the NIR region. This compound is now commercially available as "Aka Lumine®".

  16. Bioluminescence for determining energy state of plants

    Science.gov (United States)

    Ching, T. M.

    1975-01-01

    Bioluminescence produced by the luciferin-luciferase system is a very sensitive assay for ATP content in extracts of plant materials. The ATP test for seed and pollen viability and vigor is presented, along with prediction of high growth potential and productivity in new crosses and selections of breeding materials. ATP as an indicator for environmental quality, stresses, and metabolic regulation is also considered.

  17. Bubble stimulation efficiency of dinoflagellate bioluminescence.

    Science.gov (United States)

    Deane, Grant B; Stokes, M Dale; Latz, Michael I

    2016-02-01

    Dinoflagellate bioluminescence, a common source of bioluminescence in coastal waters, is stimulated by flow agitation. Although bubbles are anecdotally known to be stimulatory, the process has never been experimentally investigated. This study quantified the flash response of the bioluminescent dinoflagellate Lingulodinium polyedrum to stimulation by bubbles rising through still seawater. Cells were stimulated by isolated bubbles of 0.3-3 mm radii rising at their terminal velocity, and also by bubble clouds containing bubbles of 0.06-10 mm radii for different air flow rates. Stimulation efficiency, the proportion of cells producing a flash within the volume of water swept out by a rising bubble, decreased with decreasing bubble radius for radii less than approximately 1 mm. Bubbles smaller than a critical radius in the range 0.275-0.325 mm did not stimulate a flash response. The fraction of cells stimulated by bubble clouds was proportional to the volume of air in the bubble cloud, with lower stimulation levels observed for clouds with smaller bubbles. An empirical model for bubble cloud stimulation based on the isolated bubble observations successfully reproduced the observed stimulation by bubble clouds for low air flow rates. High air flow rates stimulated more light emission than expected, presumably because of additional fluid shear stress associated with collective buoyancy effects generated by the high air fraction bubble cloud. These results are relevant to bioluminescence stimulation by bubbles in two-phase flows, such as in ship wakes, breaking waves, and sparged bioreactors.

  18. Amplitude metrics for cellular circadian bioluminescence reporters.

    Science.gov (United States)

    St John, Peter C; Taylor, Stephanie R; Abel, John H; Doyle, Francis J

    2014-12-01

    Bioluminescence rhythms from cellular reporters have become the most common method used to quantify oscillations in circadian gene expression. These experimental systems can reveal phase and amplitude change resulting from circadian disturbances, and can be used in conjunction with mathematical models to lend further insight into the mechanistic basis of clock amplitude regulation. However, bioluminescence experiments track the mean output from thousands of noisy, uncoupled oscillators, obscuring the direct effect of a given stimulus on the genetic regulatory network. In many cases, it is unclear whether changes in amplitude are due to individual changes in gene expression level or to a change in coherence of the population. Although such systems can be modeled using explicit stochastic simulations, these models are computationally cumbersome and limit analytical insight into the mechanisms of amplitude change. We therefore develop theoretical and computational tools to approximate the mean expression level in large populations of noninteracting oscillators, and further define computationally efficient amplitude response calculations to describe phase-dependent amplitude change. At the single-cell level, a mechanistic nonlinear ordinary differential equation model is used to calculate the transient response of each cell to a perturbation, whereas population-level dynamics are captured by coupling this detailed model to a phase density function. Our analysis reveals that amplitude changes mediated at either the individual-cell or the population level can be distinguished in tissue-level bioluminescence data without the need for single-cell measurements. We demonstrate the effectiveness of the method by modeling experimental bioluminescence profiles of light-sensitive fibroblasts, reconciling the conclusions of two seemingly contradictory studies. This modeling framework allows a direct comparison between in vitro bioluminescence experiments and in silico ordinary

  19. Bioluminescence as an ecological factor during high Arctic polar night

    Science.gov (United States)

    Cronin, Heather A.; Cohen, Jonathan H.; Berge, Jørgen; Johnsen, Geir; Moline, Mark A.

    2016-11-01

    Bioluminescence commonly influences pelagic trophic interactions at mesopelagic depths. Here we characterize a vertical gradient in structure of a generally low species diversity bioluminescent community at shallower epipelagic depths during the polar night period in a high Arctic fjord with in situ bathyphotometric sampling. Bioluminescence potential of the community increased with depth to a peak at 80 m. Community composition changed over this range, with an ecotone at 20–40 m where a dinoflagellate-dominated community transitioned to dominance by the copepod Metridia longa. Coincident at this depth was bioluminescence exceeding atmospheric light in the ambient pelagic photon budget, which we term the bioluminescence compensation depth. Collectively, we show a winter bioluminescent community in the high Arctic with vertical structure linked to attenuation of atmospheric light, which has the potential to influence pelagic ecology during the light-limited polar night.

  20. Bioluminescence microscopy using a short focal-length imaging lens.

    Science.gov (United States)

    Ogoh, K; Akiyoshi, R; May-Maw-Thet; Sugiyama, T; Dosaka, S; Hatta-Ohashi, Y; Suzuki, H

    2014-03-01

    Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo-toxicity associated with fluorescence imaging and permits the long-term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.

  1. Bioluminescence-Sensing Assay for Microbial Growth Recognition

    Directory of Open Access Journals (Sweden)

    Heba Ramadan Eed

    2016-01-01

    Full Text Available The conventional methods for microbial viability quantification require cultivation and are laborious. There is consequently a widespread need for cultivation-free methods. The adenosine triphosphate (ATP bioluminescence-sensing assay is considered an extremely effective biosensor; hence ATP is the energy currency of all living microbes and can be used as a rapid indicator of microbial viability. We developed an ATP bioluminescence-sensing assay to detect microbial viability. A bioluminescent recombinant E. coli strain was used with luciferase extracted from transformed bacteria. Results showed that there is a direct correlation between the bioluminescence intensity of the ATP bioluminescence-sensing assay and the microbial viability. Bacterial counts from food samples were detected using the developed sensing assay and validated by the traditional plate-counting method. Compared with the plate-counting method, ATP bioluminescence-sensing assay is a more rapid and efficient approach for detecting microbial viability.

  2. Transformation Experiment Using Bioluminescence Genes of "Vibrio fischeri."

    Science.gov (United States)

    Slock, James

    1995-01-01

    Bioluminescence transformation experiments show students the excitement and power of recombinant DNA technology. This laboratory experiment utilizes two plasmids of "Vibrio fischeri" in a transformation experiment. (LZ)

  3. Stimulated bioluminescence by fluid shear stress associated with pipe flow

    Energy Technology Data Exchange (ETDEWEB)

    Cao Jing; Wang Jiangan; Wu Ronghua, E-mail: caojing981@126.com [Col. of Electronic Eng., Naval University of Engineering, Wuhan 430033 (China)

    2011-01-01

    Dinoflagellate can be stimulated bioluminescence by hydrodynamic agitation. Two typical dinoflagellate (Lingulodinium polyedrum and Pyrocystis noctiluca) was choosed to research stimulated bioluminescence. The bioluminescence intensity and shear stress intensity were measured using fully developed pipe flow. There is shear stress threshold to agitate organism bioluminescence. From these experiment, the response thresholds of the stimulated bioluminscence always occurred in laminar flows at a shear stress level of 0.6-3 dyn/cm{sup 2}. At the same time, the spectral characteristc of dinoflagellate was recorded, the wavelength of them is about 470nm, and the full width at half maximum is approximate 30nm.

  4. Bioluminescence as a classroom tool for scientist volunteers.

    Science.gov (United States)

    Hammer, M; Andrade, J D

    2000-01-01

    There is a great need for practicing scientists to volunteer their time and expertise in the K-12th grade science classroom. We have found that bioluminescence is a fun and exciting way to teach basic science concepts and is an excellent tool for the volunteering scientist. We have had very positive reactions from both teachers and students. The excitement of the students when they first see bioluminescence is contagious. Bioluminescent dinoflagellates are one of the easiest ways to introduce students to this fascinating topic. Many activities and experiments can be done using the bioluminescent dinoflagellates and many students and teachers could benefit from your knowledge and expertise. See you in the classroom.

  5. Bioluminescent assay for human lymphocyte blast transformation.

    Science.gov (United States)

    Bulanova, E G; Budagyan, V M; Romanova, N A; Brovko LYu; Ugarova, N N

    1995-05-01

    One of the basic tests of in vitro evaluation of immune cell functional activity is a proliferative response of lymphocytes on the action of external stimuli such as mitogenic lectines, antigens, etc. We compared two methods used to assess the lymphocyte functional status. (1) [3H]thymidine incorporation and (2) bioluminescence for determination of intracellular ATP in blast cells. Comparison has been done for healthy donors and patients with proven low immunological status. The proposed bioluminescent method for evaluation of the proliferative response was shown to be sensitive enough for diagnostic purposes. This method allows one to process a large number of samples at the same time and correlates highly with the radionuclide test use hazardous radioactive materials.

  6. A REVIEW OF ENVIRONMENTAL APPLICATIONS OF BIOLUMINESCENCE MEASUREMENTS

    Science.gov (United States)

    This review of the recent literature on environmental applications of bioluminescence systems will focus on in vivo and in vitro bioluminescence methods that have been utilized to elucidate properties of chemicals, toxic and mutagenic effects, and to estimate biomass. The unifyin...

  7. Detection of ATP and NADH: A Bioluminescent Experience.

    Science.gov (United States)

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  8. Single-cell bioluminescence and GFP in biofilm research

    Energy Technology Data Exchange (ETDEWEB)

    Palmer, R.J. Jr, Sayler, G., White, D.C. [Tennessee Univ., Knoxville, TN (United States), Ctr. Env. Biotech; Phiefer, C. [Oak Ridge National Lab., TN (United States), Environmental Sciences Div.

    1996-12-31

    Using flow cells and a combination of microscopy techniques, we can unequivocally identify single bacterial cells that express bioluminescent and fluorescent bioreporters. We have shown that, for attached cells, bioluminescence output within a bacterial strain can vary greatly from cell to cell.

  9. Dinoflagellate bioluminescence in response to mechanical stimuli in water flows

    Directory of Open Access Journals (Sweden)

    A. S. Cussatlegras

    2005-01-01

    Full Text Available Bioluminescence of plankton organisms induced by water movements has long been observed and is still under investigations because of its great complexity. In particular, the exact mechanism occurring at the level of the cell has not been yet fully understood. This work is devoted to the study of the bioluminescence of the dinoflagellates plankton species Pyrocystis noctiluca in response to mechanical stimuli generated by water flows. Several experiments were performed with different types of flows in a Couette shearing apparatus. All of them converge to the conclusion that stationary homogeneous laminar shear does not trigger massive bioluminescence, but that acceleration and shear are both necessary to stimulate together an intense bioluminescence response. The distribution of the experimental bioluminescence thresholds is finally calculated from the light emission response for the Pyrocystis noctiluca species.

  10. Moment searching algorithm for bioluminescence tomography

    Institute of Scientific and Technical Information of China (English)

    Ludong Jin; Yan Wu; Jie Tian; Heyu Huang; Xiaochao Qu

    2009-01-01

    To avoid the ill-posedness in the inverse problem of bioluminescence tomography, a moment searching algorithm fusing the finite element method (FEM) with the moment concept in theoretical mechanics is developed. In the algorithm, the source's information is mapped to the surface photon flux density by FEM, and the source's position is modified with the feedback through the algorithm of barycenter searching, which makes full use of the position information of the photon flux density on surface. The position is modified in every iterative step and will finally converge to the real source's value theoretically.

  11. Development of bioluminescent Salmonella strains for use in food safety

    Directory of Open Access Journals (Sweden)

    Bailey R Hartford

    2008-01-01

    Full Text Available Abstract Background Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, we developed bioluminescent Salmonella strains that can be used for real-time monitoring of the pathogen's growth on food products. To accomplish this, twelve Salmonella strains from the broiler production continuum were transformed with the broad host range plasmid pAKlux1, and a chicken skin attachment model was developed. Results Salmonella strains carrying pAKlux1 constitutively expressed the luxCDABE operon and were therefore detectable using bioluminescence. Strains were characterized in terms of bioluminescence properties and plasmid stability. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, we developed an attachment model using chicken skin. The effect of washing on attachment of Salmonella strains to chicken skin was tested using bioluminescent strains, which revealed the attachment properties of each strain. Conclusion This study demonstrated that bioluminescence is a sensitive and effective tool to detect Salmonella on food products in real-time. Bioluminescence imaging is a promising technology that can be utilized to evaluate new food safety measures for reducing Salmonella contamination on food products.

  12. Measuring IL-1β Processing by Bioluminescence Sensors I: Using a Bioluminescence Resonance Energy Transfer Biosensor.

    Science.gov (United States)

    Compan, Vincent; Pelegrín, Pablo

    2016-01-01

    IL-1β processing is one of the hallmarks of inflammasome activation and drives the initiation of the inflammatory response. For decades, Western blot or ELISA have been extensively used to study this inflammatory event. Here, we describe the use of a bioluminescence resonance energy transfer (BRET) biosensor to monitor IL-1β processing in real time and in living macrophages either using a plate reader or a microscope.

  13. Bioluminescence-activated deep-tissue photodynamic therapy of cancer.

    Science.gov (United States)

    Kim, Yi Rang; Kim, Seonghoon; Choi, Jin Woo; Choi, Sung Yong; Lee, Sang-Hee; Kim, Homin; Hahn, Sei Kwang; Koh, Gou Young; Yun, Seok Hyun

    2015-01-01

    Optical energy can trigger a variety of photochemical processes useful for therapies. Owing to the shallow penetration of light in tissues, however, the clinical applications of light-activated therapies have been limited. Bioluminescence resonant energy transfer (BRET) may provide a new way of inducing photochemical activation. Here, we show that efficient bioluminescence energy-induced photodynamic therapy (PDT) of macroscopic tumors and metastases in deep tissue. For monolayer cell culture in vitro incubated with Chlorin e6, BRET energy of about 1 nJ per cell generated as strong cytotoxicity as red laser light irradiation at 2.2 mW/cm(2) for 180 s. Regional delivery of bioluminescence agents via draining lymphatic vessels killed tumor cells spread to the sentinel and secondary lymph nodes, reduced distant metastases in the lung and improved animal survival. Our results show the promising potential of novel bioluminescence-activated PDT.

  14. Expanding the bioluminescent toolkit for in vivo imaging

    OpenAIRE

    Paley, Miranda Amelia

    2014-01-01

    Bioluminescence imaging (BLI) is among the most dynamic imaging modalities for visualizing whole cells and gene expression patterns in vivo. This technique captures light emission from the luciferase-catalyzed oxidation of small molecule luciferins with highly sensitive CCD cameras. While powerful, current options for multiplexed BLI in mice are limited by the number of luciferase/luciferin pairs found in nature. Our lab aims to expand the bioluminescent toolkit by pairing mutant luciferases ...

  15. Bioluminescence-Sensing Assay for Microbial Growth Recognition

    OpenAIRE

    Heba Ramadan Eed; Nora S. Abdel-Kader; Mahmoud Helmy El Tahan; Tianhong Dai; Rehab Amin

    2016-01-01

    The conventional methods for microbial viability quantification require cultivation and are laborious. There is consequently a widespread need for cultivation-free methods. The adenosine triphosphate (ATP) bioluminescence-sensing assay is considered an extremely effective biosensor; hence ATP is the energy currency of all living microbes and can be used as a rapid indicator of microbial viability. We developed an ATP bioluminescence-sensing assay to detect microbial viability. A biolumine...

  16. Real-Time Bioluminescence Imaging of Nitroreductase in Mouse Model.

    Science.gov (United States)

    Feng, Ping; Zhang, Huateng; Deng, Quankun; Liu, Wei; Yang, Linghui; Li, Guobo; Chen, Guo; Du, Lupei; Ke, Bowen; Li, Minyong

    2016-06-01

    Nitroreductase (NTR) is an endogenous reductase overexpressed in hypoxic tumors; however, its precise detection in living cells and animals remains a considerable challenge. Herein, we developed three reaction-based probes and a related bioluminescence assay for the real-time NTR detection. The high sensitivity and selectivity of probe 3, combined with its remarkable potential of bioluminescence imaging, affords a valuable approach for in vivo imaging of NTR in a tumor model mouse.

  17. BIOLUMINESCENCE: TEACHING BIOCHEMISTRY BEYOND THE UNIVERSITY WALLS

    Directory of Open Access Journals (Sweden)

    Ana Paula Jesus de Almeida

    2016-11-01

    Full Text Available INTRODUCTION: The use of video in teaching and learning processes provides a challenging environment, able to stimulate the intellect and facilitate understanding in life science studies. Videos can be of extraordinary importance in education and dissemination of knowledge, contributing to greater learning, but is rarely used and exploited properly, especially for teaching biochemistry. Biochemistry is considered complex because it involves many molecular structures and processes, especially considering the number of events and molecules involved in the metabolism. OBJECTIVES: This study aimed to introduce biochemistry for the students of basic education using the theme "Light, Science and Life" in a playful and fun way. MATERIALS AND METHODS: A video about bioluminescence was designed and prepared aiming to use it as a support for learning biochemistry by students of basic education of public schools located in Salvador, Bahia. In order to prepare the video, undergraduate students initially revised the literature in order to acquire proper knowledge, and along with their teacher advisor worked the elaboration of texts, textbook and questionnaire and applied at school. DISCUSSION AND RESULTS: Analysis the qualitative results of the experiment on the preparation and use of the video about "Bioluminescence" focused mainly on the content of biochemistry linked to theme Light, Science and Life, and demonstrated the importance of such work in the teaching-learning process. The dynamics used allowed greater interaction between students and teacher, and the teaching of biochemistry in a fun way beyond the university walls. CONCLUSION: The teaching through recreational resources, e.g. videos and other educational strategies that foster learning should be encouraged from basic education, always bearing in order to transmit through these teaching methods the main concepts covered in biochemistry.

  18. Bioluminescence imaging of Chlamydia muridarum ascending infection in mice.

    Directory of Open Access Journals (Sweden)

    Jessica Campbell

    Full Text Available Chlamydial pathogenicity in the upper genital tract relies on chlamydial ascending from the lower genital tract. To monitor chlamydial ascension, we engineered a luciferase-expressing C. muridarum. In cells infected with the luciferase-expressing C. muridarum, luciferase gene expression and enzymatic activity (measured as bioluminescence intensity correlated well along the infection course, suggesting that bioluminescence can be used for monitoring chlamydial replication. Following an intravaginal inoculation with the luciferase-expressing C. muridarum, 8 of 10 mice displayed bioluminescence signal in the lower with 4 also in the upper genital tracts on day 3 after infection. By day 7, all 10 mice developed bioluminescence signal in the upper genital tracts. The bioluminescence signal was maintained in the upper genital tract in 6 and 2 mice by days 14 and 21, respectively. The bioluminescence signal was no longer detectable in any of the mice by day 28. The whole body imaging approach also revealed an unexpected airway infection following the intravaginal inoculation. Although the concomitant airway infection was transient and did not significantly alter the genital tract infection time courses, caution should be taken during data interpretation. The above observations have demonstrated that C. muridarum can not only achieve rapid ascending infection in the genital tract but also cause airway infection following a genital tract inoculation. These findings have laid a foundation for further optimizing the C. muridarum intravaginal infection murine model for understanding chlamydial pathogenic mechanisms.

  19. Bioluminescence imaging of Chlamydia muridarum ascending infection in mice.

    Science.gov (United States)

    Campbell, Jessica; Huang, Yumeng; Liu, Yuanjun; Schenken, Robert; Arulanandam, Bernard; Zhong, Guangming

    2014-01-01

    Chlamydial pathogenicity in the upper genital tract relies on chlamydial ascending from the lower genital tract. To monitor chlamydial ascension, we engineered a luciferase-expressing C. muridarum. In cells infected with the luciferase-expressing C. muridarum, luciferase gene expression and enzymatic activity (measured as bioluminescence intensity) correlated well along the infection course, suggesting that bioluminescence can be used for monitoring chlamydial replication. Following an intravaginal inoculation with the luciferase-expressing C. muridarum, 8 of 10 mice displayed bioluminescence signal in the lower with 4 also in the upper genital tracts on day 3 after infection. By day 7, all 10 mice developed bioluminescence signal in the upper genital tracts. The bioluminescence signal was maintained in the upper genital tract in 6 and 2 mice by days 14 and 21, respectively. The bioluminescence signal was no longer detectable in any of the mice by day 28. The whole body imaging approach also revealed an unexpected airway infection following the intravaginal inoculation. Although the concomitant airway infection was transient and did not significantly alter the genital tract infection time courses, caution should be taken during data interpretation. The above observations have demonstrated that C. muridarum can not only achieve rapid ascending infection in the genital tract but also cause airway infection following a genital tract inoculation. These findings have laid a foundation for further optimizing the C. muridarum intravaginal infection murine model for understanding chlamydial pathogenic mechanisms.

  20. Stimulation of bioluminescence in Noctiluca sp. using controlled temperature changes.

    Science.gov (United States)

    Han, Jing; Li, GuiJuan; Liu, HuanYing; Hu, HaoHao; Zhang, XueGang

    2013-01-01

    Bioluminescence induced by multifarious stimuli has long been observed and is remains under investigation because of its great complexity. In particular, the exact mechanism underlying bioluminescence is not yet fully understood. This work presents a new experimental method for studying Noctiluca sp. bioluminescence under temperature change stimulation. It is a study of Noctiluca sp. bioluminescence using controlled temperature changes in a tank. A characteristic of this experiment is the large volume of water used (1 m(3) in a tank of 2 × 1 × 1 m). Temperature changes were controlled by two methods. In the first, a flask filled with hot water was introduced into the tank and in the second, a water heater was used in the tank. Temperature changes were recorded using sensors. Noctiluca sp. bioluminescence was recorded using a Canon 5D Mark II and this allowed the characteristics of Noctiluca sp. bioluminescence under temperature change stimulation to be monitored.

  1. Bioluminescence tracking of alginate micro-encapsulated cell transplants.

    Science.gov (United States)

    Tiernan, Aubrey R; Sambanis, Athanassios

    2017-02-01

    Cell-based therapies to treat loss-of-function hormonal disorders such as diabetes and Parkinson's disease are routinely coupled with encapsulation strategies, but an understanding of when and why grafts fail in vivo is lacking. Consequently, investigators cannot clearly define the key factors that influence graft success. Although bioluminescence is a popular method to track the survival of free cells transplanted in preclinical models, little is known of the ability to use bioluminescence for real-time tracking of microencapsulated cells. Furthermore, the impact that dynamic imaging distances may have, due to freely-floating microcapsules in vivo, on cell survival monitoring is unknown. This work addresses these questions by applying bioluminescence to a pancreatic substitute based on microencapsulated cells. Recombinant insulin-secreting cells were transduced with a luciferase lentivirus and microencapsulated in Ba(2+) crosslinked alginate for in vitro and in vivo studies. In vitro quantitative bioluminescence monitoring was possible and viable microencapsulated cells were followed in real time under both normoxic and anoxic conditions. Although in vivo dispersion of freely-floating microcapsules in the peritoneal cavity limited the analysis to a qualitative bioluminescence evaluation, signals consistently four orders of magnitude above background were clear indicators of temporal cell survival. Strong agreement between in vivo and in vitro cell proliferation over time was discovered by making direct bioluminescence comparisons between explanted microcapsules and parallel in vitro cultures. Broader application of this bioluminescence approach to retrievable transplants, in supplement to currently used end-point physiological tests, could improve understanding and accelerate development of cell-based therapies for critical clinical applications. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Observations and Measurements of Planktonic Bioluminescence in and Around a Milky Sea

    Science.gov (United States)

    1988-03-01

    produced by plankton subjected to mechanical stimulation) can be observed from breaking wave crests and swimming shoals of fish . The Arabian Sea is...identification: growth at 4 ’C, growth at 35 ’C, ainylase, lipase , gelatinase. growth on maltose, cellobiose, gluconate, BIOLUMINESCENCE IN MILKY SEA 57...neofluar oil -immersion objective. BIOLUMINESCENCE MEASUREMENTS Surface-water bioluminescence Surface-water bioluminescence was measured continuously during

  3. Influence of antibiotic pressure on bacterial bioluminescence, with emphasis on Staphylococcus aureus

    NARCIS (Netherlands)

    Daghighi, Seyedmojtaba; Sjollema, Jelmer; Harapanahalli, Akshay; Dijkstra, Rene J. B.; van der Mei, Henny C.; Busscher, Henk J.

    2015-01-01

    Bioluminescence imaging is used for longitudinal evaluation of bacteria in live animals. Clear relations exist between bacterial numbers and their bioluminescence. However, bioluminescence images of Staphylococcus aureus Xen29, S. aureus Xen36 and Escherichia coli Xen14 grown on tryptone soy agar in

  4. Thoughts on the diversity of convergent evolution of bioluminescence on earth

    Science.gov (United States)

    Waldenmaier, Hans E.; Oliveira, Anderson G.; Stevani, Cassius V.

    2012-10-01

    The widespread independent evolution of analogous bioluminescent systems is one of the most impressive and diverse examples of convergent evolution on earth. There are roughly 30 extant bioluminescent systems that have evolved independently on Earth, with each system likely having unique enzymes responsible for catalysing the bioluminescent reaction. Bioluminescence is a chemical reaction involving a luciferin molecule and a luciferase or photoprotein that results in the emission of light. Some independent systems utilize the same luciferin, such as the use of tetrapyrrolic compounds by krill and dinoflagellates, and the wide use of coelenterazine by marine organisms, while the enzymes involved are unique. One common thread among all the different bioluminescent systems is the requirement of molecular oxygen. Bioluminescence is found in most forms of life, especially marine organisms. Bioluminescence in known to benefit the organism by: attraction, repulsion, communication, camouflage, and illumination. The marine ecosystem is significantly affected by bioluminescence, the only light found in the pelagic zone and below is from bioluminescent organisms. Transgenic bioluminescent organisms have revolutionized molecular research, medicine and the biotechnology industry. The use of bioluminescence in studying molecular pathways and disease allows for non-invasive and real-time analysis. Bioluminescence-based assays have been developed for several analytes by coupling luminescence to many enzyme-catalysed reactions.

  5. Seasonal Changes of Bioluminescence in Photosynthetic and Heterotrophic Dinoflagellates at San Clemente Island

    Science.gov (United States)

    2012-02-01

    2 Seasonal Changes of Bioluminescence in Photosynthetic and Heterotrophic Dinoflagellates at San Clemente Island David Lapota Space and Naval...Warfare Systems Center, Pacific USA 1. Introduction A significant portion of bioluminescence in all oceans is produced by dinoflagellates . Numerous...studies have documented the ubiquitous distribution of bioluminescent dinoflagellates in near surface waters (Seliger et al., 1961; Yentsch and Laird

  6. Bioluminescence in vivo imaging of autoimmune encephalomyelitis predicts disease

    Directory of Open Access Journals (Sweden)

    Steinman Lawrence

    2008-02-01

    Full Text Available Abstract Background Experimental autoimmune encephalomyelitis is a widely used animal model to understand not only multiple sclerosis but also basic principles of immunity. The disease is scored typically by observing signs of paralysis, which do not always correspond with pathological changes. Methods Experimental autoimmune encephalomyelitis was induced in transgenic mice expressing an injury responsive luciferase reporter in astrocytes (GFAP-luc. Bioluminescence in the brain and spinal cord was measured non-invasively in living mice. Mice were sacrificed at different time points to evaluate clinical and pathological changes. The correlation between bioluminescence and clinical and pathological EAE was statistically analyzed by Pearson correlation analysis. Results Bioluminescence from the brain and spinal cord correlates strongly with severity of clinical disease and a number of pathological changes in the brain in EAE. Bioluminescence at early time points also predicts severity of disease. Conclusion These results highlight the potential use of bioluminescence imaging to monitor neuroinflammation for rapid drug screening and immunological studies in EAE and suggest that similar approaches could be applied to other animal models of autoimmune and inflammatory disorders.

  7. Detection of DNA adducts by bioluminescence

    Science.gov (United States)

    Xu, Shunqing; Tan, Xianglin; Yao, Qunfeng; He, Min; Zhou, Yikai; Chen, Jian

    2001-09-01

    Luminescent assay for detection ATP is very sensitive with limitation of 10-17 moles. ATP using styrene oxide as a model carcinogen we currently apply a luminescence technique to detect the very low levels of carcinogen-DNA adducts in vitro and in vivo. The bioluminescent assay of DNA adducts entails three consecutive steps: digestion of modified DNA to adducted dinucleoside monophosphate and normal nucleotide are hydrolyzed to nucleosides (N) by nuclease P1 and prostatic acid phosphomonesterase (PAP); incorporation of (gamma) -P of ATP into normal nucleoside(N); detection of consumption of ATP by luminescence. This assay does not require separate manipulation because of the selective property of nuclease P1. One fmol of carcinogen- DNA adducts was detected by luminescent assay. A good correlation between results of luminescent assay and 32P-postlabeling procedures has been observed. We detect 1 adduct in 108 nucleotides for 10(mu) g DNA sample. The procedures of luminescent method is very simple and low- cost. IT appears applicable to the ultra sensitive detection of low levels of DNA adducts without radioactive isotope.

  8. The Expanding Toolbox of In Vivo Bioluminescent Imaging

    Science.gov (United States)

    Xu, Tingting; Close, Dan; Handagama, Winode; Marr, Enolia; Sayler, Gary; Ripp, Steven

    2016-01-01

    In vivo bioluminescent imaging (BLI) permits the visualization of engineered bioluminescence from living cells and tissues to provide a unique perspective toward the understanding of biological processes as they occur within the framework of an authentic in vivo environment. The toolbox of in vivo BLI includes an inventory of luciferase compounds capable of generating bioluminescent light signals along with sophisticated and powerful instrumentation designed to detect and quantify these light signals non-invasively as they emit from the living subject. The information acquired reveals the dynamics of a wide range of biological functions that play key roles in the physiological and pathological control of disease and its therapeutic management. This mini review provides an overview of the tools and applications central to the evolution of in vivo BLI as a core technology in the preclinical imaging disciplines. PMID:27446798

  9. In Vivo Bioluminescence Imaging for Longitudinal Monitoring of Inflammation in Animal Models of Uveitis

    Science.gov (United States)

    Gutowski, Michal B.; Wilson, Leslie; Van Gelder, Russell N.; Pepple, Kathryn L.

    2017-01-01

    Purpose We develop a quantitative bioluminescence assay for in vivo longitudinal monitoring of inflammation in animal models of uveitis. Methods Three models of experimental uveitis were induced in C57BL/6 albino mice: primed mycobacterial uveitis (PMU), endotoxin-induced uveitis (EIU), and experimental autoimmune uveitis (EAU). Intraperitoneal injection of luminol sodium salt, which emits light when oxidized, provided the bioluminescence substrate. Bioluminescence images were captured by a PerkinElmer In Vivo Imaging System (IVIS) Spectrum and total bioluminescence was analyzed using Living Image software. Bioluminescence on day zero was compared to bioluminescence on the day of peak inflammation for each model. Longitudinal bioluminescence imaging was performed in EIU and EAU. Results In the presence of luminol, intraocular inflammation generates detectable bioluminescence in three mouse models of uveitis. Peak bioluminescence in inflamed PMU eyes (1.46 × 105 photons/second [p/s]) was significantly increased over baseline (1.47 × 104 p/s, P = 0.01). Peak bioluminescence in inflamed EIU eyes (3.18 × 104 p/s) also was significantly increased over baseline (1.09 × 104 p/s, P = 0.04), and returned to near baseline levels by 48 hours. In EAU, there was a nonsignificant increase in bioluminescence at peak inflammation. Conclusions In vivo bioluminescence may be used as a noninvasive, quantitative measure of intraocular inflammation in animal models of uveitis. Primed mycobacterial uveitis and EIU are both acute models with robust anterior inflammation and demonstrated significant changes in bioluminescence corresponding with peak inflammation. Experimental autoimmune uveitis is a more indolent posterior uveitis and generated a more modest bioluminescent signal. In vivo imaging system bioluminescence is a nonlethal, quantifiable assay that can be used for monitoring inflammation in animal models of uveitis. PMID:28278321

  10. Space application research of EMCCDs for bioluminescence imaging

    Science.gov (United States)

    Zhang, Tao

    The detection of bioluminescense is widely used on the ground, while the detection of bioluminescence in space is still at the stage of detecting bright bioluminescense. With the rapid development of research in Space Life Sciences, it will be necessary to develop a detection technology to detect weak bioluminescense. Compared to other low-light detection techniques for ground, there are more advantages of EMCCDs for space application. Build a space bioluminescence imaging detection system, analysis the feasibility and capability of its will be significant. Co-Author:Xie Zongbao,Zheng Weibo

  11. Bioluminescence as the Basis for the Detection of Trichothecenes

    Science.gov (United States)

    1986-03-17

    screened for their ability to quench bioluminescence were obtained through the courtesy of Dr. Lou Carson, of the Toxicology Division of the Food and...34 Recent Adv. Phytochem . 9, 167 (1974). 13. Lyman, J. and Fleming, R.H., "Composition of Seawater," J. Mar. Res. 3, 134 (1940). 14. Mayer, C.F., "Endemic...DIELDRIN Cl CI Cl~c C 1 2I• HEPTACHLOR EPOXIDE OCTACHLOR EPOXIDE "Fig. 11 - Pesticides screened for ability to quench bioluminescence Ir £ d, PF K.I IR 10 R 125 - I ’S * N 586 9 -q

  12. A combination of NADHP and hispidin is not essential for bioluminescence in luminous fungal living gills of Mycena chlorophos.

    Science.gov (United States)

    Teranishi, Katsunori

    2017-01-05

    The chemical mechanisms underlying visible bioluminescence in the fungus Mycena chlorophos are not clear. A combination of dihydronicotinamide adenine dinucleotide phosphate (NADPH) and hispidin, which has been reported to increase the intensity of in vitro luminescence in crude cold-water extracts prepared from the bioluminescent fruiting bodies of M. chlorophos, exhibited potential bioluminescence activation in the early bioluminescence stages, in which the bioluminescence was ultra-weak, for living gills and luminescence activation for non-bioluminescent gills, which was collapsed by freezing and subsequent thawing, at all bioluminescence stages. These abilities were not evident in considerably bioluminescent gills. These abilities were blocked by trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid, which were identified as in vivo bioluminescence-activating components. Original bioluminescence and bioluminescence produced from the addition of trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid in living gills were almost completely inhibited by 10 mM NaN3 , whereas the luminescence produced form the combination of NADPH and hispidin in thawed non-bioluminescent and living gills at the early weak bioluminescence stages was not inhibited by 10 mM NaN3 . Thus, the combination of NADPH and hispidin plays different roles in luminescence systems compared with essential bioluminescence systems, and the combination of NADPH and hispidin was not essential for visible bioluminescence in living gills.

  13. Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon.

    Science.gov (United States)

    Yagur-Kroll, Sharon; Belkin, Shimshon

    2011-05-01

    Bioluminescent bacterial bioreporters harbor a fusion of bacterial bioluminescence genes (luxCDABE), acting as the reporting element, to a stress-response promoter, serving as the sensing element. Upon exposure to conditions that activate the promoter, such as an environmental stress or the presence of an inducing chemical, the promoter::reporter fusion generates a dose-dependent bioluminescent signal. In order to improve bioluminescent bioreporter performance we have split the luxCDABE genes of Photorhabdus luminescens into two smaller functional units: luxAB, that encode for the luciferase enzyme, which catalyzes the luminescence reaction, and luxCDE that encode for the enzymatic complex responsible for synthesis of the reaction's substrate, a long-chain aldehyde. The expression of each subunit was put under the control of either an inducible stress-responsive promoter or a synthetic constitutive promoter, and different combinations of the two units were tested for their response to selected chemicals in Escherichia coli. In all cases tested, the split combinations proved to be superior to the native luxCDABE configuration, suggesting an improved efficiency in the transcription and/or translation of two small gene units instead of a larger one with the same genes. The best combination was that of an inducible luxAB and a constitutive luxCDE, indicating that aldehyde availability is limited when the five genes are expressed together in E. coli, and demonstrating that improved biosensor performance may be achieved by rearrangement of the lux operon genes.

  14. Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca

    NARCIS (Netherlands)

    Eremeeva, Elena V.; Bartsev, Sergey I.; Berkel, van Willem J.H.; Vysotski, Eugene S.

    2017-01-01

    Upon binding their metal ion cofactors, Ca2+-regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca2+-regulated

  15. Bioluminescent system for dynamic imaging of cell and animal behavior

    Energy Technology Data Exchange (ETDEWEB)

    Hara-Miyauchi, Chikako [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, Saitama 351-0198 (Japan); Department of Biophysics and Biochemistry, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Tsuji, Osahiko [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo 160-8582 (Japan); Hanyu, Aki [Division of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo 135-8550 (Japan); Okada, Seiji [Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Yasuda, Akimasa [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo 160-8582 (Japan); Fukano, Takashi [Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, Saitama 351-0198 (Japan); Akazawa, Chihiro [Department of Biophysics and Biochemistry, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakamura, Masaya [Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo 160-8582 (Japan); Imamura, Takeshi [Department of Molecular Medicine for Pathogenesis, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295 (Japan); Core Research for Evolutional Science and Technology, The Japan Science and Technology Corporation, Tokyo 135-8550 (Japan); Matsuzaki, Yumi [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Okano, Hirotaka James, E-mail: hjokano@jikei.ac.jp [Department of Physiology, Keio University School of Medicine, Tokyo 160-8582 (Japan); Division of Regenerative Medicine Jikei University School of Medicine, Tokyo 150-8461 (Japan); and others

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer We combined a yellow variant of GFP and firefly luciferase to make ffLuc-cp156. Black-Right-Pointing-Pointer ffLuc-cp156 showed improved photon yield in cultured cells and transgenic mice. Black-Right-Pointing-Pointer ffLuc-cp156 enabled video-rate bioluminescence imaging of freely-moving animals. Black-Right-Pointing-Pointer ffLuc-cp156 mice enabled tracking real-time drug delivery in conscious animals. -- Abstract: The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.

  16. Monitoring Bloom Dynamics of a Common Coastal Bioluminescent Ctenophore

    Science.gov (United States)

    2010-09-30

    watershed run-off and discharge of submarine ground-water can profoundly impact growth conditions of bioluminescent plankton on very short space and...changes in marine ecosystems (Kane, 2009). Gelatinous zooplankton, such as Mnemiopsis sp., feed on mesozooplankton, with copepods being their main food

  17. Feasibility Study for a Compact, Multi-Purpose Bioluminescence Detector

    Science.gov (United States)

    1998-09-30

    which is likely to be primarily dinoflagellates, ostracods and gelatinous zooplankton, however a faster pump speed would be desirable in a profiling...and defense sectors by providing a valuable tool for biological oceanographers interested in plankton distribution patterns as well as providing...quantifying, tracking and identifying bioluminescent plankton . IEEE J. Oceanic Engineering. Widder, E.A. (1997) In situ video recordings of

  18. Boosting bioluminescence neuroimaging: an optimized protocol for brain studies.

    Science.gov (United States)

    Aswendt, Markus; Adamczak, Joanna; Couillard-Despres, Sebastien; Hoehn, Mathias

    2013-01-01

    Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (i.v., i.p., s.c.), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000-300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.

  19. Validation of constitutively expressed bioluminescent Pseudomonas aeruginosa as a rapid microbiological quantification tool.

    Science.gov (United States)

    Shah, N; Naseby, D C

    2015-06-15

    Whole cell biosensors have been extensively used for monitoring toxicity and contamination of various compounds and xenobiotics in environmental biology and microbial ecology; their application in the pharmaceutical and cosmetics industries has been limited. According to several pharmacopoeias, pharmaceutical products must be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. However there is a lack of a validated bioluminescence method. Prototype whole cell microbial biosensors have already been developed in Pseudomonas aeruginosa ATCC 9027. Validation of the bioluminescent strains was performed in accordance with the pharmacopoeia, Parenteral Drug Association and International Organisation of Standardisation. These strains demonstrated that the bioluminescent method was accurate, precise and equivalent, as compared with plate counting at a range of 10(3)-10(7) CFU/mL. Percentage recoveries using the bioluminescent method were between 70% and 130% for all bioluminescent strains and therefore the bioluminescent method was accurate according to the criteria set in PDA technical report 33. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. The lower limit of detection was 10(3) CFU/mL. Two-way ANOVA showed no significant difference between the traditional plate counting and the novel bioluminescent method for all bioluminescent strains. The bioluminescent constructs passed/exceeded pharmacopoeia-specified criteria for range, limit of detection, accuracy, precision and equivalence.

  20. BLProt: Prediction of bioluminescent proteins based on support vector machine and relieff feature selection

    KAUST Repository

    Kandaswamy, Krishna Kumar

    2011-08-17

    Background: Bioluminescence is a process in which light is emitted by a living organism. Most creatures that emit light are sea creatures, but some insects, plants, fungi etc, also emit light. The biotechnological application of bioluminescence has become routine and is considered essential for many medical and general technological advances. Identification of bioluminescent proteins is more challenging due to their poor similarity in sequence. So far, no specific method has been reported to identify bioluminescent proteins from primary sequence.Results: In this paper, we propose a novel predictive method that uses a Support Vector Machine (SVM) and physicochemical properties to predict bioluminescent proteins. BLProt was trained using a dataset consisting of 300 bioluminescent proteins and 300 non-bioluminescent proteins, and evaluated by an independent set of 141 bioluminescent proteins and 18202 non-bioluminescent proteins. To identify the most prominent features, we carried out feature selection with three different filter approaches, ReliefF, infogain, and mRMR. We selected five different feature subsets by decreasing the number of features, and the performance of each feature subset was evaluated.Conclusion: BLProt achieves 80% accuracy from training (5 fold cross-validations) and 80.06% accuracy from testing. The performance of BLProt was compared with BLAST and HMM. High prediction accuracy and successful prediction of hypothetical proteins suggests that BLProt can be a useful approach to identify bioluminescent proteins from sequence information, irrespective of their sequence similarity. 2011 Kandaswamy et al; licensee BioMed Central Ltd.

  1. Influence of antibiotic pressure on bacterial bioluminescence, with emphasis on Staphylococcus aureus.

    Science.gov (United States)

    Daghighi, Seyedmojtaba; Sjollema, Jelmer; Harapanahalli, Akshay; Dijkstra, Rene J B; van der Mei, Henny C; Busscher, Henk J

    2015-12-01

    Bioluminescence imaging is used for longitudinal evaluation of bacteria in live animals. Clear relations exist between bacterial numbers and their bioluminescence. However, bioluminescence images of Staphylococcus aureus Xen29, S. aureus Xen36 and Escherichia coli Xen14 grown on tryptone soy agar in Etests demonstrated increased bioluminescence at sub-MICs of different antibiotics. This study aimed to further evaluate the influence of antibiotic pressure on bioluminescence in S. aureus Xen29. Bioluminescence of S. aureus Xen29, grown planktonically in tryptone soy broth, was quantified in the absence and presence of different concentrations of vancomycin, ciprofloxacin, erythromycin or chloramphenicol and was related to expression of the luxA gene under antibiotic pressure measured using real-time PCR. In the absence of antibiotics, staphylococcal bioluminescence increased over time until a maximum after ca. 6h of growth, and subsequently decreased to the detection threshold after 24h of growth owing to reduced bacterial metabolic activity. Up to MICs of the antibiotics, bioluminescence increased according to a similar pattern up to 6h of growth, but after 24h bioluminescence was higher than in the absence of antibiotics. Contrary to expectations, bioluminescence per organism (CFU) after different growth periods in the absence and at MICs of different antibiotics decreased with increasing expression of luxA. Summarising, antibiotic pressure impacts the relation between CFU and bioluminescence. Under antibiotic pressure, bioluminescence is not controlled by luxA expression but by co-factors impacting the bacterial metabolic activity. This conclusion is of utmost importance when evaluating antibiotic efficacy in live animals using bioluminescent bacterial strains.

  2. Uptake kinetics and biodistribution of C-14-D-luciferin-a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction : impact on bioluminescence based reporter gene imaging

    NARCIS (Netherlands)

    Berger, Frank; Paulmurugan, Ramasamy; Bhaumik, Srabani; Gambhir, Sanjiv Sam

    2008-01-01

    Purpose Firefly luciferase catalyzes the oxidative decarboxylation of D-luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter g

  3. Modelling dinoflagellates as an approach to the seasonal forecasting of bioluminescence in the North Atlantic

    Science.gov (United States)

    Marcinko, Charlotte L. J.; Martin, Adrian P.; Allen, John T.

    2014-11-01

    Bioluminescence within ocean surface waters is of significant interest because it can enhance the study of subsurface movement and organisms. Little is known about how bioluminescence potential (BPOT) varies spatially and temporally in the open ocean. However, light emitted from dinoflagellates often dominates the stimulated bioluminescence field. As a first step towards forecasting surface ocean bioluminescence in the open ocean, a simple ecological model is developed which simulates seasonal changes in dinoflagellate abundance. How forecasting seasonal changes in BPOT may be achieved through combining such a model with relationships derived from observations is discussed and an example is given. The study illustrates a potential new approach to forecasting BPOT through explicitly modelling the population dynamics of a prolific bioluminescent phylum. The model developed here offers a promising platform for the future operational forecasting of the broad temporal changes in bioluminescence within the North Atlantic. Such forecasting of seasonal patterns could provide valuable information for the targeting of scientific field campaigns.

  4. Impact of Anesthesia Protocols on In Vivo Bioluminescent Bacteria Imaging Results.

    Directory of Open Access Journals (Sweden)

    Thomas Chuzel

    Full Text Available Infectious murine models greatly benefit from optical imaging using bioluminescent bacteria to non-invasively and repeatedly follow in vivo bacterial infection. In this context, one of the most critical parameters is the bioluminescence sensitivity to reliably detect the smallest number of bacteria. Another critical point is the anesthetic approaches that have been demonstrated to impact the bioluminescence flux emission in studies with luciferase-transfected tumor cells. However, this impact has never been assessed on bacteria bioluminescent models. To this end, we investigated the effects of four anesthesia protocols on the bioluminescence flux in a central venous catheter murine model (SKH1-hr(hr mice infected by a bioluminescent S. aureus Xen36 strain. Bioluminescence imaging was performed on mice anesthetized by either ketamine/xylazine (with or without oxygen supplementation, or isoflurane carried with air or oxygen. Total flux emission was determined in vivo daily for 3 days and ex vivo at the end of the study together with a CFU counting of the biofilm in the catheter. Bioluminescence flux differences appear between the different anesthetic protocols. Using a ketamine/xylazine anesthesia (with air, bacteria detection was impossible since the bioluminescence signal remains in the background signal. Mice anesthetized with isoflurane and oxygen led to a signal significantly higher to the background all along the kinetics. The use of isoflurane in air presents a bioluminescence signal similar to the use of ketamine/xylazine with oxygen. These data highlight the importance of oxygen to improve bioluminescence flux by bacteria with isoflurane as well as with ketamine/xylazine anesthetics. As a conclusion, we recommend the use of isoflurane anesthetic with oxygen to increase the bioluminescence sensitivity in this kind of study.

  5. Impact of Anesthesia Protocols on In Vivo Bioluminescent Bacteria Imaging Results.

    Science.gov (United States)

    Chuzel, Thomas; Sanchez, Violette; Vandamme, Marc; Martin, Stéphane; Flety, Odile; Pager, Aurélie; Chabanel, Christophe; Magnier, Luc; Foskolos, Marie; Petit, Océane; Rokbi, Bachra; Chereul, Emmanuel

    2015-01-01

    Infectious murine models greatly benefit from optical imaging using bioluminescent bacteria to non-invasively and repeatedly follow in vivo bacterial infection. In this context, one of the most critical parameters is the bioluminescence sensitivity to reliably detect the smallest number of bacteria. Another critical point is the anesthetic approaches that have been demonstrated to impact the bioluminescence flux emission in studies with luciferase-transfected tumor cells. However, this impact has never been assessed on bacteria bioluminescent models. To this end, we investigated the effects of four anesthesia protocols on the bioluminescence flux in a central venous catheter murine model (SKH1-hr(hr) mice) infected by a bioluminescent S. aureus Xen36 strain. Bioluminescence imaging was performed on mice anesthetized by either ketamine/xylazine (with or without oxygen supplementation), or isoflurane carried with air or oxygen. Total flux emission was determined in vivo daily for 3 days and ex vivo at the end of the study together with a CFU counting of the biofilm in the catheter. Bioluminescence flux differences appear between the different anesthetic protocols. Using a ketamine/xylazine anesthesia (with air), bacteria detection was impossible since the bioluminescence signal remains in the background signal. Mice anesthetized with isoflurane and oxygen led to a signal significantly higher to the background all along the kinetics. The use of isoflurane in air presents a bioluminescence signal similar to the use of ketamine/xylazine with oxygen. These data highlight the importance of oxygen to improve bioluminescence flux by bacteria with isoflurane as well as with ketamine/xylazine anesthetics. As a conclusion, we recommend the use of isoflurane anesthetic with oxygen to increase the bioluminescence sensitivity in this kind of study.

  6. An Assessment and Annotated Bibliography of Marine Bioluminescence Research: 1979-1987.

    Science.gov (United States)

    1993-01-01

    1983). Speculations on the hydrogen peroxide and the photogenic cells are Colours of Marine Bioluminescence. Abstr., 15th associated with a brown...of the taxonomic distribution of Affinity of the Reduced Riboflavin 5’-Phosphate Site. bioluminescence among various groups of organisms Biochemistry...possible biological functions for for reduced riboflavin 5’-phosphate (FMNH,). The bioluminescence are explored. The spectral emission inhibitor was

  7. A study on bioluminescence and photoluminescence in the earthworm Eisenia lucens.

    Science.gov (United States)

    Pes, O; Midlik, A; Schlaghamersky, J; Zitnan, M; Taborsky, P

    2016-02-01

    Eisenia lucens is an earthworm living in the organic soil layer of decomposing wood. When irritated, the worm expels coelomic fluid through pores in its body wall, exhibiting blue-green bioluminescence. The mechanism of the bioluminescence, which seems to be different from other bioluminescence systems of terrestrial animals, has been studied in this work. Many lines of evidence indicate that riboflavin stored in coelomycetes plays an important role in this glowing reaction.

  8. Mechanisms of bioluminescence, chemiluminescence and of their regulation. Progress report, one year period through March 1976

    Energy Technology Data Exchange (ETDEWEB)

    Seliger, H H

    1976-01-01

    Progress is reported on a 10-yr study of the production and role of excited states in biological systems and the mechanisms involved in bioluminescence and chemoluminescence. An hypothesis of the origin of bioluminescence is presented that is based on the mixed function oxygenase reaction. Techniques of absolute measurements of light intensities and spectral composition were applied in studies of bioluminescence of marine dinoflagellates and the chemiluminescence of carcinogenic polycyclic aromatic hydrocarbons as the result of enzymatic hydroxylation. (CH)

  9. Phage-amplified bioluminescent bioreporters for the detection of foodborne pathogens

    Science.gov (United States)

    Ripp, Steven; Young, Jacque C.; Ozen, Aysu; Jegier, Patricia; Johnson, Courtney; Daumer, Kathleen; Garland, Jay; Sayler, Gary S.

    2004-06-01

    The objective of this investigation is to develop a bioluminescent bioreporter system for the detection and monitoring of pathogenic microbial species. Current detection methodologies typically rely on time-consuming sample pre-enrichment steps to elevate pathogen concentrations to detectable levels or DNA based polymerase chain reaction (PCR) techniques that require extensive user training and expensive instrumentation. Detection utilizing bioluminescent bioreporter organisms, however, can provide a simple and rapid means of monitoring foodborne pathogens. Bioluminescent bioreporters are engineered to produce light in response to specific environmental inducers. The light signal is then measured with photodetector devices to generate a quantitative assessment of inducer concentration. The immediate goal of this research effort is to integrate key quorum sensing signal transduction elements into pathogen specific bacteriophages. Upon infection of a unique pathogenic species by the bacteriophages, quorum sensing signals will be generated that will subsequently stimulate bioluminescence in neighboring bioluminescent bioreporter cells. Utilizing both bacteriophages and bioluminescent bioreporters, we realize exceptional pathogen specificity while attaining enhanced bioluminescence production. This integrative approach will lead to rapid pathogen identification without requisite sample pre-enrichment. Additionally, since the bioluminescent response is completely intrinsic to the bioreporter organism, no user interventions are required for generating light signals; the protocol requires only addition of the food sample with the bacteriophage/bioluminescent bioreporter system. Measurement of light responses can be achieved using high-throughput microtiter plate readers, hand-held photomultiplier units, or microchip luminometers.

  10. Measurement of Bacterial Bioluminescence Intensity and Spectrum: Current Physical Techniques and Principles.

    Science.gov (United States)

    Jia, Kun; Ionescu, Rodica Elena

    2016-01-01

    : Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

  11. Monitoring of Bioluminescent Lactobacillus plantarum in a Complex Food Matrix

    Science.gov (United States)

    Narbad, Arjan

    2017-01-01

    A bioluminescent Lactobacillus plantarum (pLuc2) strain was constructed. The luminescent signal started to increase during the early exponential phase and reached its maximum in the mid-exponential phase in a batch culture of the strain. The signal detection sensitivity of the strain was the highest in PBS (phosphate buffered saline), followed by milk and MRS broth, indicating that the sensitivity was influenced by the matrix effect. The strain was used in millet seed fermentation which has a complex matrix and native lactic acid bacteria (LAB). The luminescent signal was gradually increased until 9 h during fermentation and abolished at 24 h, indicating that the strain could be specifically tracked in the complex matrix and microflora. Therefore, the bioluminescent labeling system can be used for monitoring LAB in food and dairy sciences and industries. PMID:28316482

  12. Fluorescence and Bioluminescence Imaging of Orthotopic Brain Tumors in Mice.

    Science.gov (United States)

    McKinnon, Emilie; Moore, Alfred; Dixit, Suraj; Zhu, Yun; Broome, Ann-Marie

    2017-01-01

    Optical imaging strategies, such as fluorescence and bioluminescence imaging, are non-invasive, in vivo whole body imaging techniques utilized to study cancer. Optical imaging is widely used in preclinical work because of its ease of use and cost-friendliness. It also provides the opportunity to study animals and biological responses longitudinally over time. Important considerations include depth of tissue penetration, photon scattering, absorption and the choice of light emitting probe, all of which affect the resolution (image quality and data information) and the signal to noise ratio of the image. We describe how to use bioluminescence and fluorescence imaging to track a chemotherapeutic delivery nanocarrier conjugated with a fluorophore to determine its localization in vivo.

  13. Bacterial bioluminescence and Gumbel statistics: From quorum sensing to correlation

    Science.gov (United States)

    Delle Side, Domenico; Velardi, Luciano; Nassisi, Vincenzo; Pennetta, Cecilia; Alifano, Pietro; Talà, Adelfia; Salvatore Tredici, Maurizio

    2013-12-01

    We show that, in particular experimental conditions, the time course of the radiant fluxes, measured from a bioluminescent emission of a Vibrio harveyi related strain, collapse after suitable rescaling onto the Gumbel distribution of extreme value theory. We argue that the activation times of the strain luminous emission follow the universal behavior described by this statistical law, in spite of the fact that no extremal process is known to occur.

  14. Bioluminescence imaging: a shining future for cardiac regeneration

    OpenAIRE

    Roura, Santiago; Gálvez-Montón, Carolina; Bayes-Genis, Antoni

    2013-01-01

    Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described...

  15. Regulation of Bioluminescence in Photobacterium leiognathi Strain KNH6

    OpenAIRE

    Dunn, Anne K.; Rader, Bethany A.; Stabb, Eric V.; Mandel, Mark J.

    2015-01-01

    Bacterial bioluminescence is taxonomically restricted to certain proteobacteria, many of which belong to the Vibrionaceae. In the most well-studied cases, pheromone signaling plays a key role in regulation of light production. However, previous reports have indicated that certain Photobacterium strains do not use this regulatory method for controlling luminescence. In this study, we combined genome sequencing with genetic approaches to characterize the regulation of luminescence in Photobacte...

  16. A human brainstem glioma xenograft model enabled for bioluminescence imaging

    OpenAIRE

    Hashizume, Rintaro; Ozawa, Tomoko; Dinca, Eduard B.; Banerjee, Anuradha; Prados, Michael D.; James, Charles D.; Gupta, Nalin

    2009-01-01

    Despite the use of radiation and chemotherapy, the prognosis for children with diffuse brainstem gliomas is extremely poor. There is a need for relevant brainstem tumor models that can be used to test new therapeutic agents and delivery systems in pre-clinical studies. We report the development of a brainstem-tumor model in rats and the application of bioluminescence imaging (BLI) for monitoring tumor growth and response to therapy as part of this model. Luciferase-modified human glioblastoma...

  17. Bioluminescence imaging of estrogen receptor activity during breast cancer progression.

    Science.gov (United States)

    Vantaggiato, Cristina; Dell'Omo, Giulia; Ramachandran, Balaji; Manni, Isabella; Radaelli, Enrico; Scanziani, Eugenio; Piaggio, Giulia; Maggi, Adriana; Ciana, Paolo

    2016-01-01

    Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation.

  18. In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy

    Science.gov (United States)

    Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

    2016-01-01

    Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner. PMID:27231601

  19. In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy.

    Science.gov (United States)

    Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

    2016-03-01

    Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner.

  20. Synchronization of circadian bioluminescence as a group-foraging strategy in cave glowworms.

    Science.gov (United States)

    Maynard, Andrew J; Merritt, David J

    2013-07-01

    Flies of the genus Arachnocampa are sit-and-lure predators that use bioluminescence to attract flying prey to their silk webs. Some species are most common in rainforest habitat and others inhabit both caves and rainforest. We have studied the circadian regulation of bioluminescence in two species: one found in subtropical rainforest with no known cave populations and the other found in temperate rainforest with large populations in limestone caves. The rainforest species is typical of most nocturnal animals in that individuals are entrained by the light:dark (LD) cycle to be active at night; in this case, their propensity to bioluminesce is greatest at night. The dual-habitat species shows an opposite phase response to the same entrainment; its bioluminescence propensity rhythm is entrained by LD exposure to peak during the day. Nevertheless, in LD environments, individuals do not bioluminesce during the day because ambient light inhibits their bioluminescence (negative masking), pushing bioluminescence into the dark period. This unusual and unexpected phenomenon could be related to their association with caves and has been suggested to be an adaptation of the circadian system that promotes synchronization of a colony's output of bioluminescence. Here, we use controlled laboratory experiments to show that individuals do synchronize their bioluminescence rhythms when in visual contact with each other. Entrainment of the bioluminescence rhythm to the biological photophase causes colony-wide synchronization, creating a daily sinusoidal rhythm of the intensity of bioluminescence in the many thousands of individuals making up a colony. This synchronization could provide a group-foraging advantage, allowing the colony to glow most brightly when the prey are most likely to be active.

  1. An adaptive regularization parameter choice strategy for multispectral bioluminescence tomography

    Energy Technology Data Exchange (ETDEWEB)

    Feng Jinchao; Qin Chenghu; Jia Kebin; Han Dong; Liu Kai; Zhu Shouping; Yang Xin; Tian Jie [Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, P. O. Box 2728, Beijing 100190 (China); College of Electronic Information and Control Engineering, Beijing University of Technology, Beijing 100124 (China); Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, P. O. Box 2728, Beijing 100190 (China); Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, P. O. Box 2728, Beijing 100190 (China) and School of Life Sciences and Technology, Xidian University, Xi' an 710071 (China)

    2011-11-15

    Purpose: Bioluminescence tomography (BLT) provides an effective tool for monitoring physiological and pathological activities in vivo. However, the measured data in bioluminescence imaging are corrupted by noise. Therefore, regularization methods are commonly used to find a regularized solution. Nevertheless, for the quality of the reconstructed bioluminescent source obtained by regularization methods, the choice of the regularization parameters is crucial. To date, the selection of regularization parameters remains challenging. With regards to the above problems, the authors proposed a BLT reconstruction algorithm with an adaptive parameter choice rule. Methods: The proposed reconstruction algorithm uses a diffusion equation for modeling the bioluminescent photon transport. The diffusion equation is solved with a finite element method. Computed tomography (CT) images provide anatomical information regarding the geometry of the small animal and its internal organs. To reduce the ill-posedness of BLT, spectral information and the optimal permissible source region are employed. Then, the relationship between the unknown source distribution and multiview and multispectral boundary measurements is established based on the finite element method and the optimal permissible source region. Since the measured data are noisy, the BLT reconstruction is formulated as l{sub 2} data fidelity and a general regularization term. When choosing the regularization parameters for BLT, an efficient model function approach is proposed, which does not require knowledge of the noise level. This approach only requests the computation of the residual and regularized solution norm. With this knowledge, we construct the model function to approximate the objective function, and the regularization parameter is updated iteratively. Results: First, the micro-CT based mouse phantom was used for simulation verification. Simulation experiments were used to illustrate why multispectral data were used

  2. Bacterial Bioluminescence: Spectral Study of the Emitters in the In Vivo Reaction

    NARCIS (Netherlands)

    Matheson, I.B.C.; Lee, J.; Muller, F.

    1981-01-01

    Transient fluorescent species are observed in the bioluminescent reactions of three reduced flavin mononucleotides with aliphatic aldehydes and oxygen, catalyzed by bacterial luciferase. In each case the fluorescence spectral distribution is similar to that of the bioluminescence but is readily dist

  3. Evaluation of ATP bioluminescence assays for potential use in a hospital setting.

    Science.gov (United States)

    Aiken, Zoie A; Wilson, Michael; Pratten, Jonathan

    2011-05-01

    ATP bioluminescence is being applied in hospitals to measure surface contamination. We compared commercial luminometers for detecting the number Staphylococcus aureus associated with surfaces. The data showed that the ATP bioluminescence methods tested were not robust enough to generate quantitative data on bacterial numbers, especially at low concentrations.

  4. Rapid antimicrobial susceptibility determination of uropathogens in clinical urine specimens by use of ATP bioluminescence.

    Science.gov (United States)

    Ivancic, Vesna; Mastali, Mitra; Percy, Neil; Gornbein, Jeffrey; Babbitt, Jane T; Li, Yang; Landaw, Elliot M; Bruckner, David A; Churchill, Bernard M; Haake, David A

    2008-04-01

    We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

  5. Long Term Dinoflagellate Bioluminescence, Chlorophyll, And Their Environmental Correlates In Southern California Coastal Waters

    Science.gov (United States)

    2012-02-01

    1 Long Term Dinoflagellate Bioluminescence, Chlorophyll, and Their Environmental Correlates in Southern California Coastal Waters David Lapota...2012 4. TITLE AND SUBTITLE Long Term Dinoflagellate Bioluminescence, Chlorophyll, And Their Environmental Correlates In Southern California... dinoflagellates were identified to the species level when possible. Chlorophyll a was extracted from the seawater samples using standard methods (APHA 1981) and

  6. Application of ATP-based bioluminescence for bioaerosol quantification: effect of sampling method.

    Science.gov (United States)

    Han, Taewon; Wren, Melody; DuBois, Kelsey; Therkorn, Jennifer; Mainelis, Gediminas

    2015-12-01

    An adenosine triphosphate (ATP)-based bioluminescence has potential to offer a quick and affordable method for quantifying bioaerosol samples. Here we report on our investigation into how different bioaerosol aerosolization parameters and sampling methods affect bioluminescence output per bacterium, and implications of that effect for bioaerosol research. Bacillus atrophaeus and Pseudomonas fluorescens bacteria were aerosolized by using a Collison nebulizer (BGI Inc., Waltham, MA) with a glass or polycarbonate jar and then collected for 15 and 60 min with: (1) Button Aerosol Sampler (SKC Inc., Eighty Four, PA) with polycarbonate, PTFE, and cellulose nitrate filters, (2) BioSampler (SKC Inc.) with 5 and 20 mL of collection liquid, and (3) our newly developed Electrostatic Precipitator with Superhydrophobic Surface (EPSS). For all aerosolization and sampling parameters we compared the ATP bioluminescence output per bacterium relative to that before aerosolization and sampling. In addition, we also determined the ATP reagent storage and preparation conditions that that do not affect the bioluminescence signal intensity. Our results show that aerosolization by a Collison nebulizer with a polycarbonate jar yields higher bioluminescence output per bacterium compared to the glass jar. Interestingly enough, the bioluminescence output by P. fluorescens increased substantially after its aerosolization compared to the fresh liquid suspension. For both test microorganisms, the bioluminescence intensity per bacterium after sampling was significantly lower than that before sampling suggesting negative effect of sampling stress on bioluminescence output. The decrease in bioluminescence intensity was more pronounces for longer sampling times and significantly and substantially depended on the sampling method. Among the investigated method, the EPSS was the least injurious for both microorganisms and sampling times. While the ATP-based bioluminescence offers a quick bioaerosol

  7. Assessing laser-tissue damage with bioluminescent imaging.

    Science.gov (United States)

    Wilmink, Gerald J; Opalenik, Susan R; Beckham, Joshua T; Davidson, Jeffrey M; Jansen, E Duco

    2006-01-01

    Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. Traditionally, human subjects, animal models, and monolayer cell cultures have been used to study wound healing, tissue damage, and cellular effects of laser radiation. Each of these models has significant limitations, and consequently, a novel skin model is needed. To this end, a highly reproducible human skin model that enables noninvasive and longitudinal studies of gene expression was sought. In this study, we present an organotypic raft model (engineered skin) used in combination with bioluminescent imaging (BLI) techniques. The efficacy of the raft model was validated and characterized by investigating the role of heat shock protein 70 (hsp70) as a sensitive marker of thermal damage. The raft model consists of human cells incorporated into an extracellular matrix. The raft cultures were transfected with an adenovirus containing a murine hsp70 promoter driving transcription of luciferase. The model enables quantitative analysis of spatiotemporal expression of proteins using BLI. Thermal stress was induced on the raft cultures by means of a constant temperature water bath or with a carbon dioxide (CO2) laser (lambda=10.6 microm, 0.679 to 2.262 Wcm2, cw, unfocused Gaussian beam, omegaL=4.5 mm, 1 min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12 h after exposure to thermal stress. A minimum irradiance of 0.679 Wcm2 activated the hsp70 response, and a higher irradiance of 2.262 Wcm2 was associated with a severe reduction in hsp70 response due to tissue ablation. Reverse transcription polymerase chain reaction demonstrated that hsp70 mRNA levels increased with prolonged heating exposures. Enzyme-linked immunosorbent protein assays confirmed that luciferase was an accurate surrogate for hsp70 intracellular protein levels. Hematoxylin

  8. Experimental Study on Bioluminescence Tomography with Multimodality Fusion

    Directory of Open Access Journals (Sweden)

    Yujie Lv

    2007-01-01

    Full Text Available To verify the influence of a priori information on the nonuniqueness problem of bioluminescence tomography (BLT, the multimodality imaging fusion based BLT experiment is performed by multiview noncontact detection mode, which incorporates the anatomical information obtained by the microCT scanner and the background optical properties based on diffuse reflectance measurements. In the reconstruction procedure, the utilization of adaptive finite element methods (FEMs and a priori permissible source region refines the reconstructed results and improves numerical robustness and efficiency. The comparison between the absence and employment of a priori information shows that multimodality imaging fusion is essential to quantitative BLT reconstruction.

  9. BIOLUMINESCENCE TOMOGRAPHY: BIOMEDICAL BACKGROUND, MATHEMATICAL THEORY, AND NUMERICAL APPROXIMATION

    Institute of Scientific and Technical Information of China (English)

    Weimin Han; Ce Wang

    2008-01-01

    Over the last couple of years molecular imaging has been rapidly developed to study physiological and pathological processes in vivo at the cellular and molecular levels. Among molecular imaging modalities, optical imaging stands out for its unique advantages, especially performance and cost-effectiveness. Bioluminescence tomography (BLT) is an emerging optical imaging mode with promising biomedical advantages. In this survey paper, we explain the biomedical significance of BLT, summarize theoretical results on the analysis and numerical solution of a diffusion based BLT model, and comment on a few extensions for the study of BLT.

  10. Bioluminescence monitor and method for enzymatic determinations. [Patents

    Energy Technology Data Exchange (ETDEWEB)

    Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

    1981-04-28

    An on-line, nonreferenced apparatus for measuring the concentration of a biomarker species in authentic biological samples in solution comprises conduit means for conducting said sample solution from a source of said solution, stream diversion means disposed within the conduit for diverting a predetermined amount of said sample for analysis, means for introducing and independently regulating the flow of one or more reactants disposed in fluid communication with said diverted stream, incubating means within the diverted stream for reacting said reactants and biomarkers to produce a bioluminescence emission, and means disposed within the diverted stream for monitoring said emission intensity which is correlatable to said biomarker concentration.

  11. Antioxidant assay using genetically engineered bioluminescent Escherichia coli

    Science.gov (United States)

    Bartolome, Amelita; Macalino, Bernadette; Pastoral, Ian Lemuel; Sevilla, Fortunato, III

    2006-02-01

    A new antioxidant activity assay based on the reactive oxygen species (ROS)-inducible bacterial strain (E. coli DPD2511) is described. The strain harbors the plasmid pKatG::luxCDABE and responds to hydrogen peroxide treatment by increasing light emission at 490 nm. Antioxidant capacity is evaluated through the ability of an agent to inhibit the hydrogen peroxide-induced bioluminescence of E. coli DPD2511. Applicability of the developed assay in detecting levels of antioxidants in various aqueous plant extracts is demonstrated. The assay was validated against 2,2-diphenylpicrylhydrazyl (DPPH) assay, a known antioxidant assay.

  12. Assessing laser-tissue damage with bioluminescent imaging

    Science.gov (United States)

    Wilmink, Gerald J.; Opalenik, Susan R.; Beckham, Josh T.; Davidson, Jeffrey M.; Jansen, Eric D.

    2006-07-01

    Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. Traditionally, human subjects, animal models, and monolayer cell cultures have been used to study wound healing, tissue damage, and cellular effects of laser radiation. Each of these models has significant limitations, and consequently, a novel skin model is needed. To this end, a highly reproducible human skin model that enables noninvasive and longitudinal studies of gene expression was sought. In this study, we present an organotypic raft model (engineered skin) used in combination with bioluminescent imaging (BLI) techniques. The efficacy of the raft model was validated and characterized by investigating the role of heat shock protein 70 (hsp70) as a sensitive marker of thermal damage. The raft model consists of human cells incorporated into an extracellular matrix. The raft cultures were transfected with an adenovirus containing a murine hsp70 promoter driving transcription of luciferase. The model enables quantitative analysis of spatiotemporal expression of proteins using BLI. Thermal stress was induced on the raft cultures by means of a constant temperature water bath or with a carbon dioxide (CO2) laser (λ=10.6 µm, 0.679 to 2.262 W/cm2, cw, unfocused Gaussian beam, ωL=4.5 mm, 1 min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12 h after exposure to thermal stress. A minimum irradiance of 0.679 W/cm2 activated the hsp70 response, and a higher irradiance of 2.262 W/cm2 was associated with a severe reduction in hsp70 response due to tissue ablation. Reverse transcription polymerase chain reaction demonstrated that hsp70 mRNA levels increased with prolonged heating exposures. Enzyme-linked immunosorbent protein assays confirmed that luciferase was an accurate surrogate for hsp70 intracellular protein levels. Hematoxylin and

  13. Experimental Study on Bioluminescence Tomography with Multimodality Fusion

    Science.gov (United States)

    Lv, Yujie; Tian, Jie; Cong, Wenxiang; Wang, Ge

    2007-01-01

    To verify the influence of a priori information on the nonuniqueness problem of bioluminescence tomography (BLT), the multimodality imaging fusion based BLT experiment is performed by multiview noncontact detection mode, which incorporates the anatomical information obtained by the microCT scanner and the background optical properties based on diffuse reflectance measurements. In the reconstruction procedure, the utilization of adaptive finite element methods (FEMs) and a priori permissible source region refines the reconstructed results and improves numerical robustness and efficiency. The comparison between the absence and employment of a priori information shows that multimodality imaging fusion is essential to quantitative BLT reconstruction. PMID:18256736

  14. Quorum sensing influences Vibrio harveyi growth rates in a manner not fully accounted for by the marker effect of bioluminescence.

    Directory of Open Access Journals (Sweden)

    Zeena E Nackerdien

    Full Text Available BACKGROUND: The light-emitting Vibrios provide excellent material for studying the interaction of cellular communication with growth rate because bioluminescence is a convenient marker for quorum sensing. However, the use of bioluminescence as a marker is complicated because bioluminescence itself may affect growth rate, e.g. by diverting energy. METHODOLOGY/PRINCIPAL FINDINGS: The marker effect was explored via growth rate studies in isogenic Vibrio harveyi (Vh strains altered in quorum sensing on the one hand, and bioluminescence on the other. By hypothesis, growth rate is energy limited: mutants deficient in quorum sensing grow faster because wild type quorum sensing unleashes bioluminescence and bioluminescence diverts energy. Findings reported here confirm a role for bioluminescence in limiting Vh growth rate, at least under the conditions tested. However, the results argue that the bioluminescence is insufficient to explain the relationship of growth rate and quorum sensing in Vh. A Vh mutant null for all genes encoding the bioluminescence pathway grew faster than wild type but not as fast as null mutants in quorum sensing. Vh quorum sensing mutants showed altered growth rates that do not always rank with their relative increase or decrease in bioluminescence. In addition, the cell-free culture fluids of a rapidly growing Vibrio parahaemolyticus (Vp strain increased the growth rate of wild type Vh without significantly altering Vh's bioluminescence. The same cell-free culture fluid increased the bioluminescence of Vh quorum mutants. CONCLUSIONS/SIGNIFICANCE: The effect of quorum sensing on Vh growth rate can be either positive or negative and includes both bioluminescence-dependent and independent components. Bioluminescence tends to slow growth rate but not enough to account for the effects of quorum sensing on growth rate.

  15. Foraging in the darkness of the Southern Ocean: influence of bioluminescence on a deep diving predator.

    Directory of Open Access Journals (Sweden)

    Jade Vacquié-Garcia

    Full Text Available How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES (Mirounga leonina have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES's main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do so, four SES were satellite-tracked during their post-breeding foraging trip and were equipped with Time-Depth-Recorders that also recorded light levels every two seconds. A total of 3386 dives were processed through a light-treatment model that detected light events higher than ambient level, i.e. bioluminescence events. The number of bioluminescence events was related to an index of foraging intensity for SES dives deep enough to avoid the influence of natural ambient light. The occurrence of bioluminescence was found to be negatively related to depth both at night and day. Foraging intensity was also positively related to bioluminescence both during day and night. This result suggests that bioluminescence likely provides SES with valuable indications of prey occurrence and might be a key element in predator-prey interactions in deep-dark marine environments.

  16. Foraging in the darkness of the Southern Ocean: influence of bioluminescence on a deep diving predator.

    Science.gov (United States)

    Vacquié-Garcia, Jade; Royer, François; Dragon, Anne-Cécile; Viviant, Morgane; Bailleul, Frédéric; Guinet, Christophe

    2012-01-01

    How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES) (Mirounga leonina) have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES's main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do so, four SES were satellite-tracked during their post-breeding foraging trip and were equipped with Time-Depth-Recorders that also recorded light levels every two seconds. A total of 3386 dives were processed through a light-treatment model that detected light events higher than ambient level, i.e. bioluminescence events. The number of bioluminescence events was related to an index of foraging intensity for SES dives deep enough to avoid the influence of natural ambient light. The occurrence of bioluminescence was found to be negatively related to depth both at night and day. Foraging intensity was also positively related to bioluminescence both during day and night. This result suggests that bioluminescence likely provides SES with valuable indications of prey occurrence and might be a key element in predator-prey interactions in deep-dark marine environments.

  17. Bioluminescence in the ghost fungus Omphalotus nidiformis does not attract potential spore dispersing insects.

    Science.gov (United States)

    Weinstein, Philip; Delean, Steven; Wood, Tom; Austin, Andrew D

    2016-12-01

    Bioluminescence has been known from fungi since ancient times, but little work has been done to establish its potential role. There is evidence that some bioluminescent fungi differentially attract potential spore-dispersing insects, and we aimed to establish if this was the case for the ghost fungus, Omphalotus nidiformis (Agaricales,Marasmiaceae), a widespread Australian temperate zone species. We examined three corroborative lines of evidence: circadian rhythmicity of bioluminescence; field-recorded insect abundance at the time of basidiome production; and attractiveness of glowing fungi to flying insects. Basidiomes glowed continuously day and night, and were present in winter (June-July) when insect abundance was low. To assess attractiveness, we deployed sticky-traps in open woodland in the absence of light pollution, in Treatment (baited with fresh bioluminescent O. nidiformis) and Control pairs, for 480 trap-hours on moonless nights. There was no statistical difference in mean insect abundance between Treatment and Control traps (mean 0.33 and 0.54 individuals per trap night, respectively). To interpret these results, we provide a brief review of competing hypotheses for fungal bioluminescence, and conclude that for some fungi, bioluminescence may be an incidental by-product of metabolism rather than conferring any selective advantage. It is possible that the role of bioluminescence differs among evolutionary lineages of fungi and/or with attributes of their growth environments that could affect spore dispersal, such as wind and insect abundance.

  18. Reporter cell activity within hydrogel constructs quantified from oxygen-independent bioluminescence.

    Science.gov (United States)

    Lambrechts, Dennis; Roeffaers, Maarten; Kerckhofs, Greet; Hofkens, Johan; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

    2014-09-01

    By providing a three-dimensional (3D) support to cells, hydrogels offer a more relevant in vivo tissue-like environment as compared to two-dimensional cell cultures. Hydrogels can be applied as screening platforms to investigate in 3D the role of biochemical and biophysical cues on cell behaviour using bioluminescent reporter cells. Gradients in oxygen concentration that result from the interplay between molecular transport and cell metabolism can however cause substantial variability in the observed bioluminescent reporter cell activity. To assess the influence of these oxygen gradients on the emitted bioluminescence for various hydrogel geometries, a combined experimental and modelling approach was implemented. We show that the applied model is able to predict oxygen gradient independent bioluminescent intensities which correlate better to the experimentally determined viable cell numbers, as compared to the experimentally measured bioluminescent intensities. By analysis of the bioluminescence reaction dynamics we obtained a quantitative description of cellular oxygen metabolism within the hydrogel, which was validated by direct measurements of oxygen concentration within the hydrogel. Bioluminescence peak intensities can therefore be used as a quantitative measurement of reporter cell activity within a hydrogel, but an unambiguous interpretation of these intensities requires a compensation for the influence of cell-induced oxygen gradients on the luciferase activity.

  19. Evaluation of an improved bioluminescence assay for the detection of bacteria in soy milk.

    Science.gov (United States)

    Shinozaki, Yohei; Sato, Jun; Igarashi, Toshinori; Suzuki, Shigeya; Nishimoto, Kazunori; Harada, Yasuhiro

    2013-01-01

    Because soy milk is nutrient rich and nearly neutral in pH, it favors the growth of microbial contaminants. To ensure that soy milk meets food-safety standards, it must be pasteurized and have its sterility confirmed. ATP bioluminescence assay has become a widely accepted means of detecting food microorganisms. However, the high background bioluminescence intensity of soy milk has rendered it unsuitable for ATP analysis. Here, we tested the efficacy of an improved pre-treated bioluminescence assay on soy milk. By comparing background bioluminescence intensities obtained by the conventional and improved methods, we demonstrated that our method significantly reduces soy milk background bioluminescence. The dose-response curve of the assay was tested with serial dilutions of Bacillus sp. culture. An extremely strong log-linear relation between the bioluminescence intensity relative light units and colony formation units CFU/ml emerged for the tested strain. The detection limit of the assay was estimated as 5.2×10(3) CFU/ml from the dose-response curve and an imposed signal limit was three times the background level. The results showed that contaminated samples could be easily detected within 24 h using our improved bioluminescence assay.

  20. Enhanced Landweber algorithm via Bregman iterations for bioluminescence tomography

    Science.gov (United States)

    Xia, Yi; Zhang, Meng

    2014-09-01

    Bioluminescence tomography (BLT) is an important optical molecular imaging modality aimed at visualizing physiological and pathological processes at cellular and molecular levels. While the forward process of light propagation is described by the diffusion approximation to radiative transfer equation, BLT is the inverse problem to reconstruct the 3D localization and quantification of internal bioluminescent sources distribution. Due to the inherent ill-posedness of the BLT problem, regularization is generally indispensable to obtain more favorable reconstruction. In particular, total variation (TV) regularization is known to be effective for piecewise-constant source distribution which can permit sharp discontinuities and preserve edges. However, total variation regularization generally suffers from the unsatisfactory staircasing effect. In this work, we introduce the Bregman iterative regularization to alleviate this degeneration and enhance the numerical reconstruction of BLT. Based on the existing Landweber method (LM), we put forward the Bregman-LM-TV algorithm for BLT. Numerical experiments are carried out and preliminary simulation results are reported to evaluate the proposed algorithms. It is found that Bregman-LM-TV can significantly outperform the individual Landweber method for BLT when the source distribution is piecewise-constant.

  1. Smartphone-based low light detection for bioluminescence application

    Science.gov (United States)

    Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

    2017-01-01

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time.

  2. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    Science.gov (United States)

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader

    2003-07-01

    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  3. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    Science.gov (United States)

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  4. Smartphone-based low light detection for bioluminescence application

    Science.gov (United States)

    Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

    2017-01-01

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time. PMID:28067287

  5. A synthetic luciferin improves in vivo bioluminescence imaging of gene expression in cardiovascular brain regions.

    Science.gov (United States)

    Simonyan, Hayk; Hurr, Chansol; Young, Colin N

    2016-10-01

    Bioluminescence imaging is an effective tool for in vivo investigation of molecular processes. We have demonstrated the applicability of bioluminescence imaging to spatiotemporally monitor gene expression in cardioregulatory brain nuclei during the development of cardiovascular disease, via incorporation of firefly luciferase into living animals, combined with exogenous d-luciferin substrate administration. Nevertheless, d-luciferin uptake into the brain tissue is low, which decreases the sensitivity of bioluminescence detection, particularly when considering small changes in gene expression in tiny central areas. Here, we tested the hypothesis that a synthetic luciferin, cyclic alkylaminoluciferin (CycLuc1), would be superior to d-luciferin for in vivo bioluminescence imaging in cardiovascular brain regions. Male C57B1/6 mice underwent targeted delivery of an adenovirus encoding the luciferase gene downstream of the CMV promoter to the subfornical organ (SFO) or paraventricular nucleus of hypothalamus (PVN), two crucial cardioregulatory neural regions. While bioluminescent signals could be obtained following d-luciferin injection (150 mg/kg), CycLuc1 administration resulted in a three- to fourfold greater bioluminescent emission from the SFO and PVN, at 10- to 20-fold lower substrate concentrations (7.5-15 mg/kg). This CycLuc1-mediated enhancement in bioluminescent emission was evident early following substrate administration (i.e., 6-10 min) and persisted for up to 1 h. When the exposure time was reduced from 60 s to 1,500 ms, minimal signal in the PVN was detectable with d-luciferin, whereas bioluminescent images could be reliably captured with CycLuc1. These findings demonstrate that bioluminescent imaging with the synthetic luciferin CycLuc1 provides an improved physiological genomics tool to investigate molecular events in discrete cardioregulatory brain nuclei.

  6. DEVELOPMENT OF A DUAL MODALITY TOMOGRAPHIC IMAGING SYSTEM FOR BIOLUMINESCENCE AND PET

    Energy Technology Data Exchange (ETDEWEB)

    CHATZIIOANNOU, ARION

    2011-12-21

    The goal of this proposal was to develop a new hybrid imaging modality capable to simultaneously image optical bioluminescence signals, as well as radionuclide emissions from the annihilation of positrons originating from molecular imaging probes in preclinical mouse models. This new technology enables the simultaneous in-vivo measurements of both emissions that could be produced from a single or a combination of two different biomarkers. It also facilitates establishing the physical limitations of bioluminescence imaging, its tomographic and spectral image reconstruction potential and the quantification of bioluminescence signals.

  7. High resolution in vitro bioluminescence imaging using a multimodal optical system

    Science.gov (United States)

    Altabella, L.; Gigliotti, C. R.; Perani, L.; Crippa, M. P.; Boschi, F.; Spinelli, A. E.

    2016-01-01

    Bioluminescence in vitro studies are usually performed with dedicated microscopes. In this work, we developed a novel image recovery algorithm and a multimodal system prototype to perform bioluminescence microscopy. We performed a feasibility study using GEANT4 Monte Carlo (MC) simulation of bioluminescent cells acquired at low SNR frames and processed using a Super Resolution Regularization Algorithm (SRRA). The method was also tested using in vitro cell acquisition. The results obtained with MC simulations showed an improvement in the spatial resolution from 90 μ m to 10 μ m and from 110 μ m to 13 μ m for in vitro imaging of mesothelioma cells.

  8. Study of firefly luciferin oxidation and isomerism as possible inhibition pathways for firefly bioluminescence

    Science.gov (United States)

    Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

    2014-01-01

    Firefly bioluminescence presents a light emitting profile with a form of a flash, due to the firefly luciferase-catalyzed formation of inhibitory products. These impair the binding of the substrate luciferin to the active site of the enzyme. However, this luciferase catalyzed pathways may not be the only ones responsible for the flash profile. The oxidation and isomerisation of the substrate luciferin lead to the formation of compounds that are also known inhibitors of firefly bioluminescence. So, the objective of this Letter was to analyze if these reactions could be capable of interfering with the bioluminescence reaction.

  9. A novel reconstruction algorithm for bioluminescent tomography based on Bayesian compressive sensing

    Science.gov (United States)

    Wang, Yaqi; Feng, Jinchao; Jia, Kebin; Sun, Zhonghua; Wei, Huijun

    2016-03-01

    Bioluminescence tomography (BLT) is becoming a promising tool because it can resolve the biodistribution of bioluminescent reporters associated with cellular and subcellular function through several millimeters with to centimeters of tissues in vivo. However, BLT reconstruction is an ill-posed problem. By incorporating sparse a priori information about bioluminescent source, enhanced image quality is obtained for sparsity based reconstruction algorithm. Therefore, sparsity based BLT reconstruction algorithm has a great potential. Here, we proposed a novel reconstruction method based on Bayesian compressive sensing and investigated its feasibility and effectiveness with a heterogeneous phantom. The results demonstrate the potential and merits of the proposed algorithm.

  10. QM/MM study on the light emitters of aequorin chemiluminescence, bioluminescence, and fluorescence: a general understanding of the bioluminescence of several marine organisms.

    Science.gov (United States)

    Chen, Shu-Feng; Ferré, Nicolas; Liu, Ya-Jun

    2013-06-24

    Aequorea victoria is a type of jellyfish that is known by its famous protein, green fluorescent protein (GFP), which has been widely used as a probe in many fields. Aequorea has another important protein, aequorin, which is one of the members of the EF-hand calcium-binding protein family. Aequorin has been used for intracellular calcium measurements for three decades, but its bioluminescence mechanism remains largely unknown. One of the important reasons is the lack of clear and reliable knowledge about the light emitters, which are complex. Several neutral and anionic forms exist in chemiexcited, bioluminescent, and fluorescent states and are connected with the H-bond network of the binding cavity in the protein. We first theoretically investigated aequorin chemiluminescence, bioluminescence, and fluorescence in real proteins by performing hybrid quantum mechanics and molecular mechanics methods combined with a molecular dynamics method. For the first time, this study reported the origin and clear differences in the chemiluminescence, bioluminescence and fluorescence of aequorin, which is important for understanding the bioluminescence not only of jellyfish, but also of many other marine organisms (that have the same coelenterazine caved in different coelenterazine-type luciferases).

  11. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Jason Alan Gruenhagen

    2003-12-12

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K{sup +} and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized Cd

  12. U-SPECT-BioFluo: an integrated radionuclide, bioluminescence, and fluorescence imaging platform

    NARCIS (Netherlands)

    Van Oosterom, M.N.; Kreuger, R.; Buckle, T.; Mahn, W.A.; Bunschoten, A.; Josephson, L.; Van Leeuwen, F.W.B.; Beekman, F.J.

    2014-01-01

    Background: In vivo bioluminescence, fluorescence, and single-photon emission computed tomography (SPECT) imaging provide complementary information about biological processes. However, to date these signatures are evaluated separately on individual preclinical systems. In this paper, we introduce a

  13. Bioluminescent luciferase-modified magnetic nanoparticles as potential imaging agents for mammalian spermatozoa detection and tracking

    Science.gov (United States)

    Background: Nanoparticles have emerged as key materials for developing applications in nanomedicine, nanobiotechnology, bioimaging and theranostics. Existing bioimaging technologies include bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs). Despite the current use of BRET-Q...

  14. Bacterial bioluminescence response to long-term exposure to reverse osmosis treated effluents from dye industries.

    Science.gov (United States)

    Ravindran, J; Manikandan, B; Shirodkar, P V; Francis, K X; Mani Murali, R; Vethamony, P

    2014-10-01

    The bacterial bioluminescence assay is one of the novel means for toxicity detection. The bioluminescence response of 2 marine bioluminescent bacteria was tested upon their long-term exposure to 9 different reverse osmosis (RO) rejects with varying chemical composition sampled from various dye industries. Bioluminescent bacteria were cultured in the RO reject samples, at different concentrations, and their growth rate and luminescence was measured for 24 h. The RO reject samples caused sublethal effects upon exposure and retarded the growth of bacteria, confirming their toxic nature. Further, continuation of the exposure showed that the initial luminescence, though reduced, recovered and increased beyond the control cultures irrespective of cell density, and finally decreased once again. The present study emphasizes the need of evolving a long-term exposure assay and shows that the method followed in this study is suitable to evaluate the toxicants that exert delayed toxicity, using lower concentrations of toxicants as well as coloured samples.

  15. Submersible Data (Dive Trackpoints) for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Data and information collected by the Johnson Sea Link II during sixteen dives of the "Bioluminescence 2009" expedition sponsored by the National Oceanic and...

  16. Ship track for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Ship track of the R/V Seward Johnson during the "Bioluminescence 2009" expedition sponsored by the National Oceanic and Atmospheric Administration (NOAA) Office of...

  17. Submersible Data (Dive Waypoints) for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Data and information collected by the submersible Johnson Sea-Link II at waypoints along its track during seventeen dives of the 2009 "Bioluminescence" expedition...

  18. Interaction between in vivo bioluminescence and extracellular electron transfer in Shewanella woodyi via charge and discharge.

    Science.gov (United States)

    Tian, Xiaochun; Zhao, Feng; You, Lexing; Wu, Xuee; Zheng, Zhiyong; Wu, Ranran; Jiang, Yanxia; Sun, Shigang

    2017-01-18

    Extracellular electron transfer (EET) and bioluminescence are both important for microbial growth and metabolism, but the mechanism of interaction between EET and bioluminescence is poorly understood. Herein, we demonstrate an exclusively respiratory luminous bacterium, Shewanella woodyi, which possesses EET ability and electron communication at the interface of S. woodyi and solid substrates via charge and discharge methods. Using an electro-chemiluminescence apparatus, our results confirmed that the FMN/FMNH2 content and the redox status of cytochrome c conjointly regulated the bioluminescence intensity when the potential of an indium-tin oxide electrode was changed. More importantly, this work revealed that there is an interaction between the redox reaction of single cells and bioluminescence of group communication via the EET pathway.

  19. Ship Sensor Observations for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Hourly measurements made by selected ship sensors on the R/V Seward Johnson during the "Bioluminescence 2009" expedition sponsored by the National Oceanic and...

  20. Dive Activities for Bioluminescence 2009 - Office of Ocean Exploration and Research

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Information about dive activities were recorded by personnel during the "Bioluminescence 2009" expedition, July 20 through 31, 2009. Additional information was...

  1. Bioluminescence: A Potentially Convergent Signature of Life in Future Exploration of Europa's Subsurface Ocean

    Science.gov (United States)

    Flores Martinez, C. L.

    2014-02-01

    This presentation deals with theoretical and evolutionary aspects pertaining to the nature and degree of biological complexity that is expectable among putative organisms on Europa. Bioluminescence is suggested as a new type of biosignature.

  2. ATP binding cassette transporters modulate both coelenterazine- and D-luciferin- based bioluminescence imaging

    OpenAIRE

    Huang, Ruimin; Vider, Jelena; Serganova, Inna; Blasberg, Ronald G.

    2011-01-01

    Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI-readout-intensity from intact living cells.

  3. Rapid Analysis of Eukaryotic Bioluminescence to Assess Potential Groundwater Contamination Events

    Directory of Open Access Journals (Sweden)

    Zacariah L. Hildenbrand

    2015-01-01

    Full Text Available Here we present data using a bioluminescent dinoflagellate, Pyrocystis lunula, in a toxicological bioassay to rapidly assess potential instances of groundwater contamination associated with natural gas extraction. P. lunula bioluminescence can be quantified using spectrophotometry as a measurement of organismal viability, with normal bioluminescent output declining with increasing concentration(s of aqueous toxicants. Glutaraldehyde and hydrochloric acid (HCl, components used in hydraulic fracturing and shale acidization, triggered significant toxicological responses in as little as 4 h. Conversely, P. lunula was not affected by the presence of arsenic, selenium, barium, and strontium, naturally occurring heavy metal ions potentially associated with unconventional drilling activities. If exogenous compounds, such as glutaraldehyde and HCl, are thought to have been introduced into groundwater, quantification of P. lunula bioluminescence after exposure to water samples can serve as a cost-effective detection and risk assessment tool to rapidly assess the impact of putative contamination events attributed to unconventional drilling activity.

  4. Filtrage et déconvolution en imagerie de bioluminescence chez le petit animal

    OpenAIRE

    Akkoul, Smaïl

    2010-01-01

    This thesis is devoted to the analysis of bioluminescence images applied to the small animal. This kind of imaging modality is used in cancerology studies. Nevertheless, some problems are related to the diffusion and the absorption of the tissues of the light of internal bioluminescent sources. In addition, system noise and the cosmic rays noise are present. This influences the quality of the images and makes it difficult to analyze. The purpose of this thesis is to overcome these disturbing ...

  5. Roles of biogenic amines in regulating bioluminescence in the Australian glowworm Arachnocampa flava.

    Science.gov (United States)

    Rigby, Lisa M; Merritt, David J

    2011-10-01

    The glowworm Arachnocampa flava is a carnivorous fly larva (Diptera) that uses light to attract prey into its web. The light organ is derived from cells of the Malpighian tubules, representing a bioluminescence system that is unique to the genus. Bioluminescence is modulated through the night although light levels change quite slowly compared with the flashing of the better-known fireflies (Coleoptera). The existing model for the neural regulation of bioluminescence in Arachnocampa, based on use of anaesthetics and ligations, is that bioluminescence is actively repressed during the non-glowing phase and the repression is partially released during the bioluminescence phase. The effect of the anaesthetic, carbon dioxide, on the isolated light organ from the present study indicates that the repression is at least partially mediated at the light organ itself rather than less directly through the central nervous system. Blocking of neural signals from the central nervous system through ligation leads to uncontrolled release of bioluminescence but light is emitted at relatively low levels compared with under anaesthesia. Candidate biogenic amines were introduced by several methods: feeding prey items injected with test solution, injecting the whole larva, injecting a ligated section containing the light organ or bathing the isolated light organ in test solution. Using these methods, dopamine, serotonin and tyramine do not affect bioluminescence output. Exposure to elevated levels of octopamine via feeding, injection or bathing of the isolated light organ indicates that it is involved in the regulation of repression. Administration of the octopamine antagonists phentolamine or mianserin results in very high bioluminescence output levels, similar to the effect of anaesthetics, but only mianserin acts directly on the light organ.

  6. A continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

    OpenAIRE

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-01-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear...

  7. ATP-Bioluminescence as a method to evaluated microbiological quality of UHT milk

    OpenAIRE

    A.F. Cunha; A.D. Lage; M.M. Pereira e Araújo; Abreu,C.F.; Tassinari,A.R.; M.A. Ferraz; K. Davenport; Cerqueira,M.M.O.P.

    2014-01-01

    New approaches are needed to quickly indicate possible contamination of UHT milk, among them the technique of ATP-Bioluminescence. Therefore, the aim of this study was to compare the results of culture methods with the results of ATP-Bioluminescence technique of 102 UHT whole milk samples incubated at 48, 72, and 168 hours. UHT milk samples were analyzed for the presence of mesophilic and psychrotrophic aerobic microorganisms using Plate Count Agar (PCA), Brain-Heart Infusion (BHI) media and ...

  8. Confocal Bioluminescence Imaging for Living Tissues with a Caged Substrate of Luciferin.

    Science.gov (United States)

    Hattori, Mitsuru; Kawamura, Genki; Kojima, Ryosuke; Kamiya, Mako; Urano, Yasuteru; Ozawa, Takeaki

    2016-06-21

    Fluorescence imaging can elucidate morphological organization and coordinal networks, but its background luminescence degrades the image contrast. Our confocal bioluminescence imaging system uses a luciferase caged substrate, with light passing through multipinhole arrays, causing bioluminescence at a focal plane. After a charge-coupled device camera captures luminescence, the imaging system acquires confocal images of multilayered cells with depth information, supporting quantitative analysis of spatial cellular localization in living tissues.

  9. Real-Time Bioluminescent Tracking of Cellular Population Dynamics

    Science.gov (United States)

    Close, Dan; Xu, Tingling; Ripp, Steven; Sayler, Gary

    2015-01-01

    Cellular population dynamics are routinely monitored across many diverse fields for a variety of purposes. In general, these dynamics are assayed either through the direct counting of cellular aliquots followed by extrapolation to the total population size, or through the monitoring of signal intensity from any number of externally stimulated reporter proteins. While both viable methods, here we describe a novel technique that allows for the automated, non-destructive tracking of cellular population dynamics in real-time. This method, which relies on the detection of a continuous bioluminescent signal produced through expression of the bacterial luciferase gene cassette, provides a low cost, low time-intensive means for generating additional data compared to alternative methods. PMID:24166372

  10. Real-Time Bioluminescent Tracking of Cellular Population Dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Close, Dan [University of Tennessee, Knoxville (UTK); Sayler, Gary Steven [ORNL; Xu, Tingting [ORNL; Ripp, Steven Anthony [ORNL

    2014-01-01

    Cellular population dynamics are routinely monitored across many diverse fields for a variety of purposes. In general, these dynamics are assayed either through the direct counting of cellular aliquots followed by extrapolation to the total population size, or through the monitoring of signal intensity from any number of externally stimulated reporter proteins. While both viable methods, here we describe a novel technique that allows for the automated, non-destructive tracking of cellular population dynamics in real-time. This method, which relies on the detection of a continuous bioluminescent signal produced through expression of the bacterial luciferase gene cassette, provides a low cost, low time-intensive means for generating additional data compared to alternative methods.

  11. Preservative efficacy screening of pharmaceutical formulations using ATP bioluminescence.

    Science.gov (United States)

    Kramer, Mateja; Suklje-Debeljak, Helena; Kmetec, Vojko

    2008-05-01

    The preservative challenge test is a method used to determine the efficacy of a preservation system in a pharmaceutical or cosmetic formulation. However, such testing is a labor-intensive, repetitive task often requiring days before results can be generated. Several alternatives to traditional colony-count techniques have been developed. A study using pure suspensions of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Candida albicans, and Aspergillus niger showed that the accuracy, repeatability, and linearity of the Pallchek luminometer ATP bioluminescence (ATP-B) system was equivalent to the traditional colony-count method. In any case, the method proved sensitive enough to follow the effect of preservatives on a number of test microorganisms, indicating the applicability of the ATP-B method for preservative screening studies in various pharmaceutical formulations.

  12. Bioluminescence microscopy: application to ATP measurements in single living cells

    Science.gov (United States)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  13. Detection of dichloromethane with a bioluminescent (lux) bacterial bioreporter.

    Science.gov (United States)

    Lopes, Nicholas; Hawkins, Shawn A; Jegier, Patricia; Menn, Fu-Min; Sayler, Gary S; Ripp, Steven

    2012-01-01

    The focus of this research effort was to develop an autonomous, inducible, lux-based bioluminescent bioreporter for the real-time detection of dichloromethane. Dichloromethane (DCM), also known as methylene chloride, is a volatile organic compound and one of the most commonly used halogenated solvents in the U.S., with applications ranging from grease and paint stripping to aerosol propellants and pharmaceutical tablet coatings. Predictably, it is released into the environment where it contaminates air and water resources. Due to its classification as a probable human carcinogen, hepatic toxin, and central nervous system effector, DCM must be carefully monitored and controlled. Methods for DCM detection usually rely on analytical techniques such as solid-phase microextraction (SPME) and capillary gas chromatography or photoacoustic environmental monitors, all of which require trained personnel and/or expensive equipment. To complement conventional monitoring practices, we have created a bioreporter for the self-directed detection of DCM by taking advantage of the evolutionary adaptation of bacteria to recognize and metabolize chemical agents. This bioreporter, Methylobacterium extorquens DCM( lux ), was engineered to contain a bioluminescent luxCDABE gene cassette derived from Photorhabdus luminescens fused downstream to the dcm dehalogenase operon, which causes the organism to generate visible light when exposed to DCM. We have demonstrated detection limits down to 1.0 ppm under vapor phase exposures and 0.1 ppm under liquid phase exposures with response times of 2.3 and 1.3 h, respectively, and with specificity towards DCM under relevant industrial environmental monitoring conditions.

  14. Whole-cell bioluminescent bioreporter sensing of foodborne toxicants

    Science.gov (United States)

    Ripp, Steve A.; Applegate, Bruce M.; Simpson, Michael L.; Sayler, Gary S.

    2001-03-01

    The presence of biologically derived toxins in foods is of utmost significance to food safety and human health concerns. Biologically active amines, referred to as biogenic amines, serve as a noteworthy example, having been implicated as the causative agent in numerous food poisoning episodes. Of the various biogenic amines encountered, histamine, putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, spermine, and spermidine are considered to be the most significant, and can be used as hygienic-quality indicators of food. Biogenic amines can be monitored using whole-cell bioluminescent bioreporters, which represent a family of genetically engineered microorganisms that generate visible light in response to specific chemical or physical agents in their environment. The light response occurs due to transcriptional activation of a genetically incorporated lux cassette, and can be measured using standard photomultiplier devices. We have successfully engineered a lux-based bioreporter capable of detecting and monitoring the biogenic amine beta-phenylethylamine. This research represents a biologically-based sensor technology that can be readily integrated into Hazard Analysis Critical Control Point programs to provide a rugged monitoring regime that can be uniformly applied for field-based and in-house laboratory quality control analyses. Since the bioreporter and biosensing elements are completely self-contained within the sensor design, this system provides ease of use, with operational capabilities realized by simply combining the food sample with the bioreporter and allowing the sensor to process the ensuing bioluminescent signal and communicate the results. The application of this technology to the critically important issue of food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biologically active toxins in food commodities.

  15. Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Golberg, Karina, E-mail: karingo@bgu.ac.il; Elbaz, Amit [Ben-Gurion University of the Negev, Avram and Stella Goldstein-Goren Department of Biotechnology Engineering (Israel); McNeil, Ronald [The Institute of Fluorescence, University of Maryland Baltimore County (United States); Kushmaro, Ariel [Ben-Gurion University of the Negev, Avram and Stella Goldstein-Goren Department of Biotechnology Engineering (Israel); Geddes, Chris D. [The Institute of Fluorescence, University of Maryland Baltimore County (United States); Marks, Robert S., E-mail: rsmarks@bgu.ac.il [Ben-Gurion University of the Negev, Avram and Stella Goldstein-Goren Department of Biotechnology Engineering (Israel)

    2014-12-15

    We report the use of bioluminescence signal enhancement via proximity to deposited silver nanoparticles for bioactive compound discovery. This approach employs a whole-cell bioreporter harboring a plasmid-borne fusion of a specific promoter incorporated with a bioluminescence reporter gene. The silver deposition process was first optimized to provide optimal nanoparticle size in the reaction time dependence with fluorescein. The use of silver deposition of 350 nm particles enabled the doubling of the bioluminescent signal amplitude by the bacterial bioreporter when compared to an untouched non-silver-deposited microtiter plate surface. This recording is carried out in the less optimal but necessary far-field distance. SEM micrographs provided a visualization of the proximity of the bioreporter to the silver nanoparticles. The electromagnetic field distributions around the nanoparticles were simulated using Finite Difference Time Domain, further suggesting a re-excitation of non-chemically excited bioluminescence in addition to metal-enhanced bioluminescence. The possibility of an antiseptic silver effect caused by such a close proximity was eliminated disregarded by the dynamic growth curves of the bioreporter strains as seen using viability staining. As a highly attractive biotechnology tool, this silver deposition technique, coupled with whole-cell sensing, enables increased bioluminescence sensitivity, making it especially useful for cases in which reporter luminescence signals are very weak.

  16. The effect of culture conditions on the mycelial growth and luminescence of naturally bioluminescent fungi.

    Science.gov (United States)

    Weitz, H J; Ballard, A L; Campbell, C D; Killham, K

    2001-08-21

    The effects of temperature, light and pH on mycelial growth and luminescence of four naturally bioluminescent fungi were investigated. Cultures of Armillaria mellea, Mycena citricolor, Omphalotus olearius and Panellus stipticus were grown at 5 degrees C, 15 degrees C, 22 degrees C and 30 degrees C, under 24 h light, 12 h light/12 h dark and 24 h dark, and at a pH ranging from 3.5 to 7 in three separate experiments. Temperature and pH had a significant effect on mycelial growth and bioluminescence, however light did not. Bioluminescence and mycelial growth were optimum at 22 degrees C and pH 3-3.5, the exception being M. citricolor for which bioluminescence and growth were optimum at pH 5-6 and pH 4, respectively. With the exception of M. citricolor, bioluminescence and mycelial growth were greater under 24 h darkness. An understanding of the effect of culture conditions on mycelial growth and luminescence is necessary for the future application of bioluminescent fungi as biosensors.

  17. A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging.

    Science.gov (United States)

    Kuchimaru, Takahiro; Iwano, Satoshi; Kiyama, Masahiro; Mitsumata, Shun; Kadonosono, Tetsuya; Niwa, Haruki; Maki, Shojiro; Kizaka-Kondoh, Shinae

    2016-06-14

    In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.

  18. Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

    Science.gov (United States)

    Golberg, Karina; Elbaz, Amit; McNeil, Ronald; Kushmaro, Ariel; Geddes, Chris D.; Marks, Robert S.

    2014-12-01

    We report the use of bioluminescence signal enhancement via proximity to deposited silver nanoparticles for bioactive compound discovery. This approach employs a whole-cell bioreporter harboring a plasmid-borne fusion of a specific promoter incorporated with a bioluminescence reporter gene. The silver deposition process was first optimized to provide optimal nanoparticle size in the reaction time dependence with fluorescein. The use of silver deposition of 350 nm particles enabled the doubling of the bioluminescent signal amplitude by the bacterial bioreporter when compared to an untouched non-silver-deposited microtiter plate surface. This recording is carried out in the less optimal but necessary far-field distance. SEM micrographs provided a visualization of the proximity of the bioreporter to the silver nanoparticles. The electromagnetic field distributions around the nanoparticles were simulated using Finite Difference Time Domain, further suggesting a re-excitation of non-chemically excited bioluminescence in addition to metal-enhanced bioluminescence. The possibility of an antiseptic silver effect caused by such a close proximity was eliminated disregarded by the dynamic growth curves of the bioreporter strains as seen using viability staining. As a highly attractive biotechnology tool, this silver deposition technique, coupled with whole-cell sensing, enables increased bioluminescence sensitivity, making it especially useful for cases in which reporter luminescence signals are very weak.

  19. Second bioluminescence-activating component in the luminous fungus Mycena chlorophos.

    Science.gov (United States)

    Teranishi, Katsunori

    2017-03-01

    Mycena chlorophos is an oxygen-dependent bioluminescent fungus. The mechanisms underlying its light emission are unknown. A component that increased the bioluminescence intensity of the immature living gills of M. chlorophos was isolated from mature M. chlorophos gills and chemically characterized. The bioluminescence-activating component was found to be trans-3,4-dihydroxycinnamic acid and its bioluminescence activation was highly structure-specific. (13) C- and (18) O-labelling studies using the immature living gills showed that trans-3,4-dihydroxycinnamic acid was synthesized from trans-4-hydroxycinnamic acid in the gills by hydroxylation with molecular oxygen as well as by the general metabolism, and trans-3,4-dihydroxycinnamic acid did not produce hispidin (detection-limit concentration: 10 pmol/1 g wet gill). Addition of 0.01 mM hispidin to the immature living gills generated no bioluminescence activation. These results suggested that the prompt bioluminescence activation resulting from addition of trans-3,4-dihydroxycinnamic acid could not be attributed to the generation of hispidin. Copyright © 2016 John Wiley & Sons, Ltd.

  20. Discovery of a glowing millipede in California and the gradual evolution of bioluminescence in Diplopoda.

    Science.gov (United States)

    Marek, Paul E; Moore, Wendy

    2015-05-19

    The rediscovery of the Californian millipede Xystocheir bistipita surprisingly reveals that the species is bioluminescent. Using molecular phylogenetics, we show that X. bistipita is the evolutionary sister group of Motyxia, the only genus of New World bioluminescent millipedes. We demonstrate that bioluminescence originated in the group's most recent common ancestor and evolved by gradual, directional change through diversification. Because bioluminescence in Motyxia has been experimentally demonstrated to be aposematic, forewarning of the animal's cyanide-based toxins, these results are contrary to aposematic theory and empirical evidence that a warning pattern cannot evolve gradually in unpalatable prey. However, gradual evolution of a warning pattern is plausible if faint light emission served another function and was co-opted as an aposematic signal later in the diversification of the genus. Luminescence in Motyxia stem-group taxa may have initially evolved to cope with reactive oxygen stress triggered by a hot, dry environment and was repurposed for aposematism by high-elevation crown-group taxa colonizing new habitats with varying levels of predation. The discovery of bioluminescence in X. bistipita and its pivotal phylogenetic location provides insight into the independent and repeated evolution of bioluminescence across the tree of life.

  1. ATP bioluminescence rapid detection of total viable count in soy sauce.

    Science.gov (United States)

    Yan, Shou-Lei; Miao, Su-Na; Deng, Shao-Ya; Zou, Min-Juan; Zhong, Fo-Sheng; Huang, Wen-Biao; Pan, Si-Yi; Wang, Qing-Zhang

    2012-01-01

    The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10(2) -3 × 10(4) CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB-941) and 96-well plates and could analyse 50-100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce.

  2. ATP-Bioluminescence as a method to evaluated microbiological quality of UHT milk

    Directory of Open Access Journals (Sweden)

    A.F. Cunha

    2014-12-01

    Full Text Available New approaches are needed to quickly indicate possible contamination of UHT milk, among them the technique of ATP-Bioluminescence. Therefore, the aim of this study was to compare the results of culture methods with the results of ATP-Bioluminescence technique of 102 UHT whole milk samples incubated at 48, 72, and 168 hours. UHT milk samples were analyzed for the presence of mesophilic and psychrotrophic aerobic microorganisms using Plate Count Agar (PCA, Brain-Heart Infusion (BHI media and PetrifilmTM Aerobic Count (AC plates. The ATP-Bioluminescence technique was applied through the Microbial Luminescent Screening (MLS system. Significant correlations were found between counts of aerobic mesophilic microorganisms on PCA, PetrifilmTM AC, BHI and results of ATP bioluminescence technique (P≤0.05. The ATP-Bioluminescence technique had higher correlation with counting method in PCA than BHI media. At lower pass/fail limits of Relative Light Units (60, 50, 45 and 40 RLU, the number of samples identified as positive increased and statistically agreed with aerobic mesophilic microorganism counts (P>0.05. For the dairy industry, the ATP-Bioluminescence technique may become an important tool that assists the official methods to quickly monitor the microbiological quality of UHT milk though this will likely require a threshold below 150 RLU.

  3. The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro.

    Science.gov (United States)

    Moriyama, Eduardo H; Niedre, Mark J; Jarvi, Mark T; Mocanu, Joseph D; Moriyama, Yumi; Subarsky, Patrick; Li, Buhong; Lilge, Lothar D; Wilson, Brian C

    2008-06-01

    Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L(luc)). We demonstrate that the bioluminescence signal decreases at pO(2) ATP due to the reduction of mitochondrial membrane potential. Hence, the data suggest that the decrease of intracellular ATP level in vitro is the limiting factor for bioluminescence reaction and so is responsible for the reduction of bioluminescence signal in 9L(luc) cells in acute hypoxia, rather than luciferase expression or oxygen itself.

  4. Bioluminescent bacteria: lux genes as environmental biosensors Bactérias bioluminescentes: os genes lux como biosensores ambientais

    Directory of Open Access Journals (Sweden)

    Vânia da Silva Nunes-Halldorson

    2003-06-01

    Full Text Available Bioluminescent bacteria are widespread in natural environments. Over the years, many researchers have been studying the physiology, biochemistry and genetic control of bacterial bioluminescence. These discoveries have revolutionized the area of Environmental Microbiology through the use of luminescent genes as biosensors for environmental studies. This paper will review the chronology of scientific discoveries on bacterial bioluminescence and the current applications of bioluminescence in environmental studies, with special emphasis on the Microtox toxicity bioassay. Also, the general ecological significance of bioluminescence will be addressed.Bactérias que emitem bioluminescência são amplamente distribuídas em ambientes naturais. Ao longo dos anos vários pesquisadores vêm estudando a fisiologia, bioquímica e controle genético da bioluminescência. Essas descobertas têm revolucionado a Área de Microbiologia Ambiental através da utilização dos genes lux como biosensores em estudos ambientais. Esta revisão examinará a cronologia de descobertas científicas da bioluminescência bacteriana e as aplicações atuais em estudos ambientais, salientando a utilização do teste de toxicidade Microtox. A significância ecológica da bioluminescência será também examinada.

  5. Molecular detection of bioluminescent dinoflagellates in surface waters of the Patagonian shelf during early austral summer 2008.

    Science.gov (United States)

    Valiadi, Martha; Painter, Stuart C; Allen, John T; Balch, William M; Iglesias-Rodriguez, M Debora

    2014-01-01

    We investigated the distribution of bioluminescent dinoflagellates in the Patagonian Shelf region using "universal" PCR primers for the dinoflagellate luciferase gene. Luciferase gene sequences and single cell PCR tests, in conjunction with taxonomic identification by microscopy, allowed us to identify and quantify bioluminescent dinoflagellates. We compared these data to coincidental discrete optical measurements of stimulable bioluminescence intensity. Molecular detection of the luciferase gene showed that bioluminescent dinoflagellates were widespread across the majority of the Patagonian Shelf region. Their presence was comparatively underestimated by optical bioluminescence measurements, whose magnitude was affected by interspecific differences in bioluminescence intensity and by the presence of other bioluminescent organisms. Molecular and microscopy data showed that the complex hydrography of the area played an important role in determining the distribution and composition of dinoflagellate populations. Dinoflagellates were absent south of the Falkland Islands where the cold, nutrient-rich, and well-mixed waters of the Falklands Current favoured diatoms instead. Diverse populations of dinoflagellates were present in the warmer, more stratified waters of the Patagonian Shelf and Falklands Current as it warmed northwards. Here, the dinoflagellate population composition could be related to distinct water masses. Our results provide new insight into the prevalence of bioluminescent dinoflagellates in Patagonian Shelf waters and demonstrate that a molecular approach to the detection of bioluminescent dinoflagellates in natural waters is a promising tool for ecological studies of these organisms.

  6. High throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence

    Science.gov (United States)

    Shultzaberger, Ryan K.; Paddock, Mark L.; Katsuki, Takeo; Greenspan, Ralph J.; Golden, Susan S.

    2016-01-01

    The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise, and generate meaningful quantitative measurements of clock output levels for advanced analysis. PMID:25662451

  7. Bioluminescent bioreporter assays for targeted detection of chemical and biological agents

    Science.gov (United States)

    Ripp, Steven; Jegier, Pat; Johnson, Courtney; Moser, Scott; Islam, Syed; Sayler, Gary

    2008-04-01

    Bioluminescent bioreporters carrying the bacterial lux gene cassette have been well established for the sensing and monitoring of select chemical agents. Their ability to generate target specific visible light signals with no requirement for extraneous additions of substrate or other hands-on manipulations affords a real-time, repetitive assaying technique that is remarkable in its simplicity and accuracy. Although the predominant application of lux-based bioluminescent bioreporters has been towards chemical compound detection, novel genetic engineering schemes are yielding a variety of new bioreporter systems that extend the lux sensing mechanism beyond mere analyte discrimination. For example, the unique specificity of bacteriophage (bacterial viruses) has been exploited in lux bioluminescent assays for specific identification of foodborne bacterial pathogens such as Escherichia coli O157:H7. With the concurrent ability to interface bioluminescent bioreporter assays onto integrated circuit microluminometers (BBICs; bioluminescent bioreporter integrated circuits), the potential exists for the development of sentinel microchips that can function as environmental monitors for multiplexed recognition of chemical and biological agents in air, food, and water. The size and portability of BBIC biosensors may ultimately provide a deployable, interactive network sensing technology adaptable towards chem/bio defense.

  8. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

    Directory of Open Access Journals (Sweden)

    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  9. Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines

    Science.gov (United States)

    Xu, Tingting; Close, Dan M.; Webb, James D.; Price, Sarah L.; Ripp, Steven A.; Sayler, Gary S.

    2013-05-01

    Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety.

  10. Spectrally resolved bioluminescence tomography with the third-order simplified spherical harmonics approximation

    Science.gov (United States)

    Lu, Yujie; Douraghy, Ali; Machado, Hidevaldo B.; Stout, David; Tian, Jie; Herschman, Harvey; Chatziioannou, Arion F.

    2009-11-01

    Bioluminescence imaging has been extensively applied to in vivo small animal imaging. Quantitative three-dimensional bioluminescent source information obtained by using bioluminescence tomography can directly and much more accurately reflect biological changes as opposed to planar bioluminescence imaging. Preliminary simulated and experimental reconstruction results demonstrate the feasibility and promise of bioluminescence tomography. However, the use of multiple approximations, particularly the diffusion approximation theory, affects the quality of in vivo small animal-based image reconstructions. In the development of new reconstruction algorithms, high-order approximation models of the radiative transfer equation and spectrally resolved data introduce new challenges to the reconstruction algorithm and speed. In this paper, an SP3-based (the third-order simplified spherical harmonics approximation) spectrally resolved reconstruction algorithm is proposed. The simple linear relationship between the unknown source distribution and the spectrally resolved data is established in this algorithm. A parallel version of this algorithm is realized, making BLT reconstruction feasible for the whole body of small animals especially for fine spatial domain discretization. In simulation validations, the proposed algorithm shows improved reconstruction quality compared with diffusion approximation-based methods when high absorption, superficial sources and detection modes are considered. In addition, comparisons between fine and coarse mesh-based BLT reconstructions show the effects of numerical errors in reconstruction image quality. Finally, BLT reconstructions using in vivo mouse experiments further demonstrate the potential and effectiveness of the SP3-based reconstruction algorithm.

  11. Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

    Science.gov (United States)

    Close, Dan M.; Patterson, Stacey S.; Ripp, Steven; Baek, Seung J.; Sanseverino, John; Sayler, Gary S.

    2010-01-01

    Background The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Methodology/Principal Findings Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. Conclusions/Significance The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies. PMID:20805991

  12. Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines

    Science.gov (United States)

    Xu, Tingting; Close, Dan M.; Webb, James D.; Price, Sarah L.; Ripp, Steven A.; Sayler, Gary S.

    2015-01-01

    Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety. PMID:26516295

  13. Noninvasive bioluminescence imaging of dengue virus infection in the brain of A129 mice.

    Science.gov (United States)

    Li, Xiao-Feng; Deng, Yong-Qiang; Zhao, Hui; Ye, Qing; Wang, Hong-Jiang; Li, Shi-Hua; Zhu, Shun-Ya; Shi, Pei-Yong; Qin, E-De; Zhang, Bo; Qin, Cheng-Feng

    2013-05-01

    Dengue virus (DENV) infection is one of the most important public health threats globally; however, no vaccines or effective antivirals are currently available. The bioluminescence imaging technique has emerged as a powerful tool for studies on viral pathogenesis in vitro and in vivo. In this study, using a recombinant DENV that stably expressed Renilla luciferase (Rluc-DENV), we used bioluminescence for imaging of DENV infection in the brain of A129 mice that lacked type I interferon receptors. Upon intracranial inoculation with Rluc-DENV, A129 mice developed typical neurological symptoms and rapidly succumbed to viral infection. Real-time bioluminescence intensity analysis revealed the replication kinetics of Rluc-DENV in the brain of A129 mice. Linear regression analyses showed a good correlation between photon flux and viral titers (R(2) = 0.9923). Finally, the bioluminescence model was validated using a known mouse monoclonal antibody, 2A10G6, and the therapeutic effects of this neutralizing antibody were readily monitored by live imaging in the same animal. The noninvasive bioluminescence imaging of DENV infection as described here shows distinct advantages over traditional animal models and provides a powerful tool for potential antiviral or vaccine assays against DENV infection in vivo.

  14. High-throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence.

    Science.gov (United States)

    Shultzaberger, Ryan K; Paddock, Mark L; Katsuki, Takeo; Greenspan, Ralph J; Golden, Susan S

    2015-01-01

    The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

  15. In vitro influence of hypoxia on bioluminescence imaging in brain tumor cells

    Science.gov (United States)

    Moriyama, Eduardo H.; Jarvi, Mark; Niedre, Mark; Mocanu, Joseph D.; Moriyama, Yumi; Li, Buhong; Lilge, Lothar; Wilson, Brian C.

    2007-02-01

    Bioluminescence Imaging (BLI) has been employed as an imaging modality to identify and characterize fundamental processes related to cancer development and response at cellular and molecular levels. This technique is based on the reaction of luciferin with oxygen in the presence of luciferase and ATP. A major concern in this technique is that tumors are generally hypoxic, either constitutively and/or as a result of treatment, therefore the oxygen available for the bioluminescence reaction could possibly be reduced to limiting levels, and thus leading to underestimation of the actual number of luciferase-labeled cells during in vivo procedures. In this report, we present the initial in vitro results of the oxygen dependence of the bioluminescence signal in rat gliosarcoma 9L cells tagged with the luciferase gene (9L luc cells). Bioluminescence photon emission from cells exposed to different oxygen tensions was detected by a sensitive CCD camera upon exposure to luciferin. The results showed that bioluminescence signal decreased at administered pO II levels below about 5%, falling by approximately 50% at 0.2% pO II. Additional experiments showed that changes in BLI was due to the cell inability to maintain normal levels of ATP during the hypoxic period reducing the ATP concentration to limiting levels for BLI.

  16. Fre Is the Major Flavin Reductase Supporting Bioluminescence from Vibrio harveyi Luciferase in Escherichia coli.

    Science.gov (United States)

    Campbell, Zachary T; Baldwin, Thomas O

    2009-03-27

    Unlike the vast majority of flavoenzymes, bacterial luciferase requires an exogenous source of reduced flavin mononucleotide for bioluminescence activity. Within bioluminescent bacterial cells, species-specific oxidoreductases are believed to provide reduced flavin for luciferase activity. The source of reduced flavin in Escherichia coli-expressing bioluminescence is not known. There are two candidate proteins potentially involved in this process in E. coli, a homolog of the Vibrio harveyi Frp oxidoreductase, NfsA, and a luxG type oxidoreductase, Fre. Using single gene knock-out strains, we show that deletion of fre decreased light output by greater than two orders of magnitude, yet had no effect on luciferase expression in E. coli. Purified Fre is capable of supporting bioluminescence in vitro with activity comparable to that with the endogenous V. harveyi reductase (Frp), using either FMN or riboflavin as substrate. In a pull-down experiment, we found that neither Fre nor Frp co-purify with luciferase. In contrast to prior work, we find no evidence for stable complex formation between luciferase and oxidoreductase. We conclude that in E. coli, an enzyme primarily responsible for riboflavin reduction (Fre) can also be utilized to support high levels of bioluminescence.

  17. A label-free bioluminescent sensor for real-time monitoring polynucleotide kinase activity.

    Science.gov (United States)

    Du, Jiao; Xu, Qinfeng; Lu, Xiaoquan; Zhang, Chun-yang

    2014-08-19

    Polynucleotide kinase (PNK) plays a crucial role in maintaining the genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5'-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by dCTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5'-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.

  18. NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.

    Science.gov (United States)

    England, Christopher G; Ehlerding, Emily B; Cai, Weibo

    2016-05-18

    The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a nonideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system.

  19. Advancing Molecular Therapies through In Vivo Bioluminescent Imaging

    Directory of Open Access Journals (Sweden)

    Anton McCaffrey

    2003-04-01

    Full Text Available Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLI is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes.

  20. A dual-color far-red to near-infrared firefly luciferin analogue designed for multiparametric bioluminescence imaging.

    Science.gov (United States)

    Jathoul, Amit P; Grounds, Helen; Anderson, James C; Pule, Martin A

    2014-11-24

    Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λ(max)=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

  1. Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process.

    Science.gov (United States)

    Trezzani, I; Nadri, M; Dorel, C; Lejeune, P; Bellalou, J; Lieto, J; Hammouri, H; Longin, R; Dhurjati, P

    2003-01-01

    On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.

  2. Design and implementation of an optical simulation environment for bioluminescent tomography studies

    Institute of Scientific and Technical Information of China (English)

    LI Hui; TIAN Jie; LUO Jie; L(U) Yujie; CONG Wenxiang; WANG Ge

    2007-01-01

    As a challenging task for bioluminescent tomography simulation, a virtual optical environment is needed to solve the forward problem accurately, that is, to achieve a high precision for bioluminescent signal synthesis on the external body surface of a small animal. The molecular optical simulation environment named MOSE is implemented using the C + + programming language and the OpenGL techniques, including a user-friendly interface with interactive tools facilitating users' operations. The accuracy of the virtual optical environment is verified by error analysis of mesh simplification and comparison between MOSE results and experimental data. This virtual optical environment is accurate, flexible and efficient to simulate the photon propagation in complicated tissues, which has a great potential to become a software platform for bioluminescent tomography studies and other molecular imaging applications.

  3. Application of ATP bioluminescence for evaluation of surface cleanliness of milking equipment.

    Science.gov (United States)

    Vilar, M J; Rodríguez-Otero, J L; Diéguez, F J; Sanjuán, M L; Yus, E

    2008-07-31

    The ATP bioluminescence method was used to evaluate the cleanliness of milking equipment surfaces (teat cup rubbers, teat dip containers, milk receivers, and pipeline joints) in dairy farms in Galicia (northwest Spain) with parlour, pipeline tie-stall or bucket tie-stall milking systems. The cleanest surfaces were teat cup rubbers. The use of non-chlorinated water for cleaning, and of pipeline or bucket tie-stall milking systems, was associated with high ATP bioluminescence values. However, ATP bioluminescence values only explained 12% of the variability in bulk-tank bacterial count; this is attributable to the importance of other factors (notably the correct functioning of the tank cooling system) for maintenance of low bacterial count.

  4. Use of Bioluminescence Markers To Detect Pseudomonas spp. in the Rhizosphere

    Science.gov (United States)

    de Weger, Letty A.; Dunbar, Paul; Mahafee, Walter F.; Lugtenberg, Ben J. J.; Sayler, Gary S.

    1991-01-01

    The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in the rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 103 to 104 CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than β-galactosidase-based systems. Images PMID:16348610

  5. A two-hour antibiotic susceptibility test by ATP-bioluminescence.

    Science.gov (United States)

    March Rosselló, Gabriel Alberto; García-Loygorri Jordán de Urries, María Cristina; Gutiérrez Rodríguez, María Purificación; Simarro Grande, María; Orduña Domingo, Antonio; Bratos Pérez, Miguel Ángel

    2016-01-01

    The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours.

  6. Monitoring of bacterial contamination of dental unit water lines using adenosine triphosphate bioluminescence.

    Science.gov (United States)

    Watanabe, A; Tamaki, N; Yokota, K; Matsuyama, M; Kokeguchi, S

    2016-12-01

    Bacterial contamination of dental unit waterlines (DUWLs) was evaluated using ATP bioluminescence analysis and a conventional culture method. Water samples (N=44) from DUWLs were investigated for heterotrophic bacteria by culture on R2A agar, which gave counts ranging from 1.4×10(3) to 2.7×10(5) cfu/mL. The ATP bioluminescence results for DUWL samples ranged from 6 to 1189 relative light units and could be obtained within 1min; these correlated well with the culture results (r=0.727-0.855). We conclude that the results of the ATP bioluminescence assay accurately reflect the results of conventional culture-based testing. This method is potentially useful for rapid and simple monitoring of DUWL bacterial contamination.

  7. Regulated bioluminescence as a tool for bioremediation process monitoring and control of bacterial cultures

    Science.gov (United States)

    Burlage, Robert S.; Heitzer, Armin; Digrazia, Philip M.

    1991-01-01

    An effective on-line monitoring technique for toxic waste bioremediation using bioluminescent microorganisms has shown great potential for the description and optimization of biological processes. The lux genes of the bacterium Vibrio fischeri are used by this species to produce visible light. The lux genes can be genetically fused to the control region of a catabolic gene, with the result that bioluminescence is produced whenever the catabolic gene is induced. Thus the detection of light from a sample indicates that genetic expression from a specific gene is occurring. This technique was used to monitor biodegradation of specific contaminants from waste sites. For these studies, fusions between the lux genes and the operons for naphthalene and toluene/xylene degradation were constructed. Strains carrying one of these fusions respond sensitively and specifically to target substrates. Bioluminescence from these cultures can be rapidly measured in a nondestructive and noninvasive manner. The potential for this technique in this and other biological systems is discussed.

  8. Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review.

    Science.gov (United States)

    Magalhães, Carla M; Esteves da Silva, Joaquim C G; Pinto da Silva, Luís

    2016-08-04

    Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi-/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi-/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.

  9. Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain.

    Science.gov (United States)

    Ideguchi, Yamato; Oshikoshi, Yuta; Ryo, Masashi; Motoki, Shogo; Kuwano, Takashi; Tezuka, Takafumi; Aoki, Setsuyuki

    2016-01-01

    We continuously monitored bioluminescence from a wild-type reporter strain of Escherichia coli (lacp::luc+/WT), which carries the promoter of the lac operon (lacp) fused with the firefly luciferase gene (luc+). This strain showed a bioluminescence burst when shifted into the stationary growth phase. Bioluminescence profiles of other wild-type reporter strains (rpsPp::luc+ and argAp::luc+) and gene-deletion reporter strains (lacp::luc+/crp- and lacp::luc+/lacI-) indicate that transcriptional regulation is not responsible for generation of the burst. Consistently, changes in the luciferase protein levels did not recapitulate the profile of the burst. On the other hand, dissolved oxygen levels increased over the period across the burst, suggesting that the burst is, at least partially, caused by an increase in intracellular oxygen levels. We discuss limits of the firefly luciferase when used as a reporter for gene expression and its potential utility for monitoring metabolic changes in cells.

  10. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    Science.gov (United States)

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  11. Characterization of an anthraquinone fluor from the bioluminescent, pelagic polychaete Tomopteris.

    Science.gov (United States)

    Francis, Warren R; Powers, Meghan L; Haddock, Steven H D

    2014-12-01

    Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report the isolation of the fluorescent yellow-orange pigment found in the luminous exudate and in the body of the animals. Liquid chromatography-mass spectrometry revealed the mass to be 270 m/z with a molecular formula of C(15)H(10)O(5), which ultimately was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence.

  12. The use of bioluminescent dinoflagellates as an environmental risk assessment tool.

    Science.gov (United States)

    Lapota, David; Osorio, Alexandra Robayo; Liao, Connie; Bjorndal, Bryan

    2007-12-01

    A novel toxicity method to determine sublethal and lethal effects of manmade contaminants on the bioluminescence output from marine dinoflagellates has been developed and tested over the course of 16 years. The toxicity system, QwikLite, was developed for the sole purpose of evaluating the potential toxicity of various materials used in bay sediments, storm water discharges, industrial discharges from Naval facilities, and antifoulant paints. Bioluminescence inhibition was observed in the following dinoflagellates: Lingulodinium polyedrum (formerly known as Gonyaulax polyedra), Ceratocorys horrida, Pyrocystis noctiluca, Pyrocystis lunula, Pyrocystis fusiformis, and Pyrophacus steinii. Cultured cells were exposed to various concentrations of contaminants from hours through 10 days. Further application with bioluminescent dinoflagellates in a variety of toxicity testing schemes have shown that these species can be used as a screening assay organism in lieu of the more costly, labor intensive bioassays presently in use.

  13. Numerical modeling of the dynamic response of a bioluminescent bacterial biosensor.

    Science.gov (United States)

    Affi, Mahmoud; Solliec, Camille; Legentilhomme, Patrick; Comiti, Jacques; Legrand, Jack; Jouanneau, Sulivan; Thouand, Gérald

    2016-12-01

    Water quality and water management are worldwide issues. The analysis of pollutants and in particular, heavy metals, is generally conducted by sensitive but expensive physicochemical methods. Other alternative methods of analysis, such as microbial biosensors, have been developed for their potential simplicity and expected moderate cost. Using a biosensor for a long time generates many changes in the growth of the immobilized bacteria and consequently alters the robustness of the detection. This work simulated the operation of a biosensor for the long-term detection of cadmium and improved our understanding of the bioluminescence reaction dynamics of bioreporter bacteria inside an agarose matrix. The choice of the numerical tools is justified by the difficulty to measure experimentally in every condition the biosensor functioning during a long time (several days). The numerical simulation of a biomass profile is made by coupling the diffusion equation and the consumption/reaction of the nutrients by the bacteria. The numerical results show very good agreement with the experimental profiles. The growth model verified that the bacterial growth is conditioned by both the diffusion and the consumption of the nutrients. Thus, there is a high bacterial density in the first millimeter of the immobilization matrix. The growth model has been very useful for the development of the bioluminescence model inside the gel and shows that a concentration of oxygen greater than or equal to 22 % of saturation is required to maintain a significant level of bioluminescence. A continuous feeding of nutrients during the process of detection of cadmium leads to a biofilm which reduces the diffusion of nutrients and restricts the presence of oxygen from the first layer of the agarose (1 mm) and affects the intensity of the bioluminescent reaction. The main advantage of this work is to link experimental works with numerical models of growth and bioluminescence in order to provide a

  14. Photon hunting in the twilight zone: visual features of mesopelagic bioluminescent sharks.

    Science.gov (United States)

    Claes, Julien M; Partridge, Julian C; Hart, Nathan S; Garza-Gisholt, Eduardo; Ho, Hsuan-Ching; Mallefet, Jérôme; Collin, Shaun P

    2014-01-01

    The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai) and one dalatiid species (Squaliolus aliae)]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer) displays two areae within its horizontal streak that likely allows detection of conspecifics' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (λmax) fall within the range 484-491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent deep

  15. Theoretical tuning of the firefly bioluminescence spectra by the modification of oxyluciferin

    Science.gov (United States)

    Cheng, Yuan-Yuan; Zhu, Jia; Liu, Ya-Jun

    2014-01-01

    Extending the firefly bioluminescence is of practical significance for the improved visualization of living cells and the development of a multicolor reporter. Tuning the color of bioluminescence in fireflies mainly involves the modification of luciferase and luciferin. In this Letter, we theoretically studied the emission spectra of 9 firefly oxyluciferin analogs in the gas phase and in solutions. Three density functionals, including B3LYP, CAM-B3LYP and M06-2X, were employed to theoretically predict the efficiently luminescent analogs. The reliable functionals for calculating the targeted systems were suggested. The luminescence efficiency, solvent effects, and substituent effects are discussed based on the calculated results.

  16. Formulation of photon diffusion from spherical bioluminescent sources in an infinite homogeneous medium

    Directory of Open Access Journals (Sweden)

    Wang Lihong V

    2004-05-01

    Full Text Available Abstract Background The bioluminescent enzyme firefly luciferase (Luc or variants of green fluorescent protein (GFP in transformed cells can be effectively used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies can be evaluated by using bioluminescent imaging. In bioluminescent imaging, light propagates in highly scattering tissue, and the diffusion approximation is sufficiently accurate to predict the imaging signal around the biological tissue. The numerical solutions to the diffusion equation take large amounts of computational time, and the studies for its analytic solutions have attracted more attention in biomedical engineering applications. Methods Biological tissue is a turbid medium that both scatters and absorbs photons. An accurate model for the propagation of photons through tissue can be adopted from transport theory, and its diffusion approximation is applied to predict the imaging signal around the biological tissue. The solution to the diffusion equation is formulated by the convolution between its Green's function and source term. The formulation of photon diffusion from spherical bioluminescent sources in an infinite homogeneous medium can be obtained to accelerate the forward simulation of bioluminescent phenomena. Results The closed form solutions have been derived for the time-dependent diffusion equation and the steady-state diffusion equation with solid and hollow spherical sources in a homogeneous medium, respectively. Meanwhile, the relationship between solutions with a solid sphere source and ones with a surface sphere source is obtained. Conclusion We have formulated solutions for the diffusion equation with solid and hollow spherical sources in an infinite homogeneous medium. These solutions have been verified by Monte Carlo simulation for use in biomedical optical imaging studies. The closed form solution is highly accurate and more

  17. The Repetitive Detection of Toluene with Bioluminescence Bioreporter Pseudomonas putida TVA8 Encapsulated in Silica Hydrogel on an Optical Fiber

    Directory of Open Access Journals (Sweden)

    Gabriela Kuncová

    2016-06-01

    Full Text Available Living cells of the lux-based bioluminescent bioreporter Pseudomonas putida TVA8 were encapsulated in a silica hydrogel attached to the distal wider end of a tapered quartz fiber. Bioluminescence of immobilized cells was induced with toluene at high (26.5 mg/L and low (5.3 mg/L concentrations. Initial bioluminescence maxima were achieved after >12 h. One week after immobilization, a biofilm-like layer of cells had formed on the surface of the silica gel. This resulted in shorter response times and more intensive bioluminescence maxima that appeared as rapidly as 2 h after toluene induction. Considerable second bioluminescence maxima were observed after inductions with 26.5 mg toluene/L. The second and third week after immobilization the biosensor repetitively and semiquantitatively detected toluene in buffered medium. Due to silica gel dissolution and biofilm detachment, the bioluminescent signal was decreasing 20–32 days after immobilization and completely extinguished after 32 days. The reproducible formation of a surface cell layer on the wider end of the tapered optical fiber can be translated to various whole cell bioluminescent biosensor devices and may serve as a platform for in-situ sensors.

  18. Polyphyly of non-bioluminescent Vibrio fischeri sharing a lux-locus deletion.

    Science.gov (United States)

    Wollenberg, M S; Preheim, S P; Polz, M F; Ruby, E G

    2012-03-01

    This study reports the first description and molecular characterization of naturally occurring, non-bioluminescent strains of Vibrio fischeri. These 'dark' V. fischeri strains remained non-bioluminescent even after treatment with both autoinducer and aldehyde, substrate additions that typically maximize light production in dim strains of luminous bacteria. Surprisingly, the entire lux locus (eight genes) was absent in over 97% of these dark V. fischeri strains. Although these strains were all collected from a Massachusetts (USA) estuary in 2007, phylogenetic reconstructions allowed us to reject the hypothesis that these newly described non-bioluminescent strains exhibit monophyly within the V. fischeri clade. These dark strains exhibited a competitive disadvantage against native bioluminescent strains when colonizing the light organ of the model V. fischeri host, the Hawaiian bobtail squid Euprymna scolopes. Significantly, we believe that the data collected in this study may suggest the first observation of a functional, parallel locus-deletion event among independent lineages of a non-pathogenic bacterial species.

  19. Bacterial bioluminescence response to long-term exposure to reverse osmosis treated effluents from dye industries

    Digital Repository Service at National Institute of Oceanography (India)

    Ravindran, J.; Manikandan, B.; Shirodkar, P.V.; Francis, K.X.; ManiMurali, R.; Vethamony, P.

    using an electrochemical biosensor with Pseudomonas putida and a bioluminescence inhibition assay with Vibrio fischeri. Anal. Bioanal. Chem. 373: 696-703. 12. Ruiz, M.J., Lopez-Jaramillo, L., Redondo, M.J., and Font, G. 1997. Toxicity assessment...

  20. Integrated visualization of multi-angle bioluminescence imaging and micro CT

    NARCIS (Netherlands)

    Kok, P.; Dijkstra, J.; Botha, C.P.; Post, F.H.; Kaijzel, E.; Que, I.; Löwik, C.W.G.M.; Reiber, J.H.C.; Lelieveldt, B.P.F.

    2007-01-01

    This paper explores new methods to visualize and fuse multi-2D bioluminescence imaging (BLI) data with structural imaging modalities such as micro CT and MR. A geometric, back-projection-based 3D reconstruction for superficial lesions from multi-2D BLI data is presented, enabling a coarse estimate o

  1. Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo

    Directory of Open Access Journals (Sweden)

    Rasko John EJ

    2010-11-01

    Full Text Available Abstract Live animal imaging is becoming an increasingly common technique for accurate and quantitative assessment of tumor burden over time. Bioluminescence imaging systems rely on a bioluminescent signal from tumor cells, typically generated from expression of the firefly luciferase gene. However, previous reports have suggested that either a high level of luciferase or the resultant light reaction produced upon addition of D-luciferin substrate can have a negative influence on tumor cell growth. To address this issue, we designed an expression vector that allows simultaneous fluorescence and luminescence imaging. Using fluorescence activated cell sorting (FACS, we generated clonal cell populations from a human breast cancer (MCF-7 and a mouse melanoma (B16-F10 cell line that stably expressed different levels of luciferase. We then compared the growth capabilities of these clones in vitro by MTT proliferation assay and in vivo by bioluminescence imaging of tumor growth in live mice. Surprisingly, we found that neither the amount of luciferase nor biophotonic activity was sufficient to inhibit tumor cell growth, in vitro or in vivo. These results suggest that luciferase toxicity is not a necessary consideration when designing bioluminescence experiments, and therefore our approach can be used to rapidly generate high levels of luciferase expression for sensitive imaging experiments.

  2. Bioluminescent Mammalian Cells Grown in Sponge Matrices to Monitor Immune Rejection

    Directory of Open Access Journals (Sweden)

    Okechukwu Ojogho

    2007-09-01

    Full Text Available The growth and bioluminescence of cells seeded in collagen and gelatin sponge matrices were compared in vitro under different conditions, and immune rejection was quantified and visualized directly in situ based on loss of bioluminescence activity. Mammalian cells expressing a Renilla luciferase complementary deoxyribonucleic acid (cDNA were used to seed collagen and gelatin sponge matrices soaked in either polylysine or gelatin to determine optimal growth conditions in vitro. The sponges were incubated in tissue culture plates for 3 weeks and received 2, 9, or 15 injections of coelenterazine. Measurements of bioluminescence activity indicated that gelatin sponges soaked in gelatin emitted the highest levels of light emission, multiple injections of coelenterazine did not affect light emission significantly, and light emission from live cells grown in sponges could be measured qualitatively but not quantitatively. Histologic analysis of sponge matrices cultured in vitro showed that cells grew best in gelatin matrices. Visualization of subcutaneously implanted sponges in mice showed accelerated loss of light emission in immunocompetent BALB/c mice compared with immunodeficient BALB/c-scid mice, which was associated with increased cell infiltration. Our results indicate that sponge matrices carrying bioluminescent mammalian cells are a valid model system to study immune rejection in situ.

  3. Real-time monitoring of cariogenic bacteria via bioluminescent imaging: A biodontic hypothesis

    Directory of Open Access Journals (Sweden)

    Jafar Kolahi

    2016-01-01

    Full Text Available Introduction: Dental caries (tooth decay remains one of the most common chronic infectious disease in the world. Disclosure of camouflaged cariogenic bacteria will be a great motivation for better oral hygiene. The Hypothesis: At present, lux transposon cassette, Tn4001 luxABCDE Kmr, is available that could be used for stable bioluminescent transformation of a wide range of gram-positive bacteria, e.g. Streptococcus mutans and Lactobacillus. After this step, sensitive charge-coupled device (CCD camera could be used to detect the low levels of light emitted from bioluminescent cariogenic bacteria. Living imaging software would be used for analysis and three-dimensional (3D reconstruction of images. Evaluation of the Hypothesis: Entrance of transgenic organisms into the oral cavity should be done with great caution. Ethical consideration is necessary and primary animal studies are required. The main limitation of this technique will be oxygen. As mentioned previously, bioluminescent reactions need oxygen. Hence, bioluminescent imaging cannot be used for anaerobic bacteria, e.g., Streptococcus sobrinus.

  4. Hypothesis about brilliant lights by bioluminescent photons in near death experiences.

    Science.gov (United States)

    Bókkon, István; Salari, Vahid

    2012-07-01

    In near death experiences (NDEs), seeing a brilliant light may arise in the recovery period following cardiac arrest, but the subjects can think that these experiences had happened during the actual period itself. Here we hypothesize a biophysical explanation about the encounter with a brilliant light in NDEs. Accordingly, meeting brilliant light in NDEs is due to the reperfusion that induces unregulated overproduction of free radicals and excited biomolecules among them in numerous parts in the visual system. Unregulated free radicals and excited species can produce a transient increase of bioluminescent photons in different areas of the visual system. If this excess of bioluminescent photon emission exceeds a threshold, they can appear as (phosphene) lights in our mind. In other words, seeing a brilliant light in NDEs may due to bioluminescent photons simultaneously generated in the recovery phase of numerous areas of the visual system and the brain interprets these intrinsic bioluminescent photons as if they were originated from the external visual world. Although our biophysical explanation about brilliant light phenomenon in NDEs can be promising, we do not reject further potential notions.

  5. Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

    Science.gov (United States)

    Xu, Tingting; Close, Dan M.; Webb, James D.; Ripp, Steven A.; Sayler, Gary S.

    2013-01-01

    Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion. PMID:24193545

  6. Monitoring the Response of Hyperbilirubinemia in the Mouse Brain by In Vivo Bioluminescence Imaging.

    Science.gov (United States)

    Manni, Isabella; Di Rocco, Giuliana; Fusco, Salvatore; Leone, Lucia; Barbati, Saviana Antonella; Carapella, Carmine Maria; Grassi, Claudio; Piaggio, Giulia; Toietta, Gabriele

    2016-12-28

    Increased levels of unconjugated bilirubin are neurotoxic, but the mechanism leading to neurological damage has not been completely elucidated. Innovative strategies of investigation are needed to more precisely define this pathological process. By longitudinal in vivo bioluminescence imaging, we noninvasively visualized the brain response to hyperbilirubinemia in the MITO-Luc mouse, in which light emission is restricted to the regions of active cell proliferation. We assessed that acute hyperbilirubinemia promotes bioluminescence in the brain region, indicating an increment in the cell proliferation rate. Immunohistochemical detection in brain sections of cells positive for both luciferase and the microglial marker allograft inflammatory factor 1 suggests proliferation of microglial cells. In addition, we demonstrated that brain induction of bioluminescence was altered by pharmacological displacement of bilirubin from its albumin binding sites and by modulation of the blood-brain barrier permeability, all pivotal factors in the development of bilirubin-induced neurologic dysfunction. We also determined that treatment with minocycline, an antibiotic with anti-inflammatory and neuroprotective properties, or administration of bevacizumab, an anti-vascular endothelial growth factor antibody, blunts bilirubin-induced bioluminescence. Overall the study supports the use of the MITO-Luc mouse as a valuable tool for the rapid response monitoring of drugs aiming at preventing acute bilirubin-induced neurological dysfunction.

  7. Bioluminophore and Flavin Mononucleotide Fluorescence Quenching of Bacterial Bioluminescence-A Theoretical Study.

    Science.gov (United States)

    Luo, Yanling; Liu, Ya-Jun

    2016-11-02

    Bacterial bioluminescence with continuous glow has been applied to the fields of environmental toxin monitoring, drug screening, and in vivo imaging. Nonetheless, the chemical form of the bacterial bioluminophore is still a bone of contention. Flavin mononucleotide (FMN), one of the light-emitting products, and 4a-hydroxy-5-hydro flavin mononucleotide (HFOH), an intermediate of the chemical reactions, have both been assumed candidates for the light emitter because they have similar molecular structures and fluorescence wavelengths. The latter is preferred in experiments and was assigned in our previous density functional study. HFOH displays weak fluorescence in solutions, but exhibits strong bioluminescence in the bacterial luciferase. FMN shows the opposite behavior; its fluorescence is quenched when it is bound to the luciferase. This is the first example of flavin fluorescence quenching observed in bioluminescent systems and is merely an observation, both the quenching mechanism and quencher are still unclear. Based on theoretical analysis of high-level quantum mechanics (QM), combined QM and molecular mechanics (QM/MM), and molecular dynamics (MD), this paper confirms that HFOH in its first singlet excited state is the bioluminophore of bacterial bioluminescence. More importantly, the computational results indicate that Tyr110 in the luciferase quenches the FMN fluorescence via an electron-transfer mechanism.

  8. Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift.

    Science.gov (United States)

    Kheirabadi, Mitra; Sharafian, Zohreh; Naderi-Manesh, Hossein; Heineman, Udo; Gohlke, Ulrich; Hosseinkhani, Saman

    2013-12-01

    Firefly bioluminescence reaction in the presence of Mg(2+), ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350-359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7Å and 2.2Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351-359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.

  9. Detection of light and vibration modulates bioluminescence intensity in the glowworm, Arachnocampa flava.

    Science.gov (United States)

    Mills, Rebecca; Popple, Julie-Anne; Veidt, Martin; Merritt, David John

    2016-04-01

    Glowworms are larval fungus gnats that emit light from a specialised abdominal light organ. The light attracts small arthropod prey to their web-like silk snares. Larvae glow throughout the night and can modulate their bioluminescence in response to sensory input. To better understand light output regulation and its ecological significance, we examined the larvae's reaction to light exposure, vibration and sound. Exposure to a 5-min light pulse in the laboratory causes larvae to exponentially decrease their light output over 5-10 min until they completely switch off. They gradually return to pre-exposure levels but do not show a rebound. Larvae are most sensitive to ultraviolet light, then blue, green and red. Vibration of the larval snares results in a several-fold increase in bioluminescence over 20-30 s, followed by an exponential return to pre-exposure levels over 15-30 min. Under some conditions, larvae can respond to vibration by initiating bioluminescence when they are not glowing; however, the response is reduced compared to when they are glowing. We propose that inhibitory and excitatory mechanisms combine to modulate bioluminescence intensity by regulating biochemical reactions or gating the access of air to the light organ.

  10. Total evidence phylogeny and the evolution of adult bioluminescence in fireflies (Coleoptera: Lampyridae).

    Science.gov (United States)

    Martin, Gavin J; Branham, Marc A; Whiting, Michael F; Bybee, Seth M

    2017-02-01

    Fireflies are some of the most captivating organisms on the planet. They have a rich history as subjects of scientific study, especially in relation to their bioluminescent behavior. Yet, the phylogenetic relationships of fireflies are still poorly understood. Here, we present the first total evidence approach to reconstruct lampyrid phylogeny using both a molecular matrix from six loci and an extensive morphological matrix. Using this phylogeny we test the hypothesis that adult bioluminescence evolved after the origin of the firefly clade. The ancestral state of adult bioluminescence is recovered as non-bioluminescent with one to six gains and five to ten subsequent losses. The monophyly of the family, as well as the subfamilies is also tested. Ototretinae, Cyphonocerinae, Luciolinae (incl. Pristolycus), Amydetinae, "cheguevarinae" sensu Jeng 2008, and Photurinae are highly supported as monophyletic. With the exception of four taxa, Lampyrinae is also recovered as monophyletic with high support. Based on phylogenetic and morphological data Lamprohiza, Phausis, and Lamprigera are transferred to Lampyridae incertae sedis.

  11. Complete Genome Sequence of the Bioluminescent Marine Bacterium Vibrio harveyi ATCC 33843 (392 [MAV])

    OpenAIRE

    Wang, Zheng; Hervey, W. Judson; Kim, Seongwon; Lin, Baochuan; Vora, Gary J.

    2015-01-01

    Vibrio harveyi is a Gram-negative marine γ-proteobacterium that is known to be a formidable pathogen of aquatic animals and is a model organism for the study of bacterial bioluminescence and quorum sensing. In this report, we describe the complete genome sequence of the most studied strain of this species: V. harveyi ATCC 33843 (392 [MAV]).

  12. Adenosine triphosphate bioluminescence analysis for rapid screening of microbial contamination in non-sterile pharmaceutical samples.

    Science.gov (United States)

    Jimenez, Luis

    2004-01-01

    An Adenosine Triphosphate (ATP) bioluminescence system was compared and validated against standard methods for rapid microbiological monitoring of several non-sterile pharmaceutical formulations such as creams, tablets, and capsules. Results obtained using 1%, 2.5%, and 10% of product suspensions indicated that most samples that did not contain non-microbial ATP neither inhibited the bioluminescence reaction nor did something else. Ten percent product suspensions were inoculated with different concentrations of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Candida albicans, and Aspergillus niger. Samples were incubated for 24-120 h at 35 degrees C with shaking. Results indicated a strong inhibitory effect of microbial growth, as no microorganisms were detected by using the ATP bioluminescence assay. However, when 1% and 2.5% product suspensions were spiked with the same microorganisms, positive detection was confirmed. After incubation, all microorganisms were detected by the bioluminescence system within 24-72 h. All positive samples were confirmed by using standard plating media. However, to optimize detection of all microorganisms, different enrichment media were developed.

  13. Bioluminescence ATP Monitoring for the Routine Assessment of Food Contact Surface Cleanliness in a University Canteen

    Directory of Open Access Journals (Sweden)

    Andrea Osimani

    2014-10-01

    Full Text Available ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs, including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99 between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene.

  14. Bioluminescence ATP monitoring for the routine assessment of food contact surface cleanliness in a university canteen.

    Science.gov (United States)

    Osimani, Andrea; Garofalo, Cristiana; Clementi, Francesca; Tavoletti, Stefano; Aquilanti, Lucia

    2014-10-17

    ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99) between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene.

  15. Determination of Complement-Mediated Killing of Bacteria by Viability Staining and Bioluminescence

    Science.gov (United States)

    Virta, Marko; Lineri, Sanna; Kankaanpää, Pasi; Karp, Matti; Peltonen, Karita; Nuutila, Jari; Lilius, Esa-Matti

    1998-01-01

    Complement-mediated killing of bacteria was monitored by flow cytometric, luminometric, and conventional plate counting methods. A flow cytometric determination of bacterial viability was carried out by using dual staining with a LIVE/DEAD BacLight bacterial viability kit. In addition to the viable cell population, several other populations emerged in the fluorescence histogram, and there was a dramatic decrease in the total cell count in the light-scattering histogram in the course of the complement reaction. To permit luminometric measurements, Bacillus subtilis and Escherichia coli were made bioluminescent by expressing an insect luciferase gene. Addition of substrate after the complement reaction resulted in bioluminescence, the level of which was a measure of the viable cell population. All three methods gave essentially the same killing rate, suggesting that the bacteriolytic activity of serum complement can be measured rapidly and conveniently by using viability stains or bioluminescence. In principle, any bacterial strain can be used for viability staining and flow cytometric analysis. For the bioluminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a large number of samples. PMID:9464386

  16. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    Science.gov (United States)

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

  17. Bioluminescence enhancement through an added washing protocol enabling a greater sensitivity to carbofuran toxicity.

    Science.gov (United States)

    Jia, Kun; Eltzov, Evgeni; Marks, Robert S; Ionescu, Rodica E

    2013-10-01

    The effects of carbofuran toxicity on a genetically modified bacterial strain E. coli DPD2794 were enhanced using a new bioluminescent protocol which consisted of three consecutive steps: incubation, washing and luminescence reading. Specifically, in the first step, several concentrations of carbofuran aqueous solutions were incubated with different bacterial suspensions at recorded optical densities for different lengths of time. Thereafter, the resulting bacterial/toxicant mixtures were centrifuged and the aged cellular supernatant replaced with fresh medium. In the final step, the carbofuran- induced bioluminescence to the exposed E. coli DPD2794 bacteria was shown to provide a faster and higher intensity when recorded at a higher temperature at30°C which is not usually used in the literature. It was found that the incubation time and the replacement of aged cellular medium were essential factors to distinguish different concentrations of carbofuran in the bioluminescent assays. From our results, the optimum incubation time for a "light ON" bioluminescence detection of the effect of carbofuran was 6h. Thanks to the replacement of the aged cellular medium, a group of additional peaks starting around 30min were observed and we used the corresponding areas under the curve (AUC) at different contents of carbofuran to produce the calibration curve. Based on the new protocol, a carbofuran concentration of 0.5pg/mL can be easily determined in a microtiter plate bioluminescent assay, while a non-wash protocol provides an unexplainable order of curve evolutionswhich does not allow the user to determine the concentration.

  18. Analysis of neurogenesis during experimental autoimmune encephalomyelitis reveals pitfalls of bioluminescence imaging.

    Science.gov (United States)

    Ayzenberg, Ilya; Schlevogt, Sibylle; Metzdorf, Judith; Stahlke, Sarah; Pedreitturia, Xiomara; Hunfeld, Anika; Couillard-Despres, Sebastien; Kleiter, Ingo

    2015-01-01

    Bioluminescence imaging is a sensitive approach for longitudinal neuroimaging. Transgenic mice expressing luciferase under the promoter of doublecortin (DCX-luc), a specific marker of neuronal progenitor cells (NPC), allow monitoring of neurogenesis in living mice. Since the extent and time course of neurogenesis during autoimmune brain inflammation are controversial, we investigated neurogenesis in MOG-peptide induced experimental allergic encephalomyelitis (EAE) using DCX-luc reporter mice. We observed a marked, 2- to 4-fold increase of the bioluminescence signal intensity 10 days after EAE induction and a gradual decline 1-2 weeks thereafter. In contrast, immunostaining for DCX revealed no differences between EAE and control mice 2 and 4 weeks after immunization in zones of adult murine neurogenesis such as the dentate gyrus. Ex vivo bioluminescence imaging showed similar luciferase expression in brain homogenates of EAE and control animals. Apart from complete immunization including MOG-peptide also incomplete immunization with complete Freund´s adjuvant and pertussis toxin resulted in a rapid increase of the in vivo bioluminescence signal. Blood-brain barrier (BBB) leakage was demonstrated 10 days after both complete and incomplete immunization and might explain the increased bioluminescence signal in vivo. We conclude, that acute autoimmune inflammation in EAE does not alter neurogenesis, at least at the stage of DCX-expressing NPC. Effects of immunization on the BBB integrity must be considered when luciferase is used as a reporter within the CNS during the active stage of EAE. Models with stable CNS-restricted luciferase expression could serve as technically convenient way to evaluate BBB integrity in a longitudinal manner.

  19. Rapid detection of bacteria in green tea using a novel pretreatment method in a bioluminescence assay.

    Science.gov (United States)

    Shinozaki, Yohei; Harada, Yasuhiro

    2014-06-01

    Tea is one of the most popular beverages consumed in the world, and green tea has become a popular beverage in Western as well as Asian countries. A novel pretreatment method for a commercial bioluminescence assay to detect bacteria in green tea was developed and evaluated in this study. Pretreatment buffers with pH levels ranging from 6.0 to 9.0 were selected from MES (morpholineethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), or Tricine buffers. To evaluate the effect of pretreatment and the performance of the assay, serially diluted cultures of Enterobacter cloacae, Escherichia coli, Bacillus subtilis, and Staphylococcus aureus were tested. The improved methods, which consisted of a pretreatment of the sample in alkaline buffer, significantly decreased the background bioluminescence intensity of green tea samples when compared with the conventional method. Pretreatment with alkaline buffers with pH levels ranging from 8.0 to 9.0 increased the bioluminescence intensities of cultures of E. cloacae and S. aureus. Strong log-linear relationships between the bioluminescence intensities and plate counts emerged for the tested strains. Furthermore, the microbial detection limit was 15 CFU in 500 ml of bottled green tea after an 8-h incubation at 35°C and an assay time of 1 h. The results showed that contaminated samples could be detected within 1 h of operation using our improved bioluminescence assay. This method could be used to test for contamination during the manufacturing process as well as for statistical sampling for quality control.

  20. Ship track for Islands in the Stream 2002 - Pharmaceutical Discovery, Vision, and Bioluminescence - Office of Ocean Exploration

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Ship track of the R/V Seward Johnson during the 2002 "Islands in the Stream - Pharmaceutical Discovery, Vision, and Bioluminescence" expedition sponsored by the...

  1. Effects of salinity, pH and temperature on the re-establishment of bioluminescence and copper or SDS toxicity in the marine dinoflagellate Pyrocystis lunula using bioluminescence as an endpoint

    Science.gov (United States)

    Craig, J.M.; Klerks, P.L.; Heimann, K.; Waits, J.L.

    2003-01-01

    Pyrocystis lunula is a unicellular, marine, photoautotrophic, bioluminescent dinoflagellate. This organism is used in the Lumitox ?? bioassay with inhibition of bioluminescence re-establishment as the endpoint. Experiments determined if acute changes in pH, salinity, or temperature had an effect on the organisms' ability to re-establish bioluminescence, or on the bioassay's potential to detect sodium dodecyl sulfate (SDS) and copper toxicity. The re-establishment of bioluminescence itself was not very sensitive to changes in pH within the pH 6-10 range, though reducing pH from 8 to levels below 6 decreased this capacity. Increasing the pH had little effect on Cu or SDS toxicity, but decreasing the pH below 7 virtually eliminated the toxicity of either compound in the bioassay. Lowering the salinity from 33 to 27??? or less resulted in a substantial decrease in re-establishment of bioluminescence, while increasing the salinity to 43 or 48 ??? resulted in a small decline. Salinity had little influence on the bioassay's quantification of Cu toxicity, while the data showed a weak negative relationship between SDS toxicity and salinity. Re-establishment of bioluminescence showed a direct dependence on temperature, but only at 10??C did temperature have an obvious effect on the toxicity of Cu in this bioassay. ?? 2003 Elsevier Science Ltd. All rights reserved.

  2. Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

    Science.gov (United States)

    Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

    2012-06-01

    Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

  3. Improved light extraction in the bioluminescent lantern of a Photuris firefly (Lampyridae)

    Science.gov (United States)

    Bay, Annick; Cloetens, Peter; Suhonen, Heikki; Vigneron, Jean Pol

    2013-01-01

    A common problem of light sources emitting from an homogeneous high-refractive index medium into air is the loss of photons by total internal reflection. Bioluminescent organisms, as well as artificial devices, have to face this problem. It is expected that life, with its mechanisms for evolution, would have selected appropriate optical structures to get around this problem, at least partially. The morphology of the lantern of a specific firefly in the genus Photuris has been examined. The optical properties of the different parts of this lantern have been modeled, in order to determine their positive or adverse effect with regard to the global light extraction. We conclude that the most efficient pieces of the lantern structure are the misfit of the external scales (which produce abrupt roughness in air) and the lowering of the refractive index at the level of the cluster of photocytes, where the bioluminescent production takes place.

  4. Rapid identification and antibiotic susceptibility testing of Yersinia pestis using bioluminescent reporter phage.

    Science.gov (United States)

    Schofield, David A; Molineux, Ian J; Westwater, Caroline

    2012-08-01

    The rapid identification and antibiotic susceptibility testing of Yersinia pestis is paramount for a positive prognosis. We previously engineered a Y. pestis-specific 'bioluminescent' reporter phage for the identification of Y. pestis. In this study, we generated an improved reporter phage and evaluated the ability of this phage to provide direct and rapid susceptibility testing. Compared to the first generation reporter, the second generation reporter exhibited a 100-fold increase in signal strength, leading to a 10-fold increase in assay sensitivity. Y. pestis antimicrobial testing in the presence of the reporter elicited bioluminescent signals that were drug concentration-dependent, and produced susceptibility profiles that mirrored the standard CLSI method. The phage-generated susceptibility profiles, however, were obtained within hours in contrast to days with the conventional method.

  5. Robust red-emission spectra and yields in firefly bioluminescence against temperature changes

    Science.gov (United States)

    Mochizuki, Toshimitsu; Wang, Yu; Hiyama, Miyabi; Akiyama, Hidefumi

    2014-05-01

    We measured the quantitative spectra of firefly (Photinus pyralis) bioluminescence at various temperatures to investigate the temperature dependence of the luciferin-luciferase reaction at 15-34 °C. The quantitative spectra were decomposed very well into red (1.9 eV), orange (2.0 eV), and green (2.2 eV) Gaussian components. The intensity of the green component was the only temperature sensitive quantity that linearly decreased as the temperature increased at pH 7 and 8. We found the quantitative bioluminescence spectra to be robust below 2.0 eV against temperature and other experimental conditions. The revealed robustness of the red emissions should be useful for quantitative applications such as adenosine-5'-triphosphate detection.

  6. Improved light extraction in the bioluminescent lantern of a Photuris firefly (Lampyridae)

    CERN Document Server

    Bay, Annick; Suhonen, Heikki; Vigneron, Jean Pol

    2012-01-01

    A common problem of light sources emitting from an homogeneous high-refractive index medium into air is the loss of photons by total internal reflection. Bioluminescent organisms, as well as artificial devices, have to face this problem. It is expected that life, with its mechanisms for evolution, would have selected appropriate optical structures to get around this problem, at least partially. The morphology of the lantern of a specific firefly in the genus Photuris has been examined. The optical properties of the different parts of this lantern have been modelled, in order to determine their positive or adverse effect with regard to the global light extraction. We conclude that the most efficient pieces of the lantern structure are the misfit of the external scales (which produce abrupt roughness in air) and the lowering of the refractive index at the level of the cluster of photocytes, where the bioluminescent production takes place.

  7. Photon Counting System for High-Sensitivity Detection of Bioluminescence at Optical Fiber End.

    Science.gov (United States)

    Iinuma, Masataka; Kadoya, Yutaka; Kuroda, Akio

    2016-01-01

    The technique of photon counting is widely used for various fields and also applicable to a high-sensitivity detection of luminescence. Thanks to recent development of single photon detectors with avalanche photodiodes (APDs), the photon counting system with an optical fiber has become powerful for a detection of bioluminescence at an optical fiber end, because it allows us to fully use the merits of compactness, simple operation, highly quantum efficiency of the APD detectors. This optical fiber-based system also has a possibility of improving the sensitivity to a local detection of Adenosine triphosphate (ATP) by high-sensitivity detection of the bioluminescence. In this chapter, we are introducing a basic concept of the optical fiber-based system and explaining how to construct and use this system.

  8. [Quantitative specific detection of Staphylococcus aureus based on recombinant lysostaphin and ATP bioluminescence].

    Science.gov (United States)

    Li, Yuyuan; Mi, Zhiqiang; An, Xiaoping; Zhou, Yusen; Tong, Yigang

    2014-08-01

    Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.

  9. Biocidal effects of silver and zinc oxide nanoparticles on the bioluminescent bacteria

    Science.gov (United States)

    Taran, M. V.; Starodub, N. F.; Katsev, A. M.; Guidotti, M.; Khranovskyy, V. D.; Babanin, A. A.; Melnychuk, M. D.

    2013-11-01

    The effect of silver and zinc oxide nanoparticles in combination with alginate on bioluminescent Photobacterium leiognathi Sh1 bacteria was investigated. Silver nanoparticles were found to be more toxic than zinc oxide nanoparticles on bioluminescent bacteria. The nanoparticles and their ions released results in the same effect, however, it was absent in combination with alginate. The effective inhibiting concentration (EC50) for silver nanoparticles was found about 0.3 - 0.4 μg mL-1, which was up to two times larger then for zinc oxide nanoparticles. The absence of sodium chloride in the tested media prevented the formation of colloidal particles of larger size and the effective inhibition concentrations of metal derivatives were lower than in the presence of sodium chloride.

  10. Fimbrolide Natural Products Disrupt Bioluminescence of Vibrio By Targeting Autoinducer Biosynthesis and Luciferase Activity.

    Science.gov (United States)

    Zhao, Weining; Lorenz, Nicola; Jung, Kirsten; Sieber, Stephan A

    2016-01-18

    Vibrio is a model organism for the study of quorum sensing (QS) signaling and is used to identify QS-interfering drugs. Naturally occurring fimbrolides are important tool compounds known to affect QS in various organisms; however, their cellular targets have so far remained elusive. Here we identify the irreversible fimbrolide targets in the proteome of living V. harveyi and V. campbellii via quantitative mass spectrometry utilizing customized probes. Among the major hits are two protein targets with essential roles in Vibrio QS and bioluminescence. LuxS, responsible for autoinducer 2 biosynthesis, and LuxE, a subunit of the luciferase complex, were both covalently modified at their active-site cysteines leading to inhibition of activity. The identification of LuxE unifies previous reports suggesting inhibition of bioluminescence downstream of the signaling cascade and thus contributes to a better mechanistic understanding of these QS tool compounds.

  11. Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

    Science.gov (United States)

    Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

    2015-07-15

    Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

  12. Bioluminescence imaging of β cells and intrahepatic insulin gene activity under normal and pathological conditions.

    Directory of Open Access Journals (Sweden)

    Tokio Katsumata

    Full Text Available In diabetes research, bioluminescence imaging (BLI has been applied in studies of β-cell impairment, development, and islet transplantation. To develop a mouse model that enables noninvasive imaging of β cells, we generated a bacterial artificial chromosome (BAC transgenic mouse in which a mouse 200-kbp genomic fragment comprising the insulin I gene drives luciferase expression (Ins1-luc BAC transgenic mouse. BLI of mice was performed using the IVIS Spectrum system after intraperitoneal injection of luciferin, and the bioluminescence signal from the pancreatic region analyzed. When compared with MIP-Luc-VU mice [FVB/N-Tg(Ins1-lucVUPwrs/J] expressing luciferase under the control of the 9.2-kbp mouse insulin I promoter (MIP, the bioluminescence emission from Ins1-luc BAC transgenic mice was enhanced approximately 4-fold. Streptozotocin-treated Ins1-luc BAC transgenic mice developed severe diabetes concomitant with a sharp decline in the BLI signal intensity in the pancreas. Conversely, mice fed a high-fat diet for 8 weeks showed an increase in the signal, reflecting a decrease or increase in the β-cell mass. Although the bioluminescence intensity of the islets correlated well with the number of isolated islets in vitro, the intensity obtained from a living mouse in vivo did not necessarily reflect an absolute quantification of the β-cell mass under pathological conditions. On the other hand, adenovirus-mediated gene transduction of β-cell-related transcription factors in Ins1-luc BAC transgenic mice generated luminescence from the hepatic region for more than 1 week. These results demonstrate that BLI in Ins1-luc BAC transgenic mice provides a noninvasive method of imaging islet β cells and extrapancreatic activity of the insulin gene in the liver under normal and pathological conditions.

  13. Hybrid light transport model based bioluminescence tomography reconstruction for early gastric cancer detection

    Science.gov (United States)

    Chen, Xueli; Liang, Jimin; Hu, Hao; Qu, Xiaochao; Yang, Defu; Chen, Duofang; Zhu, Shouping; Tian, Jie

    2012-03-01

    Gastric cancer is the second cause of cancer-related death in the world, and it remains difficult to cure because it has been in late-stage once that is found. Early gastric cancer detection becomes an effective approach to decrease the gastric cancer mortality. Bioluminescence tomography (BLT) has been applied to detect early liver cancer and prostate cancer metastasis. However, the gastric cancer commonly originates from the gastric mucosa and grows outwards. The bioluminescent light will pass through a non-scattering region constructed by gastric pouch when it transports in tissues. Thus, the current BLT reconstruction algorithms based on the approximation model of radiative transfer equation are not optimal to handle this problem. To address the gastric cancer specific problem, this paper presents a novel reconstruction algorithm that uses a hybrid light transport model to describe the bioluminescent light propagation in tissues. The radiosity theory integrated with the diffusion equation to form the hybrid light transport model is utilized to describe light propagation in the non-scattering region. After the finite element discretization, the hybrid light transport model is converted into a minimization problem which fuses an l1 norm based regularization term to reveal the sparsity of bioluminescent source distribution. The performance of the reconstruction algorithm is first demonstrated with a digital mouse based simulation with the reconstruction error less than 1mm. An in situ gastric cancer-bearing nude mouse based experiment is then conducted. The primary result reveals the ability of the novel BLT reconstruction algorithm in early gastric cancer detection.

  14. Deep-sea bioluminescence blooms after dense water formation at the ocean surface.

    Science.gov (United States)

    Tamburini, Christian; Canals, Miquel; Durrieu de Madron, Xavier; Houpert, Loïc; Lefèvre, Dominique; Martini, Séverine; D'Ortenzio, Fabrizio; Robert, Anne; Testor, Pierre; Aguilar, Juan Antonio; Samarai, Imen Al; Albert, Arnaud; André, Michel; Anghinolfi, Marco; Anton, Gisela; Anvar, Shebli; Ardid, Miguel; Jesus, Ana Carolina Assis; Astraatmadja, Tri L; Aubert, Jean-Jacques; Baret, Bruny; Basa, Stéphane; Bertin, Vincent; Biagi, Simone; Bigi, Armando; Bigongiari, Ciro; Bogazzi, Claudio; Bou-Cabo, Manuel; Bouhou, Boutayeb; Bouwhuis, Mieke C; Brunner, Jurgen; Busto, José; Camarena, Francisco; Capone, Antonio; Cârloganu, Christina; Carminati, Giada; Carr, John; Cecchini, Stefano; Charif, Ziad; Charvis, Philippe; Chiarusi, Tommaso; Circella, Marco; Coniglione, Rosa; Costantini, Heide; Coyle, Paschal; Curtil, Christian; Decowski, Patrick; Dekeyser, Ivan; Deschamps, Anne; Donzaud, Corinne; Dornic, Damien; Dorosti, Hasankiadeh Q; Drouhin, Doriane; Eberl, Thomas; Emanuele, Umberto; Ernenwein, Jean-Pierre; Escoffier, Stéphanie; Fermani, Paolo; Ferri, Marcelino; Flaminio, Vincenzo; Folger, Florian; Fritsch, Ulf; Fuda, Jean-Luc; Galatà, Salvatore; Gay, Pascal; Giacomelli, Giorgio; Giordano, Valentina; Gómez-González, Juan-Pablo; Graf, Kay; Guillard, Goulven; Halladjian, Garadeb; Hallewell, Gregory; van Haren, Hans; Hartman, Joris; Heijboer, Aart J; Hello, Yann; Hernández-Rey, Juan Jose; Herold, Bjoern; Hößl, Jurgen; Hsu, Ching-Cheng; de Jong, Marteen; Kadler, Matthias; Kalekin, Oleg; Kappes, Alexander; Katz, Uli; Kavatsyuk, Oksana; Kooijman, Paul; Kopper, Claudio; Kouchner, Antoine; Kreykenbohm, Ingo; Kulikovskiy, Vladimir; Lahmann, Robert; Lamare, Patrick; Larosa, Giuseppina; Lattuada, Dario; Lim, Gordon; Presti, Domenico Lo; Loehner, Herbert; Loucatos, Sotiris; Mangano, Salvatore; Marcelin, Michel; Margiotta, Annarita; Martinez-Mora, Juan Antonio; Meli, Athina; Montaruli, Teresa; Moscoso, Luciano; Motz, Holger; Neff, Max; Nezri, Emma Nuel; Palioselitis, Dimitris; Păvălaş, Gabriela E; Payet, Kevin; Payre, Patrice; Petrovic, Jelena; Piattelli, Paolo; Picot-Clemente, Nicolas; Popa, Vlad; Pradier, Thierry; Presani, Eleonora; Racca, Chantal; Reed, Corey; Riccobene, Giorgio; Richardt, Carsten; Richter, Roland; Rivière, Colas; Roensch, Kathrin; Rostovtsev, Andrei; Ruiz-Rivas, Joaquin; Rujoiu, Marius; Russo, Valerio G; Salesa, Francisco; Sánchez-Losa, Augustin; Sapienza, Piera; Schöck, Friederike; Schuller, Jean-Pierre; Schussler, Fabian; Shanidze, Rezo; Simeone, Francesco; Spies, Andreas; Spurio, Maurizio; Steijger, Jos J M; Stolarczyk, Thierry; Taiuti, Mauro G F; Toscano, Simona; Vallage, Bertrand; Van Elewyck, Véronique; Vannoni, Giulia; Vecchi, Manuela; Vernin, Pascal; Wijnker, Guus; Wilms, Jorn; de Wolf, Els; Yepes, Harold; Zaborov, Dmitry; De Dios Zornoza, Juan; Zúñiga, Juan

    2013-01-01

    The deep ocean is the largest and least known ecosystem on Earth. It hosts numerous pelagic organisms, most of which are able to emit light. Here we present a unique data set consisting of a 2.5-year long record of light emission by deep-sea pelagic organisms, measured from December 2007 to June 2010 at the ANTARES underwater neutrino telescope in the deep NW Mediterranean Sea, jointly with synchronous hydrological records. This is the longest continuous time-series of deep-sea bioluminescence ever recorded. Our record reveals several weeks long, seasonal bioluminescence blooms with light intensity up to two orders of magnitude higher than background values, which correlate to changes in the properties of deep waters. Such changes are triggered by the winter cooling and evaporation experienced by the upper ocean layer in the Gulf of Lion that leads to the formation and subsequent sinking of dense water through a process known as "open-sea convection". It episodically renews the deep water of the study area and conveys fresh organic matter that fuels the deep ecosystems. Luminous bacteria most likely are the main contributors to the observed deep-sea bioluminescence blooms. Our observations demonstrate a consistent and rapid connection between deep open-sea convection and bathypelagic biological activity, as expressed by bioluminescence. In a setting where dense water formation events are likely to decline under global warming scenarios enhancing ocean stratification, in situ observatories become essential as environmental sentinels for the monitoring and understanding of deep-sea ecosystem shifts.

  15. Bioluminescence ATP Monitoring for the Routine Assessment of Food Contact Surface Cleanliness in a University Canteen

    OpenAIRE

    Andrea Osimani; Cristiana Garofalo; Francesca Clementi; Stefano Tavoletti; Lucia Aquilanti

    2014-01-01

    ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period....

  16. Deep-Sea Bioluminescence Blooms after Dense Water Formation at the Ocean Surface

    Science.gov (United States)

    Tamburini, Christian; Canals, Miquel; Durrieu de Madron, Xavier; Houpert, Loïc; Lefèvre, Dominique; Martini, Séverine; D'Ortenzio, Fabrizio; Robert, Anne; Testor, Pierre; Aguilar, Juan Antonio; Samarai, Imen Al; Albert, Arnaud; André, Michel; Anghinolfi, Marco; Anton, Gisela; Anvar, Shebli; Ardid, Miguel; Jesus, Ana Carolina Assis; Astraatmadja, Tri L.; Aubert, Jean-Jacques; Baret, Bruny; Basa, Stéphane; Bertin, Vincent; Biagi, Simone; Bigi, Armando; Bigongiari, Ciro; Bogazzi, Claudio; Bou-Cabo, Manuel; Bouhou, Boutayeb; Bouwhuis, Mieke C.; Brunner, Jurgen; Busto, José; Camarena, Francisco; Capone, Antonio; Cârloganu, Christina; Carminati, Giada; Carr, John; Cecchini, Stefano; Charif, Ziad; Charvis, Philippe; Chiarusi, Tommaso; Circella, Marco; Coniglione, Rosa; Costantini, Heide; Coyle, Paschal; Curtil, Christian; Decowski, Patrick; Dekeyser, Ivan; Deschamps, Anne; Donzaud, Corinne; Dornic, Damien; Dorosti, Hasankiadeh Q.; Drouhin, Doriane; Eberl, Thomas; Emanuele, Umberto; Ernenwein, Jean-Pierre; Escoffier, Stéphanie; Fermani, Paolo; Ferri, Marcelino; Flaminio, Vincenzo; Folger, Florian; Fritsch, Ulf; Fuda, Jean-Luc; Galatà, Salvatore; Gay, Pascal; Giacomelli, Giorgio; Giordano, Valentina; Gómez-González, Juan-Pablo; Graf, Kay; Guillard, Goulven; Halladjian, Garadeb; Hallewell, Gregory; van Haren, Hans; Hartman, Joris; Heijboer, Aart J.; Hello, Yann; Hernández-Rey, Juan Jose; Herold, Bjoern; Hößl, Jurgen; Hsu, Ching-Cheng; de Jong, Marteen; Kadler, Matthias; Kalekin, Oleg; Kappes, Alexander; Katz, Uli; Kavatsyuk, Oksana; Kooijman, Paul; Kopper, Claudio; Kouchner, Antoine; Kreykenbohm, Ingo; Kulikovskiy, Vladimir; Lahmann, Robert; Lamare, Patrick; Larosa, Giuseppina; Lattuada, Dario; Lim, Gordon; Presti, Domenico Lo; Loehner, Herbert; Loucatos, Sotiris; Mangano, Salvatore; Marcelin, Michel; Margiotta, Annarita; Martinez-Mora, Juan Antonio; Meli, Athina; Montaruli, Teresa; Motz, Holger; Neff, Max; Nezri, Emma nuel; Palioselitis, Dimitris; Păvălaş, Gabriela E.; Payet, Kevin; Payre, Patrice; Petrovic, Jelena; Piattelli, Paolo; Picot-Clemente, Nicolas; Popa, Vlad; Pradier, Thierry; Presani, Eleonora; Racca, Chantal; Reed, Corey; Riccobene, Giorgio; Richardt, Carsten; Richter, Roland; Rivière, Colas; Roensch, Kathrin; Rostovtsev, Andrei; Ruiz-Rivas, Joaquin; Rujoiu, Marius; Russo, Valerio G.; Salesa, Francisco; Sánchez-Losa, Augustin; Sapienza, Piera; Schöck, Friederike; Schuller, Jean-Pierre; Schussler, Fabian; Shanidze, Rezo; Simeone, Francesco; Spies, Andreas; Spurio, Maurizio; Steijger, Jos J. M.; Stolarczyk, Thierry; Taiuti, Mauro G. F.; Toscano, Simona; Vallage, Bertrand; Van Elewyck, Véronique; Vannoni, Giulia; Vecchi, Manuela; Vernin, Pascal; Wijnker, Guus; Wilms, Jorn; de Wolf, Els; Yepes, Harold; Zaborov, Dmitry; De Dios Zornoza, Juan; Zúñiga, Juan

    2013-01-01

    The deep ocean is the largest and least known ecosystem on Earth. It hosts numerous pelagic organisms, most of which are able to emit light. Here we present a unique data set consisting of a 2.5-year long record of light emission by deep-sea pelagic organisms, measured from December 2007 to June 2010 at the ANTARES underwater neutrino telescope in the deep NW Mediterranean Sea, jointly with synchronous hydrological records. This is the longest continuous time-series of deep-sea bioluminescence ever recorded. Our record reveals several weeks long, seasonal bioluminescence blooms with light intensity up to two orders of magnitude higher than background values, which correlate to changes in the properties of deep waters. Such changes are triggered by the winter cooling and evaporation experienced by the upper ocean layer in the Gulf of Lion that leads to the formation and subsequent sinking of dense water through a process known as “open-sea convection”. It episodically renews the deep water of the study area and conveys fresh organic matter that fuels the deep ecosystems. Luminous bacteria most likely are the main contributors to the observed deep-sea bioluminescence blooms. Our observations demonstrate a consistent and rapid connection between deep open-sea convection and bathypelagic biological activity, as expressed by bioluminescence. In a setting where dense water formation events are likely to decline under global warming scenarios enhancing ocean stratification, in situ observatories become essential as environmental sentinels for the monitoring and understanding of deep-sea ecosystem shifts. PMID:23874425

  17. In Vivo Bioluminescent Imaging (BLI): Noninvasive Visualization and Interrogation of Biological Processes in Living Animals

    OpenAIRE

    Steven Ripp; Sayler, Gary S.; Tingting Xu; Close, Dan M.

    2010-01-01

    In vivo bioluminescent imaging (BLI) is increasingly being utilized as a method for modern biological research. This process, which involves the noninvasive interrogation of living animals using light emitted from luciferase-expressing bioreporter cells, has been applied to study a wide range of biomolecular functions such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis, cancer treatment, and disease progressio...

  18. Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds

    Science.gov (United States)

    Sanseverino, John; Gupta, Rakesh K.; Layton, Alice C.; Patterson, Stacey S.; Ripp, Steven A.; Saidak, Leslie; Simpson, Michael L.; Schultz, T. Wayne; Sayler, Gary S.

    2005-01-01

    An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17β-estradiol over the concentration range of 1.2 × 10−8 through 5.6 × 10−12 M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) × 10−10 and (2.4 ± 1.0) × 10−10 M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 × 10−11 to 2.8 × 10−9 M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment. PMID:16085836

  19. In vivo bioluminescence tomography based on multi-view projection and 3D surface reconstruction

    Science.gov (United States)

    Zhang, Shuang; Wang, Kun; Leng, Chengcai; Deng, Kexin; Hu, Yifang; Tian, Jie

    2015-03-01

    Bioluminescence tomography (BLT) is a powerful optical molecular imaging modality, which enables non-invasive realtime in vivo imaging as well as 3D quantitative analysis in preclinical studies. In order to solve the inverse problem and reconstruct inner light sources accurately, the prior structural information is commonly necessary and obtained from computed tomography or magnetic resonance imaging. This strategy requires expensive hybrid imaging system, complicated operation protocol and possible involvement of ionizing radiation. The overall robustness highly depends on the fusion accuracy between the optical and structural information. In this study we present a pure optical bioluminescence tomographic system (POBTS) and a novel BLT method based on multi-view projection acquisition and 3D surface reconstruction. The POBTS acquired a sparse set of white light surface images and bioluminescent images of a mouse. Then the white light images were applied to an approximate surface model to generate a high quality textured 3D surface reconstruction of the mouse. After that we integrated multi-view luminescent images based on the previous reconstruction, and applied an algorithm to calibrate and quantify the surface luminescent flux in 3D.Finally, the internal bioluminescence source reconstruction was achieved with this prior information. A BALB/C mouse with breast tumor of 4T1-fLuc cells mouse model were used to evaluate the performance of the new system and technique. Compared with the conventional hybrid optical-CT approach using the same inverse reconstruction method, the reconstruction accuracy of this technique was improved. The distance error between the actual and reconstructed internal source was decreased by 0.184 mm.

  20. Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence.

    Science.gov (United States)

    Modestova, Yulia; Koksharov, Mikhail I; Ugarova, Natalia N

    2014-09-01

    Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in the C-domain were described. Recently we reported a strong red-shifting mutation E457K in the C-domain; the bioluminescence spectra of this enzyme were independent of temperature or pH. In the present study we investigated the role of the residue E457 in the enzyme using the Luciola mingrelica luciferase with a thermostabilized N-domain as a parent enzyme for site-directed mutagenesis. We obtained a set of mutants and studied their catalytic properties, thermal stability and bioluminescence spectra. Experimental spectra were represented as a sum of two components (bioluminescence spectra of putative "red" and "green" emitters); λmax of these components were constant for all the mutants, but the ratio of these emitters was defined by temperature and mutations in the C-domain. We suggest that each emitter is stabilized by a specific conformation of the active site; thus, enzymes with two forms of the active site coexist in the reactive media. The rigid structure of the C-domain is crucial for maintaining the conformation corresponding to the "green" emitter. We presume that the emitters are the keto- and enol forms of oxyluciferin.

  1. Ultrasensitive bioluminescent determinations of adenosine triphosphate (ATP) for investigating the energetics of host-grown microbes

    Science.gov (United States)

    Hanks, J. H.; Dhople, A. M.

    1975-01-01

    Stability and optimal concentrations of reagents were studied in bioluminescence assay of ATP levels. Luciferase enzyme was prepared and purified using Sephadex G-100. Interdependencies between enzyme and luciferin concentrations in presence of optimal Mg are illustrated. Optimal ionic strength was confirmed to be 0.05 M for the four buffers tested. Adapted features of the R- and H-systems are summarized, as well as the percentages of ATP pools released from representative microbes by heat and chloroform.

  2. Quantum Yield Determination Based on Photon Number Measurement, Protocols for Firefly Bioluminescence Reactions.

    Science.gov (United States)

    Niwa, Kazuki

    2016-01-01

    Quantum yield (QY), which is defined as the probability of photon production by a single bio/chemiluminescence reaction, is an important factor to characterize luminescence light intensity emitted diffusively from the reaction solution mixture. Here, methods to measure number of photons to determine QY according to the techniques of national radiometry standards are described. As an example, experiments using firefly bioluminescence reactions are introduced.

  3. Deep-sea bioluminescence blooms after dense water formation at the ocean surface.

    Directory of Open Access Journals (Sweden)

    Christian Tamburini

    Full Text Available The deep ocean is the largest and least known ecosystem on Earth. It hosts numerous pelagic organisms, most of which are able to emit light. Here we present a unique data set consisting of a 2.5-year long record of light emission by deep-sea pelagic organisms, measured from December 2007 to June 2010 at the ANTARES underwater neutrino telescope in the deep NW Mediterranean Sea, jointly with synchronous hydrological records. This is the longest continuous time-series of deep-sea bioluminescence ever recorded. Our record reveals several weeks long, seasonal bioluminescence blooms with light intensity up to two orders of magnitude higher than background values, which correlate to changes in the properties of deep waters. Such changes are triggered by the winter cooling and evaporation experienced by the upper ocean layer in the Gulf of Lion that leads to the formation and subsequent sinking of dense water through a process known as "open-sea convection". It episodically renews the deep water of the study area and conveys fresh organic matter that fuels the deep ecosystems. Luminous bacteria most likely are the main contributors to the observed deep-sea bioluminescence blooms. Our observations demonstrate a consistent and rapid connection between deep open-sea convection and bathypelagic biological activity, as expressed by bioluminescence. In a setting where dense water formation events are likely to decline under global warming scenarios enhancing ocean stratification, in situ observatories become essential as environmental sentinels for the monitoring and understanding of deep-sea ecosystem shifts.

  4. Development of bacteriophage-based bioluminescent bioreporters for monitoring of microbial pathogens

    Science.gov (United States)

    Ozen, Aysu; Montgomery, Kacey; Jegier, Pat; Patterson, Stacey; Daumer, Kathleen A.; Ripp, Steven A.; Garland, Jay L.; Sayler, Gary S.

    2004-03-01

    Microorganisms pose numerous problems when present in human occupied enclosed environments. Primary among these are health related hazards, manifested as infectious diseases related to contaminated drinking water, food, or air circulation systems or non-infectious allergy related complications associated with microbial metabolites (sick building syndrome). As a means towards rapid detection of microbial pathogens, we are attempting to harness the specificity of bacterial phage for their host with a modified quorum sensing amplification signal to produce quantifiable bioluminescent (lux) detection on a silicon microluminometer. The bacteriophage itself is metabolically inactive, only achieving replicative capabilities upon infection of its specific host bacterium. Bacteriophage bioluminescent bioreporters contain a genomically inserted luxI component. During an infection event, the phage genes and accompanying luxI construct are taken up by the host bacterium and transcribed, resulting in luxI expression and subsequent activation of a homoserine lactone inducible bioluminescent bioreporter. We constructed a vector carrying the luxI gene under the control of a strong E. coli promoter and cloned it into E. coli. We have shown that it can induce luminescence up to 14,000 counts per second when combined with the bioreporter strain. In their final embodiment, these sensors will be fully independent microelectronic monitors for microbial contamination, requiring only exposure of the biochip to the sample, with on-chip signal processing downloaded directly to the local area network of the environmental control system.

  5. An ebCMOS camera system for marine bioluminescence observation: The LuSEApher prototype

    Energy Technology Data Exchange (ETDEWEB)

    Dominjon, A., E-mail: a.dominjon@ipnl.in2p3.fr [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Ageron, M. [CNRS/IN2P3, Centre de Physique des Particules de Marseille, Marseille, F-13288 (France); Barbier, R. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Universite de Lyon, Universite Lyon 1, Lyon F-69003 (France); Billault, M.; Brunner, J. [CNRS/IN2P3, Centre de Physique des Particules de Marseille, Marseille, F-13288 (France); Cajgfinger, T. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Universite de Lyon, Universite Lyon 1, Lyon F-69003 (France); Calabria, P. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Chabanat, E. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France); Universite de Lyon, Universite Lyon 1, Lyon F-69003 (France); Chaize, D.; Doan, Q.T.; Guerin, C.; Houles, J.; Vagneron, L. [CNRS/IN2P3, Institut de Physique Nucleaire de Lyon, Villeurbanne F-69622 (France)

    2012-12-11

    The ebCMOS camera, called LuSEApher, is a marine bioluminescence recorder device adapted to extreme low light level. This prototype is based on the skeleton of the LUSIPHER camera system originally developed for fluorescence imaging. It has been installed at 2500 m depth off the Mediterranean shore on the site of the ANTARES neutrino telescope. The LuSEApher camera is mounted on the Instrumented Interface Module connected to the ANTARES network for environmental science purposes (European Seas Observatory Network). The LuSEApher is a self-triggered photo detection system with photon counting ability. The presentation of the device is given and its performances such as the single photon reconstruction, noise performances and trigger strategy are presented. The first recorded movies of bioluminescence are analyzed. To our knowledge, those types of events have never been obtained with such a sensitivity and such a frame rate. We believe that this camera concept could open a new window on bioluminescence studies in the deep sea.

  6. Factors Influencing Quantification of in Vivo Bioluminescence Imaging: Application to Assessment of Pancreatic Islet Transplants

    Directory of Open Access Journals (Sweden)

    John Virostko

    2004-10-01

    Full Text Available The aim of this study is to determine and characterize factors influencing in vivo bioluminescence imaging (BLI and apply them to the specific application of imaging transplanted pancreatic islets. Noninvasive quantitative assessment of transplanted pancreatic islets poses a formidable challenge. Murine pancreatic islets expressing firefly luciferase were transplanted under the renal capsule or into the portal vein of nonobese diabetic–severe combined immunodeficiency mice and the bioluminescence was quantified with a cooled charge coupled device camera and digital photon image analysis. The important, but often neglected, effects of wound healing, mouse positioning, and transplantation site on bioluminescence measurements were investigated by imaging a constant emission, isotropic light-emitting bead (λ = 600 implanted at the renal or hepatic site. The renal beads emitted nearly four times more light than hepatic beads with a smaller spot size, indicating that light absorption and scatter are greatly influenced by the transplant site and must be accounted for in BLI measurements. Detected luminescence decreased with increasing angle between the mouse surface normal and optical axis. By defining imaging parameters such as postsurgical effects, animal positioning, and light attenuation as a function of transplant site, this study develops BLI as a useful imaging modality for quantitative assessment of islets post-transplantation.

  7. Bioluminescent imaging: a critical tool in pre-clinical oncology research.

    LENUS (Irish Health Repository)

    O'Neill, Karen

    2010-02-01

    Bioluminescent imaging (BLI) is a non-invasive imaging modality widely used in the field of pre-clinical oncology research. Imaging of small animal tumour models using BLI involves the generation of light by luciferase-expressing cells in the animal following administration of substrate. This light may be imaged using an external detector. The technique allows a variety of tumour-associated properties to be visualized dynamically in living models. The increasing use of BLI as a small-animal imaging modality has led to advances in the development of xenogeneic, orthotopic, and genetically engineered animal models expressing luciferase genes. This review aims to provide insight into the principles of BLI and its applications in cancer research. Many studies to assess tumour growth and development, as well as efficacy of candidate therapeutics, have been performed using BLI. More recently, advances have also been made using bioluminescent imaging in studies of protein-protein interactions, genetic screening, cell-cycle regulators, and spontaneous cancer development. Such novel studies highlight the versatility and potential of bioluminescent imaging in future oncological research.

  8. Organization and comparative analysis of the mitochondrial genomes of bioluminescent Elateroidea (Coleoptera: Polyphaga).

    Science.gov (United States)

    Amaral, Danilo T; Mitani, Yasuo; Ohmiya, Yoshihiro; Viviani, Vadim R

    2016-07-25

    Mitochondrial genome organization in the Elateroidea superfamily (Coleoptera), which include the main families of bioluminescent beetles, has been poorly studied and lacking information about Phengodidae family. We sequenced the mitochondrial genomes of Neotropical Lampyridae (Bicellonycha lividipennis), Phengodidae (Brasilocerus sp.2 and Phrixothrix hirtus) and Elateridae (Pyrearinus termitilluminans, Hapsodrilus ignifer and Teslasena femoralis). All species had a typical insect mitochondrial genome except for the following: in the elaterid T. femoralis genome there is a non-coding region between NADH2 and tRNA-Trp; in the phengodids Brasilocerus sp.2 and P. hirtus genomes we did not find the tRNA-Ile and tRNA-Gln. The P. hirtus genome showed a ~1.6kb non-coding region, the rearrangement of tRNA-Tyr, a new tRNA-Leu copy, and several regions with higher AT contents. Phylogenetics analysis using Bayesian and ML models indicated that the Phengodidae+Rhagophthalmidae are closely related to Lampyridae family, and included Drilus flavescens (Drilidae) as an internal clade within Elateridae. This is the first report that compares the mitochondrial genomes organization of the three main families of bioluminescent Elateroidea, including the first Neotropical Lampyridae and Phengodidae. The losses of tRNAs, and translocation and duplication events found in Phengodidae mt genomes, mainly in P. hirtus, may indicate different evolutionary rates in these mitochondrial genomes. The mitophylogenomics analysis indicates the monophyly of the three bioluminescent families and a closer relationship between Lampyridae and Phengodidae/Rhagophthalmidae, in contrast with previous molecular analysis.

  9. Specific and Quantitative Assessment of Naphthalene and Salicylate Bioavailability by Using a Bioluminescent Catabolic Reporter Bacterium

    Science.gov (United States)

    Heitzer, Armin; Webb, Oren F.; Thonnard, Janeen E.; Sayler, Gary S.

    1992-01-01

    A bioassay was developed and standardized for the rapid, specific, and quantitative assessment of naphthalene and salicylate bioavailability by use of bioluminescence monitoring of catabolic gene expression. The bioluminescent reporter strain Pseudomonas fluorescens HK44, which carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism, was used. The physiological state of the reporter cultures as well as the intrinsic regulatory properties of the naphthalene degradation operon must be taken into account to obtain a high specificity at low target substrate concentrations. Experiments have shown that the use of exponentially growing reporter cultures has advantages over the use of carbon-starved, resting cultures. In aqueous solutions for both substrates, naphthalene and salicylate, linear relationships between initial substrate concentration and bioluminescence response were found over concentration ranges of 1 to 2 orders of magnitude. Naphthalene could be detected at a concentration of 45 ppb. Studies conducted under defined conditions with extracts and slurries of experimentally contaminated sterile soils and identical uncontaminated soil controls demonstrated that this method can be used for specific and quantitative estimations of target pollutant presence and bioavailability in soil extracts and for specific and qualitative estimations of napthalene in soil slurries. PMID:16348717

  10. Comparison between bioluminescence imaging technique and CFU count for the study of oropharyngeal candidiasis in mice.

    Science.gov (United States)

    Gabrielli, Elena; Roselletti, Elena; Luciano, Eugenio; Sabbatini, Samuele; Mosci, Paolo; Pericolini, Eva

    2015-05-01

    We recently described a bioluminescence in vivo imaging technique, representing a powerful tool to test the real-time progression of oropharyngeal candidiasis, hence potentially useful to evaluate the efficacy of antifungal therapies. In this study, the in vivo imaging technique was compared with CFU measurement of target organs (tongue, esophagus and stomach) for monitoring and quantifying oropharyngeal candidiasis. We have correlated these two analytical methods at different times post-infection using engineered, luminescent Candida albicans in mice rendered susceptible to oral candidiasis by cortisone-acetate. Scatter plots, Pearson correlation and Student's t test were used to compare the methods. We observed that the bioluminescence in vivo imaging technique was more reliable than CFU counts in detecting early infection of, and its extent in, the oral cavity of the mouse. This was also evident following the introduction of a variable such as treatment with fluconazole. The results described in this study could validate the bioluminescence in vivo imaging technique as a method to monitor and quantify oropharyngeal candidiasis and to assess early discovery of active compounds in vivo.

  11. Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain.

    Science.gov (United States)

    Adams, Spencer T; Mofford, David M; Reddy, G S Kiran Kumar; Miller, Stephen C

    2016-04-11

    Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

  12. Development of a rapid optic bacteria detecting system based on ATP bioluminescence

    Science.gov (United States)

    Liu, Jun Tao; Luo, JinPing; Liu, XiaoHong; Cai, XinXia

    2014-12-01

    A rapid optic bacteria detecting system based on the principle of Adenosine triphosphate(ATP) bioluminescence was presented in this paper. This system consisted of bioluminescence-based biosensor and the high-sensitivity optic meter. A photon counting photomultiplier tube (PMT) module was used to improve the detection sensitivity, and a NIOS II/f processor based on a Field Programmable Gate Array(FPGA) was used to control the system. In this work, Micrococcus luteus were chosen as the test sample. Several Micrococcus luteus suspension with different concentration was tested by both T2011 and plate counting method. By comparing the two group results, an calibration curve was obtained from the bioluminescence intensity for Micrococcus luteus in the range of 2.3×102 ~ 2.3×106 CFU/mL with a good correlation coefficient of 0.960. An impacting Air microorganism sampler was used to capture Airborne Bacteria, and 8 samples were collected in different place. The TBC results of 8 samples by T2011 were between 10 ~ 2×103 cfu/mL, consistent with that of plate counting method, which indicated that 8 samples were between 10 ~ 3×103 cfu/mL. For total airborne bacteria count was small, correlation coefficient was poor. Also no significant difference was found between T2011 and plate counting method by statistical analyses.

  13. Flow injection analysis with bioluminescence-based fiber-optic biosensors

    Science.gov (United States)

    Blum, Loic J.; Gautier, Sabine; Coulet, Pierre R.

    1991-09-01

    Fiber optic biosensors based on the firefly and the bacterial bioluminescence reactions have been constructed and incorporated in a specially designed flow-cell for the sensitive determination of ATP and NADH, respectively. The bioluminescence enzymes were immobilized on preactivated polyamide membranes which were placed in close contact with the surface on one end of a glass-fiber bundle, the other end being connected to the photomultiplier tube of a luminometer. When using the continuous-flow device with the firefly luciferase or the bacterial system immobilized separately on different membranes, the detection limit for ATP and NADH were 0.25 and 2 pmol, respectively. The versatility of the fiber optic probe has been improved by co-immobilizing the bacterial bioluminescent system and the firefly luciferase on the same support enabling the use of a single sensor for the selective, specific, and alternate determination of these two analytes. Compatible reaction conditions preserving the activity of each co-immobilized enzyme without impairing its stability were found. The selection of the appropriate reaction medium was done using a four port valve. Alternate quantification of ATP and NADH could then be performed in the linear ranges 0.25 pmol - 3 nmol and 5 pmol - 1 nmol, respectively with a RSD of 4.0 - 4.5%.

  14. Liquid-chromatographic separation and on-line bioluminescence detection of creatine kinase isoenzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

    1980-01-01

    Isoenzymes of creatine kinase were separated by anion-exchange chromatography, with use of an elution gradient containing lithium acetate (0.1 to 0.6 mol/L). A stream splitter was used to divert a 5% side stream of column effluent, which was subsequently mixed with the reagents necessary for bioluminescence assay of the separated isoenzymes. The use of the stream splitter greatly decreased the rate of consumption of reagent and, when combined with a peristaltic pumping system, permitted independent control of the side-stream flow rate. Thus both the residence interval in a delay coil in which the ATP reaction product is formed and the bioluminescence emission was monitored in a flow-through fluorometer without use of an external light source or filters. Separation and detection of the isoenzymes of creatine kinase were rapid, sensitive, and highly selective. The incremental decrease of bioluminescence response owing to inhibition by the ions in the eluent was less than 31% across the entire gradient.

  15. Disposable bioluminescence-based biosensor for detection of bacterial count in food.

    Science.gov (United States)

    Luo, Jinping; Liu, Xiaohong; Tian, Qing; Yue, Weiwei; Zeng, Jing; Chen, Guangquan; Cai, Xinxia

    2009-11-01

    A biosensor for rapid detection of bacterial count based on adenosine 5'-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin-luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10(3) to 10(8) colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n=22) within 5min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.

  16. A new rapid and sensitive bioluminescence assay for antibiotics that inhibit protein synthesis.

    Science.gov (United States)

    Naveh, A; Potasman, I; Bassan, H; Ulitzur, S

    1984-06-01

    A new sensitive, rapid and simple bioluminescence assay for antibiotics inhibiting protein synthesis is described. In this assay the ability of the tested antibiotic to inhibit the de novo synthesis of the enzymes participating in the bacterial luminescence system is determined by means of a dark variant of a luminous bacterium that undergoes prompt induction of the luminescence system with certain DNA-intercalating agents. Upon induction, the in vivo luminescence of the dark variant is increased more than 50-fold within 30 min. Antibiotics that block the de novo synthesis of protein limit the development of luminescence at a level that was found to be a function of the antibiotic concentration. The minimum detectable concentration of antibiotics in the bioluminescence test, after 45-60 min of incubation, was 0.1 microgram/ml for streptomycin, gentamicin, kanamycin, lincomycin and chloramphenicol and 0.3 microgram/ml for neomycin, clindamycin and spectinomycin. The new bioluminescence test has been used to assay these antibiotics in serum.

  17. Molecular Origin of Color Variation in Firefly (Beetle) Bioluminescence: A Chemical Basis for Biological Imaging.

    Science.gov (United States)

    Hirano, Takashi

    2016-01-01

    Firefly shows bioluminescence by "luciferin-luciferase" (L-L) reaction using luciferin, luciferase, ATP and O2. The chemical photon generation by an enzymatic reaction is widely utilized for analytical methods including biological imaging in the life science fields. To expand photondetecting analyses with firefly bioluminescence, it is important for users to understand the chemical basis of the L-L reaction. In particular, the emission color variation of the L-L reaction is one of the distinguishing characteristics for multicolor luciferase assay and in vivo imaging. From the viewpoint of fundamental chemistry, this review explains the recent progress in the studies on the molecular mechanism of emission color variation after showing the outline of the reaction mechanism of the whole L-L reaction. On the basis of the mechanism, the progresses in organic synthesis of luciferin analogs modulating their emission colors are also presented to support further developments of red/near infrared in vivo biological imaging utility of firefly bioluminescence.

  18. Spectrally resolved bioluminescence tomography with adaptive finite element analysis: methodology and simulation

    Energy Technology Data Exchange (ETDEWEB)

    Lv Yujie [Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, PO Box 2728, Beijing 100080 (China); Tian Jie [Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, PO Box 2728, Beijing 100080 (China); Cong Wenxiang [Division of Biomedical Imaging, VT-WFU School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 (United States); Wang Ge [Division of Biomedical Imaging, VT-WFU School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 (United States); Yang Wei [Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, PO Box 2728, Beijing 100080 (China); Qin Chenghu [Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, PO Box 2728, Beijing 100080 (China); Xu Min [Medical Image Processing Group, Institute of Automation, Chinese Academy of Sciences, PO Box 2728, Beijing 100080 (China)

    2007-08-07

    As a molecular imaging technique, bioluminescence tomography (BLT) with its highly sensitive detection and facile operation can significantly reveal molecular and cellular information in vivo at the whole-body small animal level. However, because of complex photon transportation in biological tissue and boundary detection data with high noise, bioluminescent sources in deeper positions generally cannot be localized. In our previous work, we used achromatic or monochromatic measurements and an a priori permissible source region strategy to develop a multilevel adaptive finite-element algorithm. In this paper, we propose a spectrally solved tomographic algorithm with a posteriori permissible source region selection. Multispectral measurements, and anatomical and optical information first deal with the nonuniqueness of BLT and constrain the possible solution of source reconstruction. The use of adaptive mesh refinement and permissible source region based on a posteriori measures not only avoids the dimension disaster arising from the multispectral measured data but also reduces the ill-posedness of BLT and therefore improves the reconstruction quality. Reconsideration of the optimization method and related modifications further enhance reconstruction robustness and efficiency. We also incorporate into the method some improvements for reducing computational burdens. Finally, using a whole-body virtual mouse phantom, we demonstrate the capability of the proposed BLT algorithm to reconstruct accurately bioluminescent sources in deeper positions. In terms of optical property errors and two sources of discernment in deeper positions, this BLT algorithm represents the unique predominance for BLT reconstruction.

  19. A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part I: design and optimization of bioluminescent bacterial strains

    Energy Technology Data Exchange (ETDEWEB)

    Charrier, Thomas; Durand, Marie-Jose; Jouanneau, Sulivan; Thouand, Gerald [UMR CNRS 6144 GEPEA, CBAC, Nantes University, PRES UNAM, Campus de la Courtaisiere-IUT, La Roche-sur-Yon cedex (France); Dion, Michel [UMR CNRS 6204, Nantes University, PRES UNAM, Biotechnologie, Biocatalyse, Bioregulation, 2, Rue de la Houssiniere, BP 92208, Nantes cedex 3 (France); Pernetti, Mimma; Poncelet, Denis [ONIRIS-ENITIAA, UMR CNRS GEPEA, Rue de la Geraudiere, BP 82225, Nantes cedex 3 (France)

    2011-05-15

    This study describes the construction of inducible bioluminescent strains via genetic engineering along with their characterization and optimization in the detection of heavy metals. Firstly, a preliminary comparative study enabled us to select a suitable carbon substrate from pyruvate, glucose, citrate, diluted Luria-Bertani, and acetate. The latter carbon source provided the best induction ratios for comparison. Results showed that the three constructed inducible strains, Escherichia coli DH1 pBzntlux, pBarslux, and pBcoplux, were usable when conducting a bioassay after a 14-h overnight culture at 30 C. Utilizing these sensors gave a range of 12 detected heavy metals including several cross-detections. Detection limits for each metal were often close to and sometimes lower than the European standards for water pollution. Finally, in order to maintain sensitive bacteria within the future biosensor-measuring cell, the agarose immobilization matrix was compared to polyvinyl alcohol (PVA). Agarose was selected because the detection limits of the bioluminescent strains were not affected, in contrast to PVA. Specific detection and cross-detection ranges determined in this study will form the basis of a multiple metals detection system by the new multi-channel Lumisens3 biosensor. (orig.)

  20. ATP - bioluminescence as a technique to evaluate the microbiological quality of water in food industry

    Directory of Open Access Journals (Sweden)

    Patrícia Dolabela Costa

    2004-07-01

    Full Text Available ATP-bioluminescence was used to evaluate the microbiological quality of water samples collected from the water supply, the water treatment system and from a dairy plant, including ammonia-cooling water and industrial water. For industrial water, there was relation between the ATP-bioluminescence technique and microbial count. There were no differences (p>0.05 between water supply and ammonia-cooling water samples for total and free ATP concentrations nor for the microbial counts. Different microbial ATP concentrations were found for these water samples. The results suggested that the physical chemical quality of ammonia cooling water decreased the RLU measurements slightly. It could be concluded that the total ATP concentration was the most effective technique to evaluate the microbiological quality of water used in the food indsutry by ATP-bioluminescence.A qualidade microbiológica da água do manancial de captação para tratamento na ETA/UFV, da água de resfriamento de amônia e da água industrial de uso em um laticínio foi avaliada pela técnica de ATP-bioluminescência. Nas águas, foram efetuadas, também, as contagens de mesófilos aeróbios, expressos em UFC.mL-1 e coliformes totais, expressos em NMP.100 mL-1. Foi utilizado um luminômetro para os testes de determinação de ATP total e livre, expressos em Unidades Relativas de Luz (URL, nas diversas águas. O ATP microbiano foi determinado pela diferença entre o ATP total e livre. Em relação à água industrial, houve a concordância entre os métodos de bioluminescência e de contagem de mesófilos aeróbios e coliformes totais. As amostras de água de manancial e de resfriamento não apresentaram diferença (p>0,05, pelo teste de Tukey, para as quantidades de ATP total, livre e também para as contagens microbianas. Constataram-se concentrações diferentes de ATP microbiano para essas amostras de água. Os resultados indicam que o teste de ATP total é o mais recomendado para

  1. Characterization of sevoflurane effects on Per2 expression using ex vivo bioluminescence imaging of the suprachiasmatic nucleus in transgenic rats.

    Science.gov (United States)

    Matsuo, Izumi; Iijima, Norio; Takumi, Ken; Higo, Shimpei; Aikawa, Satoko; Anzai, Megumi; Ishii, Hirotaka; Sakamoto, Atsuhiro; Ozawa, Hitoshi

    2016-06-01

    The inhalation anesthetic sevoflurane suppresses Per2 expression in the suprachiasmatic nucleus (SCN) in rodents. Here, we investigated the intra-SCN regional specificity, time-dependency, and pharmacological basis of sevoflurane-effects. Bioluminescence image was taken from the SCN explants of mPer2 promoter-destabilized luciferase transgenic rats, and each small regions of interest (ROI) of the image was analyzed. Sevoflurane suppressed bioluminescence in all ROIs, suggesting that all regions in the SCN are sensitive to sevoflurane. Clear time-dependency in sevoflurane effects were also observed; application during the trough phase of the bioluminescence cycle suppressed the subsequent increase in bioluminescence and resulted in a phase delay of the cycle; sevoflurane applied during the middle of the ascending phase induced a phase advance; sevoflurane on the descending phase showed no effect. These results indicate that the sevoflurane effect may depend on the intrinsic state of circadian machinery. Finally, we examined the involvement of GABAergic signal transduction in the sevoflurane effect. Co-application of both GABAA and GABAB receptor antagonists completely blocked the effect of sevoflurane on the bioluminescence rhythm, suggesting that sevoflurane inhibits Per2 expression via GABAergic signal transduction. Current study elucidated the anesthetic effects on the molecular mechanisms of circadian rhythm.

  2. Effects of binary mixtures of inducers (toluene analogs) and of metals on bioluminescence induction of a recombinant bioreporter strain.

    Science.gov (United States)

    Kong, In Chul

    2014-10-13

    This paper investigated the effects of binary mixtures of bioluminescence inducers (toluene, xylene isomers, m-toluate) and of metals (Cu, Cd, As(III), As(V), and Cr) on bioluminescence activity of recombinant (Pm-lux) strain KG1206. Different responses and sensitivities were observed depending on the types and concentrations of mixtures of inducers or metals. In the case of inducer mixtures, antagonistic and synergistic modes of action were observed, whereas metal mixtures showed all three modes of action. Antagonistic mode of action was most common for mixtures of indirect inducers, which showed bioluminescence ranging from 29% to 62% of theoretically expected effects (P(E)). On the other hand, synergistic mode of action was observed for mixtures of direct and indirect inducers, which showed bioluminescence between 141% and 243% of P(E). In the case of binary metal mixtures, bioluminescence activities were ranged from 62% to 75% and 113% to 164% of P(E) for antagonistic and synergistic modes of action, respectively (p-values 0.0001-0.038). Therefore, mixture effects could not be generalized since they were dependent on both the types and concentrations of chemicals, suggesting that biomonitoring may constitute a better strategy by investigating types and concentrations of mixture pollutants at contaminated sites.

  3. Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation.

    Science.gov (United States)

    Takeuchi, Masaki; Nagaoka, Yasutaka; Yamada, Toshimichi; Takakura, Hideo; Ozawa, Takeaki

    2010-11-15

    Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.

  4. Bioluminescence imaging reveals dynamics of beta cell loss in the non-obese diabetic (NOD) mouse model.

    Science.gov (United States)

    Virostko, John; Radhika, Armandla; Poffenberger, Greg; Dula, Adrienne N; Moore, Daniel J; Powers, Alvin C

    2013-01-01

    We generated a mouse model (MIP-Luc-VU-NOD) that enables non-invasive bioluminescence imaging (BLI) of beta cell loss during the progression of autoimmune diabetes and determined the relationship between BLI and disease progression. MIP-Luc-VU-NOD mice displayed insulitis and a decline in bioluminescence with age which correlated with beta cell mass, plasma insulin, and pancreatic insulin content. Bioluminescence declined gradually in female MIP-Luc-VU-NOD mice, reaching less than 50% of the initial BLI at 10 weeks of age, whereas hyperglycemia did not ensue until mice were at least 16 weeks old. Mice that did not become diabetic maintained insulin secretion and had less of a decline in bioluminescence than mice that became diabetic. Bioluminescence measurements predicted a decline in beta cell mass prior to the onset of hyperglycemia and tracked beta cell loss. This model should be useful for investigating the fundamental processes underlying autoimmune diabetes and developing new therapies targeting beta cell protection and regeneration.

  5. Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch-activated ion channels.

    Science.gov (United States)

    Jin, Kelly; Klima, Jason C; Deane, Grant; Dale Stokes, Malcolm; Latz, Michael I

    2013-08-01

    Dinoflagellate bioluminescence serves as a whole-cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage-sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 μM Gadolinium III (Gd(3+) ), a nonspecific blocker of stretch-activated and some voltage-gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 μM nifedipine, an inhibitor of L-type voltage-gated Ca(2+) channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol-stimulated bioluminescence was inhibited by Gd(3+) but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch-activated and voltage-gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch-activated component at the plasma membrane.

  6. A genetic screen for bioluminescence genes in the fungus Armillaria mellea, through the use of Agrobacterium tumefaciens-mediated random insertional mutagenesis

    Science.gov (United States)

    Bioluminescence is reported from 71 saprobic species of fungi from four, distant lineages in the order Agaricales. Analyses of the fungal luminescent chemistry shows that all four lineages share a functionally conserved substrate and luciferase, indicating that the bioluminescent pathway is likely c...

  7. Effect of concentrating and exposing the bioluminescent bacteria to the non-luminescent allo-bacterial extracellular products on their luminescence.

    Science.gov (United States)

    Ravindran, J; Geetha Priya, G; Kannapiran, E

    2011-01-01

    Bioluminescence is a biochemical process occurring in many organisms. Bacterial bioluminescence has been investigated extensively that lead to many applications of such knowledge. Quorum sensing in the bioluminescent bacteria is a chemical signal process to recognize the strength of its own population to start luminescence in harmony. There is a mechanism in these bacteria to also recognize inter-species strength. When there is a higher number of these bacteria, the possibility and frequency of cell-cell physical contact will be high. In this study, the physical proximity was artificially enhanced between cells and the effect on luminescence in the concentrated cells in the normal culture medium and in the presence of other non-bacterial cell-free supernatants was investigated. The role of such physical contact in the quorum sensing in the bioluminescence is not known. Increase in the luminescence of V. fischeri when concentrated shows that the presence of physical proximity facilitates the quorum sensing for their bioluminescence.

  8. Observations of in situ deep-sea marine bioluminescence with a high-speed, high-resolution sCMOS camera

    Science.gov (United States)

    Phillips, Brennan T.; Gruber, David F.; Vasan, Ganesh; Roman, Christopher N.; Pieribone, Vincent A.; Sparks, John S.

    2016-05-01

    Observing and measuring marine bioluminescence in situ presents unique challenges, characterized by the difficult task of approaching and imaging weakly illuminated bodies in a three-dimensional environment. To address this problem, a scientific complementary-metal-oxide-semiconductor (sCMOS) microscopy camera was outfitted for deep-sea imaging of marine bioluminescence. This system was deployed on multiple platforms (manned submersible, remotely operated vehicle, and towed body) in three oceanic regions (Western Tropical Pacific, Eastern Equatorial Pacific, and Northwestern Atlantic) to depths up to 2500 m. Using light stimulation, bioluminescent responses were recorded at high frame rates and in high resolution, offering unprecedented low-light imagery of deep-sea bioluminescence in situ. The kinematics of light production in several zooplankton groups was observed, and luminescent responses at different depths were quantified as intensity vs. time. These initial results signify a clear advancement in the bioluminescent imaging methods available for observation and experimentation in the deep-sea.

  9. Using the bioluminescence and microbiological contact methods in sustaining a proper hygienic level in food processing plants

    Directory of Open Access Journals (Sweden)

    Dorota Cais-Sokolińska

    2008-12-01

    Full Text Available The efficiency of bioluminescence applied to monitor the state of hygiene in a dairy processing plant was assessed in relation to the results of conventional microbiological methods. The used blotting tests were Envirocheck Contact DC with Agar CASO medium with added neutralizers. The analysed object was the surface of a beam stirrer in a fermentation tank. Swabs were collected following tank washing and disinfection after the completion of 15 production cycles. A high degree of correlation r = 0.91 was obtained at the reliability of comparison β = 0.906. The analysis of probability of distribution confirmed the feasibility of bioluminescence. Boundary values (112 and 171 RLU were determined for bioluminescence for three object cleanliness ranges, based on microbial counts (cfu/cm2. Over 13% surfaces were classified as conditionally clean (Alert.

  10. Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

    Science.gov (United States)

    Guzmán Luna, Carolina; Costán-Longares, Ana; Lucena, Francisco; Jofre, Joan

    2009-10-01

    The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

  11. [Determination of minimal concentrations of biocorrosion inhibitors by a bioluminescence method in relation to bacteria, participating in biocorrosion].

    Science.gov (United States)

    Efremenko, E N; Azizov, R E; Makhlis, T A; Abbasov, V M; Varfolomeev, S D

    2005-01-01

    By using a bioluminescence ATP assay, we have determined the minimal concentrations of some biocorrosion inhibitors (Katon, Khazar, VFIKS-82, Nitro-1, Kaspii-2, and Kaspii-4) suppressing most common microbial corrosion agents: Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Pseudomonas putida, Pseudomonas fluorescens, and Acidithiobacillus ferrooxidans. The cell titers determined by the bioluminescence method, including not only dividing cells but also their dormant living counterparts, are two- to sixfold greater than the values determined microbiologically. It is shown that the bioluminescence method can be applied to determination of cell titers in samples of oil-field waters in the presence of iron ions (up to 260 mM) and iron sulfide (to 186 mg/l) and in the absence or presence of biocidal corrosion inhibitors.

  12. Bioluminescence of beetle luciferases with 6'-amino-D-luciferin analogues reveals excited keto-oxyluciferin as the emitter and phenolate/luciferin binding site interactions modulate bioluminescence colors.

    Science.gov (United States)

    Viviani, Vadim R; Neves, Deimison Rodrigues; Amaral, Danilo Trabuco; Prado, Rogilene A; Matsuhashi, Takuto; Hirano, Takashi

    2014-08-19

    Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar

  13. Expression of a humanized viral 2A-mediated lux operon efficiently generates autonomous bioluminescence in human cells.

    Directory of Open Access Journals (Sweden)

    Tingting Xu

    Full Text Available Expression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines.The lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations.Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity

  14. A novel lux operon in the cryptically bioluminescent fish pathogen Vibrio salmonicida is associated with virulence.

    Science.gov (United States)

    Nelson, Eric J; Tunsjø, Hege S; Fidopiastis, Pat M; Sørum, Henning; Ruby, Edward G

    2007-03-01

    The cold-water-fish pathogen Vibrio salmonicida expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the lux operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the V. salmonicida lux operon revealed that homologs of all of the genes required for luminescence are present: luxAB (luciferase) and luxCDE (aliphatic-aldehyde synthesis). The arrangement and sequence of these structural lux genes are conserved compared to those in related species of luminous bacteria. However, V. salmonicida strains have a novel arrangement and number of homologs of the luxR and luxI quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the luxA gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the luxA mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the lux operon. Nevertheless, these findings document for the first time an association between a mutation in a structural lux gene and virulence, as well as provide a new molecular system to study Vibrio pathogenesis in a natural host.

  15. In vivo bioluminescence imaging of Burkholderia mallei respiratory infection and treatment in the mouse model

    Directory of Open Access Journals (Sweden)

    Shane eMassey

    2011-08-01

    Full Text Available Bioluminescent imaging (BLI technology is a powerful tool for monitoring infectious disease progression and treatment approaches. BLI is particularly useful for tracking fastidious intracellular pathogens that might be difficult to recover from certain organs. Burkholderia mallei, the causative agent of glanders, is a facultative intracellular pathogen and has been classified by the CDC as a Category B select agent due to its highly infectious nature and potential use as a biological weapon. Very little is known regarding pathogenesis or treatment of glanders. We investigated the use of bioluminescent reporter constructs to monitor the dynamics of infection as well as the efficacy of therapeutics for B. mallei in real time. A stable luminescent reporter B. mallei strain was created using the pUTmini-Tn5::luxKm2 plasmid and used to monitor glanders in the BALB/c murine model. Mice were infected via the intranasal route with 5x103 bacteria and monitored by BLI at 24, 48 and 72 h. We verified that our reporter construct maintained similar virulence and growth kinetics compared to wild-type B. mallei and confirmed that it maintains luminescent stability in the presence or absence of antibiotic selection. The luminescent signal was initially seen in the lungs, and progressed to the liver and spleen over the course of infection. We demonstrated that antibiotic treatment 24 h post-infection resulted in reduction of bioluminescence that can be attributed to decreased bacterial burden in target organs. These findings suggest that BLI can be used to monitor disease progression and efficacy of therapeutics during glanders infections. Finally, we report an alternative method to mini-Tn5::luxKm2 transposon using mini-Tn7-lux elements that insert site-specifically at known genomic attachment sites and that can also be used to tag bacteria.

  16. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Directory of Open Access Journals (Sweden)

    Olga A Gandelman

    Full Text Available BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a

  17. Ex vivo bioluminescence detection of alcelaphine herpesvirus 1 infection during malignant catarrhal fever.

    Science.gov (United States)

    Dewals, Benjamin; Myster, Françoise; Palmeira, Leonor; Gillet, Laurent; Ackermann, Mathias; Vanderplasschen, Alain

    2011-07-01

    Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD-MCF) when cross-species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be reproduced in rabbits. WD-MCF is described as a combination of lymphoproliferation and degenerative lesions in virtually all organs and is caused by unknown mechanisms. Recently, we demonstrated that WD-MCF is associated with the proliferation of CD8(+) cells supporting a latent type of infection in lymphoid tissues. Here, we investigated the macroscopic distribution of AlHV-1 infection using ex vivo bioluminescence imaging in rabbit to determine whether it correlates with the distribution of lesions in lymphoid and nonlymphoid organs. To reach that goal, a recombinant AlHV-1 strain was produced by insertion of a luciferase expression cassette (luc) in an intergenic region. In vitro, the reconstituted AlHV-1 luc(+) strain replicated comparably to the parental strain, and luciferase activity was detected by bioluminescence imaging. In vivo, rabbits infected with the AlHV-1 luc(+) strain developed WD-MCF comparably to rabbits infected with the parental wild-type strain, with hyperthermia and increases of both CD8(+) T cell frequencies and viral genomic charge over time in peripheral blood mononuclear cells and in lymph nodes at time of euthanasia. Bioluminescent imaging revealed that AlHV-1 infection could be detected ex vivo in lymphoid organs but also in lung, liver, and kidney during WD-MCF, demonstrating that AlHV-1 infection is prevalent in tissue lesions. Finally, we show that the infiltrating mononuclear leukocytes in nonlymphoid organs are mainly CD8(+) T cells and that latency is predominant during WD-MCF.

  18. Vision in lanternfish (Myctophidae): Adaptations for viewing bioluminescence in the deep-sea

    Science.gov (United States)

    Turner, J. R.; White, E. M.; Collins, M. A.; Partridge, J. C.; Douglas, R. H.

    2009-06-01

    The sensitivity hypothesis seeks to explain the correlation between the wavelength of visual pigment absorption maxima ( λmax) and habitat type in fish and other marine animals in terms of the maximisation of photoreceptor photon catch. In recent years its legitimacy has been called into question as studies have either not tested data against the output of a predictive model or are confounded by the wide phylogeny of species used. We have addressed these issues by focussing on the distribution of λmax values in one family of marine teleosts, the lanternfish (Myctophidae). Visual pigment extract spectrophotometry has shown that 54 myctophid species have a single pigment in their retinae with a λmax falling within the range 480-492 nm. A further 4 species contain two visual pigments in their retinae. The spectral distribution of these visual pigments seems relatively confined when compared to other mesopelagic fishes. Mathematical modelling based on the assumptions of the sensitivity hypothesis shows that, contrary to the belief that deep-sea fishes' visual pigments are shortwave shifted to maximise their sensitivity to downwelling sunlight, the visual pigments of myctophids instead seem better placed for the visualisation of bioluminescence. The predicted maximum visualisation distance of a blue/green bioluminescent point source by a myctophid was up to 30 m under ideal conditions. Two species ( Myctophum nitidulum and Bolinichthys longipes) have previously been shown to have longwave-shifted spectral sensitivities and we show that they could theoretically detect stomiid far-red bioluminescence from as far as ca. 7 m.

  19. Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Holliday, Nicholas D; Hansen, Jakob L

    2007-01-01

    To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive muta......To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase...

  20. Effect of Iron Fe (II and Fe (III in a Binary System Evaluated Bioluminescent Method

    Directory of Open Access Journals (Sweden)

    Elena Sorokina

    2013-01-01

    Full Text Available The effect of iron ions Fe2+ and Fe3+ on the bioluminescent recombinant strain of Escherichia coli in a single-component and binary system. Found that for the bacteria E. coli Fe3+ ions are more toxic than Fe2+. Under the combined effect of iron toxicity increases, the percentage of luminescence quenching increases, but the value is much less than the sum of the indicator for the Fe2+ and Fe3+. The biological effect of insertion of iron is not proportional to their content in the mixture.

  1. Novel peptide chemistry in terrestrial animals: natural luciferin analogues from the bioluminescent earthworm Fridericia heliota.

    Science.gov (United States)

    Dubinnyi, Maxim A; Tsarkova, Aleksandra S; Petushkov, Valentin N; Kaskova, Zinaida M; Rodionova, Natalja S; Kovalchuk, Sergey I; Ziganshin, Rustam H; Baranov, Mikhail S; Mineev, Konstantin S; Yampolsky, Ilia V

    2015-03-02

    We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine-derived chromophores, CompX or CompY and a set of amino acids, including threonine, gamma-aminobutyric acid, homoarginine, and unsymmetrical N,N-dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin.

  2. In Vivo Bioluminescent Imaging (BLI: Noninvasive Visualization and Interrogation of Biological Processes in Living Animals

    Directory of Open Access Journals (Sweden)

    Steven Ripp

    2010-12-01

    Full Text Available In vivo bioluminescent imaging (BLI is increasingly being utilized as a method for modern biological research. This process, which involves the noninvasive interrogation of living animals using light emitted from luciferase-expressing bioreporter cells, has been applied to study a wide range of biomolecular functions such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis, cancer treatment, and disease progression. This article will review the various bioreporter/biosensor integrations of BLI and discuss how BLI is being applied towards a new visual understanding of biological processes within the living organism.

  3. In Vivo Bioluminescent Imaging (BLI): Noninvasive Visualization and Interrogation of Biological Processes in Living Animals

    Science.gov (United States)

    Close, Dan M.; Xu, Tingting; Sayler, Gary S.; Ripp, Steven

    2011-01-01

    In vivo bioluminescent imaging (BLI) is increasingly being utilized as a method for modern biological research. This process, which involves the noninvasive interrogation of living animals using light emitted from luciferase-expressing bioreporter cells, has been applied to study a wide range of biomolecular functions such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis, cancer treatment, and disease progression. This article will review the various bioreporter/biosensor integrations of BLI and discuss how BLI is being applied towards a new visual understanding of biological processes within the living organism. PMID:22346573

  4. Toxicity assessment of Hanford Site wastes by bacterial bioluminescence. [Photobacter phosphoreum:a3

    Energy Technology Data Exchange (ETDEWEB)

    Rebagay, T.V.; Dodd, D.A.; Voogd, J.A.

    1991-09-01

    This paper examines the toxicity of the nonradioactive component of low-level wastes stored in tanks on the Hanford reservation. The use of a faster, cheaper bioassay to replace the 96 hour fish acute toxicity test is examined. The new bioassay is based on loss of bioluminescence of {und Photobacter phosphoreum} (commonly called Microtox) following exposure to toxic materials. This bioassay is calibrated and compares well to the standard fish acute toxicity test for characterization of Hanford Wastes. 4 refs., 11 figs., 11 tabs. (MHB)

  5. Quantum/molecular mechanics study of firefly bioluminescence on luciferase oxidative conformation

    Science.gov (United States)

    Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

    2014-07-01

    This is the first report of a computational study of the color tuning mechanism of firefly bioluminescence, using the oxidative conformation of luciferase. The results of these calculations demonstrated that the electrostatic field generated by luciferase is fundamental both for the emission shift and efficiency. Further calculations indicated that a shift in emission is achieved by modulating the energy, at different degrees, of the emissive and ground states. These differences in energy modulation will then lead to changes in the energy gap between the states.

  6. A measurement-based analytical approach to the bioluminescence tomography problem

    Science.gov (United States)

    Erkol, Hakan; Demirkiran, Aytac; Kipergil, Esra-Aytac; Uluc, Nasire; Unlu, Mehmet B.

    2014-03-01

    This work presents an analytical approach for the solution of the tissue diffusion equation based on the bound- ary measurements. We consider a bioluminescent point source in both homogeneous and heterogeneous circular turbid media. The point source is described by the Dirac delta function. Analytical expressions for the strength and position of the point source are obtained introducing boundary measurements and then applying appropriate boundary conditions. In addition, numerical simulations are performed for the position of the source. Calculations show that that the analytical results are in a good accordance with the numerical results.

  7. [An adenosine triphosphate bioluminescence assay for detecting the number of living cells].

    Science.gov (United States)

    Liu, S; Peng, Z; Wang, H; Lou, J; He, B; Tang, Q; Qiu, D

    2000-06-01

    The method for detecting the number of living cells was studied. Using an adenosine triphosphate (ATP) bioluminescence assay, the present authors reported a perfect linear relationship between lg ATP concentrations and lg luminescence counts (r = 0.9963) as well as a relationship between lg number of cells and lg ATP luminescence counts (r = 0.9922). The detectable cells ranged from 10(2) to 10(6) cells/ml, the coefficients of variation 1-3%. This method is simple, accurate and sensitive and has a high reproducibility.

  8. Bioluminescence Microscopy as a Method to Measure Single Cell Androgen Receptor Activity Heterogeneous Responses to Antiandrogens

    Science.gov (United States)

    Jain, Pallavi; Neveu, Bertrand; Velot, Lauriane; Wu, Lily; Fradet, Yves; Pouliot, Frédéric

    2016-01-01

    Cancer cell heterogeneity is well-documented. Therefore, techniques to monitor single cell heterogeneous responses to treatment are needed. We developed a highly translational and quantitative bioluminescence microscopy method to measure single cell androgen receptor (AR) activity modulation by antiandrogens from fluid biopsies. We showed that this assay can detect heterogeneous cellular response to drug treatment and that the sum of single cell AR activity can mirror the response in the whole cell population. This method may thus be used to monitor heterogeneous dynamic treatment responses in cancer cells. PMID:27678181

  9. Improved Reconstruction Quality of Bioluminescent Images by Combining SP3 Equations and Bregman Iteration Method

    Directory of Open Access Journals (Sweden)

    Qiang Wu

    2013-01-01

    Full Text Available Bioluminescence tomography (BLT has a great potential to provide a powerful tool for tumor detection, monitoring tumor therapy progress, and drug development; developing new reconstruction algorithms will advance the technique to practical applications. In the paper, we propose a BLT reconstruction algorithm by combining SP3 equations and Bregman iteration method to improve the quality of reconstructed sources. The numerical results for homogeneous and heterogeneous phantoms are very encouraging and give significant improvement over the algorithms without the use of SP3 equations and Bregman iteration method.

  10. A Bioluminescence Assay System for Imaging Metal Cationic Activities in Urban Aerosols.

    Science.gov (United States)

    Kim, Sung-Bae; Naganawa, Ryuichi; Murata, Shingo; Nakayama, Takayoshi; Miller, Simon; Senda, Toshiya

    2016-01-01

    A bioluminescence-based assay system was fabricated for an efficient determination of the activities of air pollutants. The following four components were integrated into this assay system: (1) an 8-channel assay platform uniquely designed for simultaneously sensing multiple optical samples, (2) single-chain probes illuminating toxic chemicals or heavy metal cations from air pollutants, (3) a microfluidic system for circulating medium mimicking the human body, and (4) the software manimulating the above system. In the protocol, we briefly introduce how to integrate the components into the system and the application to the illumination of the metal cationic activities in air pollutants.

  11. Bacteria as part of bioluminescence emission at the deep ANTARES station (North-Western Mediterranean Sea) during a one-year survey

    Science.gov (United States)

    Martini, S.; Michotey, V.; Casalot, L.; Bonin, P.; Guasco, S.; Garel, M.; Tamburini, C.

    2016-10-01

    Bioluminescent bacteria have been studied during a one-year survey in 2011 at the deep ANTARES site (Northwestern Mediterranean Sea, 2000 m depth). The neutrino underwater telescope ANTARES, located at this station, has been used to record the bioluminescence at the same depth. Together with these data, environmental variables (potential temperature, salinity, nutrients, dissolved organic carbon and oxygen) have been characterized in water samples. The year 2011 was characterized by relatively stable conditions, as revealed by minor variability in the monitored oceanographic variables, by low bioluminescence and low current speed. This suggests weak eukaryote participation and mainly non-stimulated light emission. Hence, no processes of dense water have affected the ANTARES station during this survey. Abundance of bioluminescent bacteria belonging to Photobacterium genus, measured by qPCR of the luxF gene, ranged from 1.4×102 to 7.2×102 genes mL-1. Their effective activity was confirmed through mRNA luxF quantification. Our results reveal that bioluminescent bacteria appeared more active than the total counterpart of bacteria, suggesting an ecological benefit of this feature such as favoring interaction with macro-organisms. Moreover, these results show that part of the bioluminescence, recorded at 2000 m depth over one year, could be due to bioluminescent bacteria in stable hydrological conditions.

  12. Diversity of the luciferin binding protein gene in bioluminescent dinoflagellates--insights from a new gene in Noctiluca scintillans and sequences from gonyaulacoid genera.

    Science.gov (United States)

    Valiadi, Martha; Iglesias-Rodriguez, Maria Debora

    2014-01-01

    Dinoflagellate bioluminescence systems operate with or without a luciferin binding protein, representing two distinct modes of light production. However, the distribution, diversity, and evolution of the luciferin binding protein gene within bioluminescent dinoflagellates are not well known. We used PCR to detect and partially sequence this gene from the heterotrophic dinoflagellate Noctiluca scintillans and a group of ecologically important gonyaulacoid species. We report an additional luciferin binding protein gene in N. scintillans which is not attached to luciferase, further to its typical combined bioluminescence gene. This supports the hypothesis that a profound re-organization of the bioluminescence system has taken place in this organism. We also show that the luciferin binding protein gene is present in the genera Ceratocorys, Gonyaulax, and Protoceratium, and is prevalent in bioluminescent species of Alexandrium. Therefore, this gene is an integral component of the standard molecular bioluminescence machinery in dinoflagellates. Nucleotide sequences showed high within-strain variation among gene copies, revealing a highly diverse gene family comprising multiple gene types in some organisms. Phylogenetic analyses showed that, in some species, the evolution of the luciferin binding protein gene was different from the organism's general phylogenies, highlighting the complex evolutionary history of dinoflagellate bioluminescence systems.

  13. Identification of a vacuolar proton channel that triggers the bioluminescent flash in dinoflagellates

    Science.gov (United States)

    Rodriguez, Juan D.; Haq, Saddef; Bachvaroff, Tsvetan; Nowak, Kristine F.; Nowak, Scott J.; Morgan, Deri; Cherny, Vladimir V.; Sapp, Maredith M.; Bernstein, Steven; Bolt, Andrew; DeCoursey, Thomas E.; Place, Allen R.; Smith, Susan M. E.

    2017-01-01

    In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel (HV1) that opens in response to depolarizing voltage across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thereby causing a pH drop that triggers light emission. HV1 channels were subsequently identified and demonstrated to have important functions in a multitude of eukaryotic cells. Here we report a predicted protein from Lingulodinium polyedrum that displays hallmark properties of bona fide HV1, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. Western blotting and fluorescence confocal microscopy of isolated L. polyedrum scintillons immunostained with antibody to LpHV1 confirm LpHV1’s predicted organellar location. Proteomics analysis demonstrates that isolated scintillon preparations contain peptides that map to LpHV1. Finally, Zn2+ inhibits both LpHV1 proton current and the acid-induced flash in isolated scintillons. These results implicate LpHV1 as the voltage gated proton channel that triggers bioluminescence in L. polyedrum, confirming Hastings’ hypothesis. The same channel likely mediates the action potential that communicates the signal along the tonoplast to the scintillon. PMID:28178296

  14. Coregulation of lux genes and riboflavin genes in bioluminescent bacteria of Photobacterium phosphoreum.

    Science.gov (United States)

    Sung, Nack-Do; Lee, Chan Yong

    2004-09-01

    Investigation of the expression of the riboflavin (rib) genes, which are found immediately downstream of luxG in the lux operon in Photobacterium phosphoreum, provides more information relevant to the evolution of bioluminescence, as well as to the regulation of supply of flavin substrate for bacterial bioluminescence reactions. In order to answer the question of whether or not the transcriptions of lux and rib genes are integrated, a transcriptional termination assay was performed with P. phosphoreum DNA, containing the possible stem-loop structures, located in the intergenic region of luxF and luxE (OmegaA), of luxG and ribE (OmegaB), and downstream of ribA (OmegaC). The expression of the CAT (Chloramphenicol Acetyl Transferase) reporter gene was remarkably decreased upon the insertion of the stem-loop structure (OmegaC) into the strong lux promoter and the reporter gene. However, the insertion of the structure (OmegaB) into the intergenic region of the lux and the rib genes caused no significant change in expression from the CAT gene. In addition, the single stranded DNA in the same region was protected by the P. phosphoreum mRNA from the S1 nuclease protection assay. These results suggest that lux genes and rib genes are part of the same operon in P. phosphoreum.

  15. Improved AFEM algorithm for bioluminescence tomography based on dual-mesh alternation strategy

    Institute of Scientific and Technical Information of China (English)

    Wei Li; Heng Zhao; Xiaochao Qu; Yanbin Hou; Xueli Chen; Duofang Chen; Xiaowei He; Qitan Zhang; Jimin Liang

    2012-01-01

    Adaptive finite element method (AFEM) is broadly adopted to recover the internal source in biological tissues.In this letter,a novel dual-mesh alternation strategy (dual-mesh AFEM) is developed for bioluminescence tomography.By comprehensively considering the error estimation of the finite element method solution on each mesh,two different adaptive strategies based on the error indicator of the reconstructed source and the photon flux density are used alternately in the process.Combined with the constantly adjusted permissible region in the adaptive process,the new algorithm can achieve a more accurate source location compared with the AFEM in the previous experiments.%Adaptive finite element method (AFEM) is broadly adopted to recover the internal source in biological tissues. In this letter, a novel dual-mesh alternation strategy (dual-mesh AFEM) is developed for biolumi-nescence tomography. By comprehensively considering the error estimation of the finite element method solution on each mesh, two different adaptive strategies based on the error indicator of the reconstructed source and the photon flux density are used alternately in the process. Combined with the constantly adjusted permissible region in the adaptive process, the new algorithm can achieve a more accurate source location compared with the AFEM in the previous experiments.

  16. Toxicological study of pesticides in air and precipitations of Paris by means of a bioluminescence method.

    Science.gov (United States)

    Trajkovska, S; Mbaye, M; Gaye Seye, M D; Aaron, J J; Chevreuil, M; Blanchoud, H

    2009-06-01

    A detailed toxicological study on several pesticides, including chlorothalonil, cyprodynil, dichlobénil, pendimethaline, trifluraline, and alpha-endosulfan, present at trace levels in air and total atmospheric precipitations of Paris is presented. The pesticides contained in the atmospheric samples, collected during sampling campaigns in February-March 2007, are identified and quantified by a high-performance liquid chromatographic (HPLC)-UV detection method. The toxicity measurements are performed by means of the Microtox bioluminescence method, based on the evaluation of the bioluminescence inhibition of the Vibrio fischeri marine bacteria at two exposure times to the pesticide solutions. The specific toxicity, corresponding to the particular toxicity of the compound under study and represented by the EC(50) parameter, is determined for these pesticides. Also, the global toxicity, which is the toxicity of all micro-pollutants present in the sample under study, is estimated for the extracts of air and atmospheric precipitation (rainwater) samples. The specific toxicities strongly vary with the nature of the pesticide, the EC(50) parameter values being comprised between 0.17 and 0.83 mg/mL and 0.15 and 0.66 mg/mL, respectively, for exposure times of 5 and 15 min. The importance of the atmospheric samples' global toxicity and the respective contribution of the toxic potency of the various pesticides contained in these samples are discussed.

  17. In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila.

    Science.gov (United States)

    Martin, Jean-René; Rogers, Kelly L; Chagneau, Carine; Brûlet, Philippe

    2007-03-07

    Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain.

  18. In vivo Bioluminescence Imaging of Ca2+ Signalling in the Brain of Drosophila

    Science.gov (United States)

    Chagneau, Carine; Brûlet, Philippe

    2007-01-01

    Many different cells' signalling pathways are universally regulated by Ca2+ concentration [Ca2+] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca2+ signals involved in neurophysiological functions. New methods for in vivo imaging of Ca2+ signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca2+ imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca2+ reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca2+] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca2+ response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca2+] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca2+] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca2+ stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca2+ signalling pathways and for functional mapping of neurophysiological processes in the fly brain. PMID:17342209

  19. Molecular phylogeny and node time estimation of bioluminescent Lantern Sharks (Elasmobranchii: Etmopteridae).

    Science.gov (United States)

    Straube, Nicolas; Iglésias, Samuel P; Sellos, Daniel Y; Kriwet, Jürgen; Schliewen, Ulrich K

    2010-09-01

    Deep-sea Lantern Sharks (Etmopteridae) represent the most speciose family within Dogfish Sharks (Squaliformes). We compiled an extensive DNA dataset to estimate the first molecular phylogeny of the family and to provide node age estimates for the origin and diversification for this enigmatic group. Phylogenetic inferences yielded consistent and well supported hypotheses based on 4685bp of both nuclear (RAG1) and mitochondrial genes (COI, 12S-partial 16S, tRNAVal and tRNAPhe). The monophyletic family Etmopteridae originated in the early Paleocene around the C/T boundary, and split further into four morphologically distinct lineages supporting three of the four extant genera. The exception is Etmopterus which is paraphyletic with respect to Miroscyllium. Subsequent rapid radiation within Etmopterus in the Oligocene/early Miocene was accompanied by divergent evolution of bioluminescent flank markings which morphologically characterize the four lineages. Higher squaliform interrelationships could not be satisfactorily identified, but convergent evolution of bioluminescence in Dalatiidae and Etmopteridae is supported.

  20. A look at some systemic properties of self-bioluminescent emission

    Science.gov (United States)

    Creath, Katherine

    2008-08-01

    Self-bioluminescent emission (SBE) is a type of biological chemiluminescence where photons are emitted as part of chemical reactions occurring during metabolic processes. This emission is also known as biophoton emission, ultraweak photon emission and ultraweak bioluminescence. This paper outlines research over the past century on some systemic properties of SBE as measured with biological detectors, photomultiplier detectors and ultra-sensitive imaging arrays. There is an apparent consensus in the literature that emission in the deep blue and ultraviolet (150-450nm) is related to DNA / RNA processes while emission in the red and near infrared (600-1000nm) is related to mitochondria and oxidative metabolisms involving reactive oxygen species, singlet oxygen and free radicals in plant, animal and human cells along with chlorophyll fluorescent decay in plants. Additionally, there are trends showing that healthy, unstressed and uninjured samples have less emission than samples that are unhealthy, stressed or injured. Mechanisms producing this emission can be narrowed down by isolating the wavelength region of interest and waiting for short-term fluorescence to decay leaving the ultraweak long-term metabolic emission. Examples of imaging this emission in healthy versus unhealthy, stressed versus unstressed, and injured versus uninjured plant parts are shown. Further discussion poses questions still to be answered related to properties such as coherence, photon statistics, and methodological means of isolating mechanisms.

  1. Development of a Fully Automated Flow Injection Analyzer Implementing Bioluminescent Biosensors for Water Toxicity Assessment

    Directory of Open Access Journals (Sweden)

    Constantinos Georgiou

    2010-07-01

    Full Text Available This paper describes the development of an automated Flow Injection analyzer for water toxicity assessment. The analyzer is validated by assessing the toxicity of heavy metal (Pb2+, Hg2+ and Cu2+ solutions. One hundred μL of a Vibrio fischeri suspension are injected in a carrier solution containing different heavy metal concentrations. Biosensor cells are mixed with the toxic carrier solution in the mixing coil on the way to the detector. Response registered is % inhibition of biosensor bioluminescence due to heavy metal toxicity in comparison to that resulting by injecting the Vibrio fischeri suspension in deionised water. Carrier solutions of mercury showed higher toxicity than the other heavy metals, whereas all metals show concentration related levels of toxicity. The biosensor’s response to carrier solutions of different pHs was tested. Vibrio fischeri’s bioluminescence is promoted in the pH 5–10 range. Experiments indicate that the whole cell biosensor, as applied in the automated fluidic system, responds to various toxic solutions.

  2. Saccharomyces cerevisiae BLYAS, a New Bioluminescent Bioreporter for Detection of Androgenic Compounds▿

    Science.gov (United States)

    Eldridge, Melanie L.; Sanseverino, John; Layton, Alice C.; Easter, James P.; Schultz, T. Wayne; Sayler, Gary S.

    2007-01-01

    A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp), yielded a bioluminescent bioreporter responsive to androgenic chemicals. Using dihydrotestosterone (DHT) as a standard, the response time and the 50% effective concentration values were 3 to 4 h and (9.7 ± 4.6) × 10−9 M, respectively. The lower limit of detection in response to DHT was 2.5 × 10−9 M, and in response to testosterone it was 2.5 × 10−10 M. This strain is suitable for high-throughput screening of chemicals with potential for remote environmental monitoring systems because of the assay speed, sensitivity, and self-containment. PMID:17675419

  3. In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila.

    Directory of Open Access Journals (Sweden)

    Jean-René Martin

    Full Text Available Many different cells' signalling pathways are universally regulated by Ca(2+ concentration [Ca(2+] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+ signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+ signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+ imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+ reporter GFP-aequorin (GA in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+ response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+] transients in the Mushroom Bodies (MBs following nicotine stimulation were accompanied by a delayed secondary [Ca(2+] rise (up to 15 min. later in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+ stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+ signalling pathways and for functional mapping of neurophysiological processes in the fly brain.

  4. Near seafloor bioluminescence, macrozooplankton and macroparticles at the Mid-Atlantic Ridge

    Science.gov (United States)

    Craig, Jessica; Youngbluth, Marsh; Jamieson, Alan J.; Priede, Imants G.

    2015-04-01

    The benthic boundary layer is a region often perceived to be high in faunal abundance and biomass. In this study, we investigated the distribution of near seafloor bioluminescent zooplankton (BL), macrozooplankton (>1 cm) and macroparticles (>430 μm) at the Northern Mid-Atlantic Ridge at ca. 2500 m depth. At sites south of 52°N, the Charlie Gibbs Fracture Zone, BL density increased weakly towards the seafloor. This trend was driven by small bioluminescent crustaceans, comprising ca. 90% of the total BL density. Macroparticle density was coherent with BL density, exhibiting a small increase towards the seafloor. Appendicularians (animals as well as occupied and discarded houses) were the most abundant macrozooplankton, and the only group to show a significant increase in density towards the seafloor. The absence of pronounced increases in BL and macroparticle density, and no increase in macrozooplankton (except appendicularians), towards the seafloor do not support the conventional view of high concentrations of particulate organic matter and zooplankton biomass in the benthic boundary layer relative to overlying waters.

  5. Accounting for filter bandwidth improves the quantitative accuracy of bioluminescence tomography

    Science.gov (United States)

    Taylor, Shelley L.; Mason, Suzannah K. G.; Glinton, Sophie L.; Cobbold, Mark; Dehghani, Hamid

    2015-09-01

    Bioluminescence imaging is a noninvasive technique whereby surface weighted images of luminescent probes within animals are used to characterize cell count and function. Traditionally, data are collected over the entire emission spectrum of the source using no filters and are used to evaluate cell count/function over the entire spectrum. Alternatively, multispectral data over several wavelengths can be incorporated to perform tomographic reconstruction of source location and intensity. However, bandpass filters used for multispectral data acquisition have a specific bandwidth, which is ignored in the reconstruction. In this work, ignoring the bandwidth is shown to introduce a dependence of the recovered source intensity on the bandwidth of the filters. A method of accounting for the bandwidth of filters used during multispectral data acquisition is presented and its efficacy in increasing the quantitative accuracy of bioluminescence tomography is demonstrated through simulation and experiment. It is demonstrated that while using filters with a large bandwidth can dramatically decrease the data acquisition time, if not accounted for, errors of up to 200% in quantitative accuracy are introduced in two-dimensional planar imaging, even after normalization. For tomographic imaging, the use of this method to account for filter bandwidth dramatically improves the quantitative accuracy.

  6. Accounting for systematic errors in bioluminescence imaging to improve quantitative accuracy

    Science.gov (United States)

    Taylor, Shelley L.; Perry, Tracey A.; Styles, Iain B.; Cobbold, Mark; Dehghani, Hamid

    2015-07-01

    Bioluminescence imaging (BLI) is a widely used pre-clinical imaging technique, but there are a number of limitations to its quantitative accuracy. This work uses an animal model to demonstrate some significant limitations of BLI and presents processing methods and algorithms which overcome these limitations, increasing the quantitative accuracy of the technique. The position of the imaging subject and source depth are both shown to affect the measured luminescence intensity. Free Space Modelling is used to eliminate the systematic error due to the camera/subject geometry, removing the dependence of luminescence intensity on animal position. Bioluminescence tomography (BLT) is then used to provide additional information about the depth and intensity of the source. A substantial limitation in the number of sources identified using BLI is also presented. It is shown that when a given source is at a significant depth, it can appear as multiple sources when imaged using BLI, while the use of BLT recovers the true number of sources present.

  7. Nanoluciferase signal brightness using furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy.

    Science.gov (United States)

    Kim, Jiho; Grailhe, Regis

    2016-08-01

    Fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques are powerful tools for studying protein-protein interactions in cellular assays. In contrast to fluorescent proteins, chemiluminescent proteins do not require excitation light, known to trigger autofluorescence, phototoxicity, and photobleaching. Regrettably, low signal intensity of luciferase systems restricts their usage as they require specialized microscopes equipped with ultra low-light imaging cameras. In this study, we report that bioluminescence quantification in living cells using a standard widefield automated microscope dedicated to screening and high content analysis is possible with the newer luciferase systems, Nanoluciferase (Nluc). With such equipment, we showed that robust intramolecular BRET can be measured using a combination of Nluc and yellow fluorescent protein (YFP). Using the human Superoxide Dismutase 1 (SOD1) dimer model, we next validated that intermolecular BRET could be quantified at a single cell level. The enhanced signal brightness of Nluc enabling BRET imaging to widefield microscopy shows strong potential to open up single cell protein-protein interactions studies to a wider audience. © 2016 International Society for Advancement of Cytometry.

  8. A bioluminescence reporter mouse that monitors expression of constitutively active β-catenin

    Science.gov (United States)

    Kommagani, Ramakrishna; Peavey, Mary C.; Hai, Lan; Lonard, David M.; Lydon, John P.

    2017-01-01

    This short technical report describes the generation and characterization of a bioluminescence reporter mouse that is engineered to detect and longitudinally monitor the expression of doxycycline-induced constitutively active β-catenin. The new responder transgenic mouse contains the TetO-ΔN89β-CatTMILA transgene, which consists of the tet-operator followed by a bicistronic sequence encoding a stabilized form of active β-catenin (ΔN89β-catenin), an internal ribosome entry site, and the firefly luciferase gene. To confirm that the transgene operates as designed, TetO-ΔN89β-CatTMILA transgenic mouse lines were crossed with an effector mouse that harbors the mouse mammary tumor virus-reverse tetracycline transactivator (MMTV-rtTA) transgene (termed MTB hereon), which primarily targets rtTA expression to the mammary epithelium. Following doxycycline administration, the resultant MTB/CatTMILA bigenic reporter exhibited precocious lobuloalveologenesis, ductal hyperplasia, and mammary adenocarcinomas, which were visualized and monitored by in vivo bioluminescence detection. Therefore, we predict that the TetO-ΔN89β-CatTMILA transgenic responder mouse—when crossed with the appropriate effector transgenic—will have wide-applicability to non-invasively monitor the influence of constitutively active β-catenin expression on cell-fate specification, proliferation, differentiation, and neoplastic transformation in a broad spectrum of target tissues. PMID:28253313

  9. Relation between deep bioluminescence and oceanographic variables: A statistical analysis using time-frequency decompositions

    Science.gov (United States)

    Martini, S.; Nerini, D.; Tamburini, C.

    2014-09-01

    We consider the statistical analysis of a 1.7-year high-frequency sampled time series, between 2009 and 2010, recorded at the ANTARES observatory in the deep NW Mediterranean Sea (2475 m depth). The objective was to estimate relationships between bioluminescence and environmental time series (temperature, salinity and current speed). As this entire dataset is characterized by non-linearity and non-stationarity, two time-frequency decomposition methods (wavelet and Hilbert-Huang) were used. These mathematical methods are dedicated to the analysis of a signal at various time and frequencies scales. This work propose some statistical tools dedicated to the study of relationships between two time series. Our study highlights three events of high bioluminescence activity in March 2009, December 2009 and March 2010. We demonstrate that the two events occurring in March 2009 and 2010 are correlated to the arrival of newly formed deep water masses at frequencies of approximately 4.8×10-7 (period of 24.1 days). In contrast, the event in December 2009 is only correlated with current speed at frequencies of approximately 1.9×10-6 (period of 6.0 days). The use of both wavelet and Hilbert-Huang transformations has proven to be successful for the analysis of multivariate time series. These methods are well-suited in a context of the increasing number of long time series recorded in oceanography.

  10. Quantitative Imaging of D-2-Hydroxyglutarate in Selected Histological Tissue Areas by a Novel Bioluminescence Technique.

    Science.gov (United States)

    Voelxen, Nadine F; Walenta, Stefan; Proescholdt, Martin; Dettmer, Katja; Pusch, Stefan; Mueller-Klieser, Wolfgang

    2016-01-01

    Patients with malignant gliomas have a poor prognosis with average survival of less than 1 year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG), a metabolite that was discovered first in this tumor entity. D2HG is generated in large amounts due to various "gain-of-function" mutations in the isocitrate dehydrogenases IDH1 and IDH2. Meanwhile, D2HG has been detected in several other tumor entities, including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography/mass spectrometry. The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0-10 μmol/g tissue (wet weight). In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension.

  11. Antimicrobial properties of cyclodextrin-antiseptics-complexes determined by microplate laser nephelometry and ATP bioluminescence assay.

    Science.gov (United States)

    Finger, Susanne; Wiegand, Cornelia; Buschmann, Hans-Jürgen; Hipler, Uta-Christina

    2012-10-15

    Antimicrobial effects of substances can be determined with different methods that measure distinct parameters. Thus, a comparison of the results obtained can be difficult. In this study, two in vitro methods were employed to determine concentration and time dependent effects of cyclodextrin (CD)-complexes with the antiseptics chlorhexidine diacetate (CHX), iodine (IOD) and polihexanide (PHMB) on Candida albicans and Malassezia pachydermatis. Using both, microplate laser nephelometry and the ATP bioluminescence assay, it could be shown that CD-antiseptics-complexes tested exhibited significant antifungal effects with the exception of γ-CD-CHX in the case of C. albicans. Microplate laser nephelometry (MLN) is an optical method and enables a quantitative determination of particle concentrations in solution. By means of this method, microbial growth under influence of potential antimicrobial substances can be monitored over a prolonged time period. In addition, the antimicrobial activity was analyzed by measurement of the microbial adenosine triphosphate (ATP) content with a bioluminescent assay. The luminescent signal is directly proportional to the amount of ATP, and thus, a linear function of the number of living microbial cells present. Both methods were compared according to the half maximal inhibitory concentration (IC(50)) calculated and the statistical evaluation of Pearson's correlation coefficient (r). In summary, it could be demonstrated that both methods yield similar results although they differ in the parameter.

  12. Interference sources in ATP bioluminescence assay of silica nanoparticle toxicity to activated sludge.

    Science.gov (United States)

    Sibag, Mark; Kim, Seung Hwan; Kim, Choah; Kim, Hee Jun; Cho, Jinwoo

    2015-06-01

    ATP measurement provides an overview of the general state of microbial activity, and thus it has proven useful for the evaluation of nanoparticle toxicity in activated sludge. ATP bioluminescence assay, however, is susceptible to interference by the components of activated sludge other than biomass. This paper presents the interference identified specific to the use of this assay after activated sludge respiration inhibition test of silica nanoparticles (OECD 209). We observed a high degree of interference (90%) in the presence of 100 mg/L silica nanoparticles and a low level of ATP being measured (0.01 μM); and 30% interference by the synthetic medium regardless of silica nanoparticle concentration and ATP level in the samples. ATP measurement in activated sludge with different MLSS concentrations revealed interference of high biomass content. In conclusion, silica nanoparticles, synthetic medium and activated sludge samples themselves interfere with ATP bioluminescence; this will need to be considered in the evaluation of silica nanoparticle toxicity to activated sludge when this type of assay is used.

  13. The reaction process of firefly bioluminescence triggered by photolysis of caged-ATP.

    Science.gov (United States)

    Kageyama, Takeshi; Tanaka, Masatoshi; Sekiya, Takao; Ohno, Shin-Ya; Wada, Naohisa

    2011-01-01

    The reaction process of firefly bioluminescence was studied by photolyzing caged-ATP to adenosine triphosphate (ATP) within 100 ms. The intensity of luminescence increases markedly to reach a maximum within 1 s, maintains almost the same intensity up to 5 s and then decays monotonically. The rise γ(1) and decay γ(2) rate constants were determined to be about 5 s(-1) and 1 × 10(-2) s(-1), respectively, so as to phenomenologically fit the time course. A second luminescence peak appears after around 350 s. The dependence of the rate constants on the concentrations of reactants and a viscous reagent revealed that two kinds of reaction contribute the observed time course: (1) an intrinsic reaction by ATP photolyzed from caged-ATP that is already trapped in luciferase; and (2) a diffusion-controlled reaction by free ATP in the buffer solution outside luciferase. Numerical analysis based on reaction kinetics related γ(1) and γ(2) to the rate constants of a three-step reaction model, and accurately described the effects of concentration of reactants and a viscous reagent on the time courses of bioluminescence. Thus, it has been clearly concluded that the binding mode of caged-ATP at the catalytic center of luciferase is very different from that of ATP.

  14. Application of bioluminescence ATP measurement for evaluation of fungal viability of foxing spots on old documents.

    Science.gov (United States)

    Rakotonirainy, Malalanirina Sylvia; Dubar, Pauline

    2013-01-01

    An adenosine triphosphate (ATP) bioluminescence-based protocol was tested to assess the viability of fungal species in old documents damaged by foxing. Foxing appears as scattered yellow brownish-red stains, damaging the aesthetics of documents and their long-term readability. In the field of cultural heritage conservation, the debate over the mechanism of foxing is ongoing. Previous studies found evidence of mold-like structures in some coloured areas; however, many species have not yet been identified and their role in the phenomenon is not understood. To better understand their involvement in this type of paper decay, we focused our attention first on their viability. We demonstrated the reliability and sensitivity of the ATP bioluminescence assay compared with conventional methods based on cultivation, which has rarely given rise to in vitro growth from foxed papers. From nine books dating back from the 19th and 20th centuries, the mean ATP amount of foxed spots ranged from 0.29 to 3.63 ng/cm(2), suggesting the presence of strains inside the brownish spots and providing evidence of their viability. Outside the spots, ATP content was considered negligible, with a mean ATP amount of 0 to 0.03 ng/cm(2). ATP assay appears to be a useful and robust method for the detection and quantification of viable elements in foxing spots.

  15. Combining biofunctional magnetic nanoparticles and ATP bioluminescence for rapid detection of Escherichia coli.

    Science.gov (United States)

    Cheng, Yuxiao; Liu, Yajun; Huang, Jingjing; Li, Kang; Zhang, Wen; Xian, Yuezhong; Jin, Litong

    2009-02-15

    A rapid, specific and sensitive method for assay of Escherichia coli (E. coli) using biofunctional magnetic nanoparticles (BMNPs) in combination with adenosine triphosphate (ATP) bioluminescence was proposed. The BMNPs were fabricated by immobilizing a specific anti-E. coli antibody on the surface of amine-functionalized magnetic nanoparticles (about 20nm in diameter), and then was applied to capture the target bacteria E. coli from samples. The BMNPs exhibited high capture efficiency to E. coli. Transmission electron microscope (TEM) images showed that the BMNPs were bound to the surface of entire E. coli cells. The target bacteria became magnetic so that could be isolated easily from the sample solution by employing an external magnetic field. The concentration of E. coli captured by the BMNPs was then detected by an ATP bioluminescence method. The optimization of ATP measurement was carried out to improve the detection sensitivity. The proposed method was applied to detect the E. coli inoculated into pasteurized milk with low detection limit (20 cfu/mL) and short detection time (about 1h).

  16. Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, L.E.; Hoffner, S.E.; Ansehn, S.

    1988-08-01

    Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria.

  17. Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Byers, D.M.; Meighen, E.A.

    1985-09-01

    Pulse-chase experiments with (/sup 3/H)tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a /sup 3/H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi (/sup 3/H)acylprotein and (/sup 3/H)palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (34K) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.

  18. [Studies on detection of E. coli O157:H7 by ATP bioluminescence].

    Science.gov (United States)

    Tang, Qian-Qian; Wang, Jian-Ping; Gai, Lin; Ye, Zun-Zhong; Ying, Yi-Bin

    2009-02-01

    In the present paper, a quantitative linear model betweena series of concentrations of E. coliO157 : H7 and counts by BPCL ultra weak luminescence analyzer was built up. And the influences of four different buffers with the same pH (pH = 7.4), Tris-HCl, PBS, KH2PO4-NaOH and Na2 HPO4-C6H8O7, and five different chemical substances with the same mass concentration (10 g x L), NaCl, KCl, NaOH, MgCl2 and NaH2PO4 on ATP bioluminescence were compared. The results showed that Tris-HCl was a suitable buffer for dilution, since it could distinguish well between different concentrations and had the lowest background signals. And MgCl2 could intensify luminescence distinctly, while the other four chemical substances decreased luminescence, of which NaOH decreased luminescence most obviously. Moreover, ATP bioluminescence was correlated well with conventional culture methods (r = 0.96), and the detection limit was 10(3) cells x mL(-1).

  19. In Situ ATP Bioluminescent Measurements in Subglacial Environments - The Engabreen Glacier in the Norwegian Arctic

    Science.gov (United States)

    Cullen, D. C.; Wadham, J. L.; Pancost, R.; Kelly, S.; Barnett, M. J.; Jackson, M.

    2007-12-01

    Engabreen is a northern outlet glacier from the western Svartisen ice cap on the Nordland coast of Norway just inside the Arctic Circle. A unique feature of the glacier is a man-made tunnel system within the bedrock beneath the glacier that offers scientists direct access to the glacier-bedrock interface. This unique facility - called the Engabreen Subglacial Laboratory - is ideal to test developments of new in situ analytical techniques. We have used the facility to perform the first in situ detection of microbial life in a subglacial environment using standard off-the-shelf ATP bioluminescence detection technology and therefore using ATP levels as a proxy of microbial life. Measurements were performed both in melt-waters in the tunnels and from melted ice samples directly from the glacier-bedrock interface. Levels of ATP above background were detected and appeared to be associated with suspended sediment particles rather than in the water or ice component. This indicated the presence of microbial life. Development of protocols for in situ sample processing and use of in situ ATP measurements in the directing and choice of sampling points for other techniques was explored. This study has shown that off-the-shelf portable ATP bioluminescence can be used to perform in situ measurements within sub-glacial environments but that further development work is required to optimize experimental protocols and to correlate findings with other life detection and enumeration techniques.

  20. Antibacterial properties of cyclodextrin-antiseptics-complexes determined by microplate laser nephelometry and ATP bioluminescence assay.

    Science.gov (United States)

    Finger, Susanne; Wiegand, Cornelia; Buschmann, Hans-Jürgen; Hipler, Uta-Christina

    2013-08-16

    Cyclodextrins (CDs) are able to form inclusion complexes with other molecules, thereby, protecting these guest molecules from degradation, enhancing their biocompatibility or influencing their physiological distribution while retaining their activity. Here, antibacterial effects of CD-complexes with the antiseptics chlorhexidine diacetate (CHX), iodine (IOD) and polihexanide (PHMB) were determined using two different in vitro methods, microplate laser nephelometry and an ATP bioluminescence assay. Laser nephelometry is a direct method for monitoring and evaluating growth of micro-organisms by measurement of the turbidity of the solution. In contrast, the ATP bioluminescence assay determines specifically the amount of metabolic active bacterial cells. The antibacterial effects of CD-antiseptics-complexes were examined for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis and the results of both methods were compared in respect of calculated means of half maximal inhibitory concentrations (IC50) and statistical evaluated Pearson's correlation coefficients (r). It could be demonstrated that both methods showed a high comparability although they differ in the parameters tested. This study revealed that CD-complexes with CHX and PHMB were most effective against E. coli and the tested staphylococci. While CD-IOD-complexes obtained high activity against K. pneumoniae, P. aeruginosa was distinctly more resistant compared to the other bacteria.

  1. Assessment of hospital daily cleaning practices using ATP bioluminescence in a developing country

    Directory of Open Access Journals (Sweden)

    Alejandra A. Zambrano

    2014-12-01

    Full Text Available Visual assessment of surfaces may not be enough to document the level of cleanliness in the hospital setting. It is necessary to introduce quantitative methods to document the results of this practice.Objective:To evaluate the efficacy of hospital terminal cleaning procedures, using an adenosine triphosphate (ATP bioluminescence method in a teaching hospital.Method:During 2008 we conducted an evaluation using ATP bioluminescence LIGHTNING MVP™ (Arquimed of external and internal housekeeping service. After conducting an initial evaluation we implemented education of cleaning practices and finally we did a post intervention evaluation. Using chi-square method we compared prior versus after cleaning, quality of cleaning performed by external versus internal personnel, single versus double terminal cleaning procedures and prior versus after intervention. A finding of three RLU or less was considered a clean surface.Results:We performed 198 evaluations in 33 patient units and nine OR. Internal personnel accomplished 25.37% of clean surfaces before and 80% after the education intervention (p = 0.01. In contrast, external personnel obtained 68.8% before and 73.33% after intervention (p = 0.3.Conclusions:This study suggests that visual assessment is not enough to ensure quality of the process and it is necessary to document the level of cleanliness by quantitative methods.

  2. In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin.

    Science.gov (United States)

    Lark, Arianna R; Kitamoto, Toshihiro; Martin, Jean-René

    2016-01-08

    Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca(2+)-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and - with the addition of its cofactor coelenterazine - emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca(2+)-transients and Ca(2+)-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca(2+)-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

  3. Bacillus anthracis diagnostic detection and rapid antibiotic susceptibility determination using 'bioluminescent' reporter phage.

    Science.gov (United States)

    Schofield, David A; Sharp, Natasha J; Vandamm, Joshua; Molineux, Ian J; Spreng, Krista A; Rajanna, Chythanya; Westwater, Caroline; Stewart, George C

    2013-11-01

    Genetically modified phages have the potential to detect pathogenic bacteria from clinical, environmental, or food-related sources. Herein we assess an engineered 'bioluminescent' reporter phage (Wß::luxAB) as a clinical diagnostic tool for Bacillus anthracis, the etiological agent of anthrax. Wß::luxAB is able to rapidly (within minutes) detect a panel of B. anthracis strains by transducing a bioluminescent phenotype. The reporter phage displays species specificity by its inability, or significantly reduced ability, to detect members of the closely related Bacillus cereus group and other common bacterial pathogens. Using spiked clinical specimens, Wß::luxAB detects B. anthracis within 5 h at clinically relevant concentrations, and provides antibiotic susceptibility information that mirrors the CLSI method, except that data are obtained at least 5-fold faster. Although anthrax is a treatable disease, a positive patient prognosis is dependent on timely diagnosis and appropriate therapy. Wß::luxAB rapidly detects B. anthracis and determines antibiotic efficacy, properties that will help patient outcome.

  4. Development of a Filtration-Based Bioluminescence Assay for Detection of Microorganisms in Tea Beverages.

    Science.gov (United States)

    Shinozaki, Yohei; Igarashi, Toshinori; Harada, Yasuhiro

    2016-03-01

    The market for tea drinks as healthy beverages has been steadily expanding, and ready-to-drink beverages in polyethylene terephthalate bottles have been popular. To more rapidly and accurately test tea beverages bottled in polyethylene terephthalate for microbial contamination, a newly developed filtration device and a washing method with a commercial bioluminescence assay were combined to detect low numbers of bacterial spores, fungal conidia, and ascospores. Washing buffers were formulated with nonionic detergents from the Tween series. Commercially available tea beverages were used to evaluate the filtration capacity of the filtration device, the effect of washing buffers, and the performance of the assay. The assay was tested with serially diluted suspensions of colonies of two bacterial strains, spores of three Bacillus strains, conidia of five fungal strains, and ascospores of four fungal strains. The filtration device enabled filtration of a large sample volume (100 to 500 ml), and the washing buffer significantly decreased the background bioluminescence intensity of tea samples when compared with the no-washing method. Low numbers (1 to 10 CFU/100 ml) of the tested strains of bacteria were detected within 8 to 18 h of cultivation, and fungi were detected within 24 to 48 h. Furthermore, a whole bottle (500 ml) of mixed tea was filtered through the filtration device and microbes were detected. This method could be used for quality control of bottled beverages without preincubation.

  5. The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium

    Directory of Open Access Journals (Sweden)

    Hannah M. Read

    2016-06-01

    Full Text Available Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC and enterohaemorrhagic E. coli (EHEC infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169 in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments.

  6. Bioluminescence : the potential of a non-invasive bio-optical imaging technique and improvement of animal research

    NARCIS (Netherlands)

    Hesselink, J. W.; van Dam, G. M.

    2007-01-01

    Bioluminescence is an optical imaging technique that exploits the emission of photons at specific wavelengths based on energy-dependent reactions catalysed by luciferases. The technique makes it possible to monitor measure, and track biological processes in living animals. A short review is presente

  7. Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real-time bioluminescence imaging

    DEFF Research Database (Denmark)

    Bartkova, Simona; Kokotovic, Branko; Dalsgaard, Inger

    2017-01-01

    Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here...

  8. Fast monitoring of indoor bioaerosol concentrations with ATP bioluminescence assay using an electrostatic rod-type sampler.

    Directory of Open Access Journals (Sweden)

    Ji-Woon Park

    Full Text Available A culture-based colony counting method is the most widely used analytical technique for monitoring bioaerosols in both indoor and outdoor environments. However, this method requires several days for colony formation. In this study, our goal was fast monitoring (Sampling: 3 min, Detection: < 1 min of indoor bioaerosol concentrations with ATP bioluminescence assay using a bioaerosol sampler. For this purpose, a novel hand-held electrostatic rod-type sampler (110 mm wide, 115 mm long, and 200 mm tall was developed and used with a commercial luminometer, which employs the Adenosine triphosphate (ATP bioluminescence method. The sampler consisted of a wire-rod type charger and a cylindrical collector, and was operated with an applied voltage of 4.5 kV and a sampling flow rate of 150.7 lpm. Its performance was tested using Staphylococcus epidermidis which was aerosolized with an atomizer. Bioaerosol concentrations were measured using ATP bioluminescence method with our sampler and compared with the culture-based method using Andersen cascade impactor under controlled laboratory conditions. Indoor bioaerosol concentrations were also measured using both methods in various indoor environments. A linear correlation was obtained between both methods in lab-tests and field-tests. Our proposed sampler with ATP bioluminescence method may be effective for fast monitoring of indoor bioaerosol concentrations.

  9. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P

    2002-01-01

    -induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations...

  10. Draft genome sequence of the polycyclic aromatic hydrocarbon-degrading, genetically engineered bioluminescent bioreporter Pseudomonas fluorescens HK44.

    Science.gov (United States)

    Chauhan, Archana; Layton, Alice C; Williams, Daniel E; Smartt, Abby E; Ripp, Steven; Karpinets, Tatiana V; Brown, Steven D; Sayler, Gary S

    2011-09-01

    Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of ∼6.1 Mb of sequence indicates that 30% of the traits are unique and distributed over five genomic islands, a prophage, and two plasmids.

  11. Bioluminescence-based method for measuring assimilable organic carbon in pretreatment water for reverse osmosis membrane desalination.

    Science.gov (United States)

    Weinrich, Lauren A; Schneider, Orren D; LeChevallier, Mark W

    2011-02-01

    A bioluminescence-based assimilable organic carbon (AOC) test was developed for determining the biological growth potential of seawater within the reverse osmosis desalination pretreatment process. The test uses Vibrio harveyi, a marine organism that exhibits constitutive luminescence and is nutritionally robust. AOC was measured in both a pilot plant and a full-scale desalination plant pretreatment.

  12. Draft Genome Sequence of the Polycyclic Aromatic Hydrocarbon-Degrading, Genetically Engineered Bioluminescent Bioreporter Pseudomonas fluorescens HK44 ▿

    Science.gov (United States)

    Chauhan, Archana; Layton, Alice C.; Williams, Daniel E.; Smartt, Abby E.; Ripp, Steven; Karpinets, Tatiana V.; Brown, Steven D.; Sayler, Gary S.

    2011-01-01

    Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of ∼6.1 Mb of sequence indicates that 30% of the traits are unique and distributed over five genomic islands, a prophage, and two plasmids. PMID:21742869

  13. Draft Genome Sequence of the Polycyclic Aromatic Hydrocarbon-Degrading, Genetically Engineered Bioluminescent Bioreporter Pseudomonas fluorescens HK44

    Energy Technology Data Exchange (ETDEWEB)

    Chauhan, Archana [ORNL; Layton, Alice [University of Tennessee, Knoxville (UTK); Williams, Daniel W [ORNL; Smart, Abby E. [University of Tennessee, Knoxville (UTK); Ripp, Steven Anthony [ORNL; Karpinets, Tatiana V [ORNL; Brown, Steven D [ORNL; Sayler, Gary Steven [ORNL

    2011-01-01

    Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of {approx}6.1 Mb sequence indicates that 30% of the traits are unique and distributed over 5 genomic islands, a prophage and two plasmids.

  14. Fast monitoring of indoor bioaerosol concentrations with ATP bioluminescence assay using an electrostatic rod-type sampler.

    Science.gov (United States)

    Park, Ji-Woon; Park, Chul Woo; Lee, Sung Hwa; Hwang, Jungho

    2015-01-01

    A culture-based colony counting method is the most widely used analytical technique for monitoring bioaerosols in both indoor and outdoor environments. However, this method requires several days for colony formation. In this study, our goal was fast monitoring (Sampling: 3 min, Detection: bioluminescence assay using a bioaerosol sampler. For this purpose, a novel hand-held electrostatic rod-type sampler (110 mm wide, 115 mm long, and 200 mm tall) was developed and used with a commercial luminometer, which employs the Adenosine triphosphate (ATP) bioluminescence method. The sampler consisted of a wire-rod type charger and a cylindrical collector, and was operated with an applied voltage of 4.5 kV and a sampling flow rate of 150.7 lpm. Its performance was tested using Staphylococcus epidermidis which was aerosolized with an atomizer. Bioaerosol concentrations were measured using ATP bioluminescence method with our sampler and compared with the culture-based method using Andersen cascade impactor under controlled laboratory conditions. Indoor bioaerosol concentrations were also measured using both methods in various indoor environments. A linear correlation was obtained between both methods in lab-tests and field-tests. Our proposed sampler with ATP bioluminescence method may be effective for fast monitoring of indoor bioaerosol concentrations.

  15. Inertial bioluminescence rhythms at the Capo Passero (KM3NeT-Italia) site, Central Mediterranean Sea

    Science.gov (United States)

    Aguzzi, J.; Fanelli, E.; Ciuffardi, T.; Schirone, A.; Craig, J.; Aiello, S.; Ameli, F.; Anghinolfi, M.; Barbarino, G.; Barbarito, E.; Beverini, N.; Biagi, S.; Biagioni, A.; Bouhadef, B.; Bozza, C.; Cacopardo, G.; Calamai, M.; Calì, C.; Capone, A.; Caruso, F.; Cecchini, S.; Ceres, A.; Chiarusi, T.; Circella, M.; Cocimano, R.; Coniglione, R.; Costa, M.; Cuttone, G.; D’Amato, C.; D’Amico, A.; De Bonis, G.; De Luca, V.; Deniskina, N.; Distefano, C.; Di Mauro, L. S.; Fermani, P.; Ferrara, G.; Flaminio, V.; Fusco, L. A.; Garufi, F.; Giordano, V.; Gmerk, A.; Grasso, R.; Grella, G.; Hugon, C.; Imbesi, M.; Kulikovskiy, V.; Larosa, G.; Lattuada, D.; Leismüller, K. P.; Leonora, E.; Litrico, P.; Lonardo, A.; Longhitano, F.; Presti, D. Lo; Maccioni, E.; Margiotta, A.; Marinelli, A.; Martini, A.; Masullo, R.; Mele, R.; Migliozzi, P.; Migneco, E.; Miraglia, A.; Mollo, C. M.; Mongelli, M.; Morganti, M.; Musico, P.; Musumeci, M.; Nicolau, C. A.; Orlando, A.; Orzelli, A.; Papaleo, R.; Pellegrino, C.; Pellegriti, M. G.; Perrina, C.; Piattelli, P.; Poma, E.; Pulvirenti, S.; Raffaelli, F.; Randazzo, N.; Riccobene, G.; Rovelli, A.; Sanguineti, M.; Sapienza, P.; Sciacca, V.; Sgura, I.; Simeone, F.; Sipala, V.; Speziale, F.; Spitaleri, A.; Spurio, M.; Stellacci, S. M.; Taiuti, M.; Terreni, G.; Trasatti, L.; Trovato, A.; Versari, F.; Vicini, P.; Viola, S.; Vivolo, D.

    2017-01-01

    In the deep sea, the sense of time is dependent on geophysical fluctuations, such as internal tides and atmospheric-related inertial currents, rather than day-night rhythms. Deep-sea neutrino telescopes instrumented with light detecting Photo-Multiplier Tubes (PMT) can be used to describe the synchronization of bioluminescent activity of abyssopelagic organisms with hydrodynamic cycles. PMT readings at 8 different depths (from 3069 to 3349 m) of the NEMO Phase 2 prototype, deployed offshore Capo Passero (Sicily) at the KM3NeT-Italia site, were used to characterize rhythmic bioluminescence patterns in June 2013, in response to water mass movements. We found a significant (p bioluminescence signal, corresponding to inertial fluctuations. Waveform and Fourier analyses of PMT data and tower orientation were carried out to identify phases (i.e. the timing of peaks) by subdividing time series on the length of detected inertial periodicity. A phase overlap between rhythms and cycles suggests a mechanical stimulation of bioluminescence, as organisms carried by currents collide with the telescope infrastructure, resulting in the emission of light. A bathymetric shift in PMT phases indicated that organisms travelled in discontinuous deep-sea undular vortices consisting of chains of inertially pulsating mesoscale cyclones/anticyclones, which to date remain poorly known. PMID:28332561

  16. Development of a high-throughput opsonophagocytic assay for the determination of functional antibody activity against Streptococcus pyogenes using bioluminescence.

    Science.gov (United States)

    Lorenz, Natalie; Loh, Jacelyn M S; Moreland, Nicole J; Proft, Thomas

    2017-03-01

    The lack of standardised protocols for the assessment of functional antibodies has hindered Streptococcus pyogenes research and the development of vaccines. A robust, high throughput opsonophagocytic bactericidal assay to determine protective antibodies in human and rabbit serum has been developed that utilises bioluminescence as a rapid read out.

  17. Detection of nitrate/nitrite bioavailability in wastewater using a luxCDABE-based Klebsiella oxytoca bioluminescent bioreporter.

    Science.gov (United States)

    Abd-El-Haleem, Desouky; Ripp, Steven; Zaki, Sahar; Sayler, Gary S

    2007-08-01

    In the present study, we have constructed a bioluminescent bioreporter for the assessment of nitrate/nitrite bioavailability in wastewater. Specifically, an approximately 500-bp DNA fragment containing a nitrate/nitrite-activated nasR-like promoter (regulating expression of genes encoding nitrite reductase in the genus Klebsiella) was fused upstream of the Vibrio fischeri luxCDABE gene cassette in a modified mini-Tn5 vector. Characterization of this strain, designated W6-1, yielded dose-dependent increased bioluminescence coincident with increased nitrate, nitrite, and ammonium added to the growth medium from 1 to 11 ppm. Bioluminescence in response to nitrogen species addition was light dependent up to 10, 7, and 8 ppm with nitrate, nitrite, and ammonium, respectively. This response was linear in the range from 1 to 8 ppm for nitrate (R2 = 0.98), 1 to 6 ppm for nitrite (R2 = 0.99), and 1 to 7 ppm for ammonium (R2 = 0.99). A significant bioluminescent response was also recorded when strain W6-1 was incubated with slurries from aged, nitrate/nitrite contaminated wastewater. Thus, bioreporter strain W6-1 can be used to elucidate factors that constrain the use of nitrate/nitrite in wastewaters.

  18. Spatiotemporal expression of heme oxygenase-1 detected by in vivo bioluminescence after hepatic ischemia in HO-1/luc mice

    NARCIS (Netherlands)

    Su, Huawei; van Dam, Gooitzen M.; Buis, Carlijn I.; Visser, Dorien S.; Hesselink, Jan Willem; Schuurs, Theo A.; Leuvenink, Henri G. D.; Contag, Christopher H.; Porte, Robert J.

    2006-01-01

    Upregulation of heme oxygenase-1 (HO-1) has been proposed as a critical mechanism protecting against cellular stress during liver transplantation, providing a potential target for new therapeutic interventions. We investigated the feasibility of in vivo bioluminescence imaging (BLI) to noninvasively

  19. Bioluminescence-based visualization of CD4 T cell dynamics using a T lineage-specific luciferase transgenic model1

    Directory of Open Access Journals (Sweden)

    Zinn Kurt R

    2009-08-01

    Full Text Available Abstract Background Rapid clonal expansion of T cells occurs in response to antigenic challenges. The kinetics of the T cell response has previously been described using tissue-based studies performed at defined time points. Luciferase bioluminescence has recently been utilized for non-invasive analysis of in vivo biologic processes in real-time. Results We have created a novel transgenic mouse model (T-Lux using a human CD2 mini-gene to direct luciferase expression specifically to the T cell compartment. T-Lux T cells demonstrated normal homing patterns within the intact mouse and following adoptive transfer. Bioluminescent signal correlated with T cell numbers in the whole body images as well as within specific organ regions of interest. Following transfer into lymphopenic (RAG2-/- recipients, homeostatic proliferation of T-Lux T cells was visualized using bioluminescent imaging. Real-time bioluminescent analysis of CD4+ T cell antigen-specific responses enabled real-time comparison of the kinetics and magnitude of clonal expansion and contraction in the inductive lymph node and tissue site of antigen injection. T cell expansion was dose-dependent despite the presence of supraphysiologic numbers of OVA-specific OT-II transgenic TCR T-Lux T cells. CD4+ T cells subsequently underwent a rapid (3–4 day contraction phase in the draining lymph node, with a delayed contraction in the antigen delivery site, with bioluminescent signal diminished below initial levels, representing TCR clonal frequency control. Conclusion The T-Lux mouse provides a novel, efficient model for tracking in vivo aspects of the CD4+ T cell response to antigen, providing an attractive approach for studies directed at immunotherapy or vaccine design.

  20. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

    Directory of Open Access Journals (Sweden)

    Daniell Henry

    2011-06-01

    Full Text Available Abstract Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun" delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI methods. Results Plasmid DNA carrying the firefly luciferase (LUC reporter gene under the control of the human Cytomegalovirus (CMV promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter using different DNA Loading Ratios (DLRs, and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50 at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results

  1. Effect of concentrating and exposing the bioluminescent bacteria to the non-luminescent allo-bacterial extracellular products on their luminescence

    Digital Repository Service at National Institute of Oceanography (India)

    Ravindran, J.; Priya, G.G.; Kannapiran, E.

    animals and in light organs of squid and fishes. The non- bioluminescent bacterium isolated was identified to be Vibrio paraheamolyticus. RLU in the concentrated water sample initially increased and the subsequently decreased. RLU of the water sample...

  2. Discussion of ATP bioluminescence detection system%三磷酸腺苷生物荧光检测系统应用管理

    Institute of Scientific and Technical Information of China (English)

    张亮

    2015-01-01

    Objective: Through the introduction of the application background of ATP bioluminescence detection system, the technical principle and the factors of ATP bioluminescence detection system should be taken into account in the procurement, prompted the personnel understand the necessity of ATP bioluminescence detection system using, help procurement staffs purchasing suitable ATP bioluminescence detection system. Methods: introduces the application of ATP bioluminescence detection system background, technical principle and ATP bioluminescence detection system detection accuracy related factors, and gives reasonable purchase suggestions. Results:Improved procurement staffs’ understand to ATP bioluminescence detection system. Conclusion:ATP bioluminescence detection system as a new kind of detection system, with high sensitivity, simple and quick operation, become auxiliary tool of the various departments to conduct self-detection, hospital infection management department to check, understand the principle of technology of ATP bioluminescence detection system and ATP bioluminescence detection system detection accuracy related factors to help procurement staffs purchase the most suitable ATP bioluminescence detection system for clinical.%目的:通过介绍三磷酸腺苷(ATP)生物荧光检测系统的应用背景、技术原理及设备引进中需参考的重要技术参数,了解ATP生物荧光检测系统使用的必要性,帮助医院选择合适的ATP生物荧光检测系统。方法:介绍ATP生物荧光检测系统的应用背景、技术原理及检测准确性相关的因素,给出合理建议。结果:提高了设备管理部门对ATP生物荧光检测系统的认识。结论:ATP生物荧光检系统作为一种新型的检测系统,以高灵敏度、简单快捷的操作,成为医院感染控制的有效技术手段。了解其技术原理及检测准确性相关的因素,能帮助设备管理部门选择最适合

  3. Continuous and real-time bioaerosol monitoring by combined aerosol-to-hydrosol sampling and ATP bioluminescence assay.

    Science.gov (United States)

    Park, Ji-Woon; Kim, Hyeong Rae; Hwang, Jungho

    2016-10-19

    We present a methodology for continuous and real-time bioaerosol monitoring wherein an aerosol-to-hydrosol sampler is integrated with a bioluminescence detector. Laboratory test was conducted by supplying an air flow with entrained test bacteria (Staphylococcus epidermidis) to the inlet of the sampler. High voltage was applied between the discharge electrode and the ground electrode of the sampler to generate air ions by corona discharge. The bacterial aerosols were charged by the air ions and sampled in a flowing liquid containing both a cell lysis buffer and adenosine triphosphate (ATP) bioluminescence reagents. While the liquid was delivered to the bioluminescence detector, sampled bacteria were dissolved by the cell lysis buffer and ATP was extracted. The ATP was reacted with the ATP bioluminescence reagents, causing light to be emitted. When the concentration of bacteria in the aerosols was varied, the ATP bioluminescence signal in relative light units (RLUs) closely tracked the concentration in particles per unit air volume (# cm(-3)), as measured by an aerosol particle sizer. The total response time required for aerosol sampling and ATP bioluminescence detection increased from 30 s to 2 min for decreasing liquid sampling flow rate from 800 to 200 μLPM, respectively. However, lower concentration of S. epidermidis aerosols was able to be detected with lower liquid sampling flow rate (1 RLU corresponded to 6.5 # cm(-3) of S. epidermidis aerosols at 200 μLPM and 25.5 # cm(-3) at 800 μLPM). After obtaining all data sets of concentration of S. epidermidis aerosols and concentration of S. epidermidis particles collected in the flowing liquid, it was found that with our bioluminescence detector, 1 RLU corresponded to 1.8 × 10(5) (±0.2 × 10(5)) # mL(-1) of S. epidermidis in liquid. After the lab-test with S. epidermidis, our bioaerosol monitoring device was located in the lobby of a building. Air sampling was conducted continuously for 90

  4. Seasonal variation of deep-sea bioluminescence in the Ionian Sea

    Energy Technology Data Exchange (ETDEWEB)

    Craig, Jessica, E-mail: j.craig@abdn.ac.u [University of Aberdeen, Oceanlab, Main Street, Newburgh, Aberdeenshire, AB41 6AA (United Kingdom); Jamieson, Alan J.; Bagley, Philip M.; Priede, Imants G. [University of Aberdeen, Oceanlab, Main Street, Newburgh, Aberdeenshire, AB41 6AA (United Kingdom)

    2011-01-21

    The ICDeep (Image Intensified Charge Coupled Device for Deep sea research) profiler was used to measure the density of deep bioluminescent animals (BL) through the water column in the east, west and mid-Ionian Sea and in the Algerian Basin. A west to east decrease in BL density was found. Generalized additive modelling was used to investigate seasonal variation in the east and west Ionian Sea (NESTOR and NEMO neutrino telescope sites, respectively) from BL measurements in autumn 2008 and spring 2009. A significant seasonal effect was found in the west Ionian Sea (p<0.001), where a deep autumnal peak in BL density occurred between 500 and 2400 m. No significant seasonal variation in BL density was found in the east Ionian Sea (p=0.07). In both spring and autumn, significant differences in BL density were found through the water column between the east and west Ionian Sea (p<0.001).

  5. Application of Bioluminescence Imaging for In Vivo Monitoring of Fungal Infections

    Directory of Open Access Journals (Sweden)

    Matthias Brock

    2012-01-01

    Full Text Available Fungi can cause severe invasive infections especially in the immunocompromised host. Patient populations at risk are increasing due to ongoing developments in cancer treatment and transplantation medicine. Only limited diagnostic tools and few antifungals are available, rendering a significant number of invasive fungal infections life threatening. To reduce mortality rates, a better understanding of the infection processes is urgently required. Bioluminescence imaging (BLI is a powerful tool for such purposes, since it allows visualisation of temporal and spatial progression of infections in real time. BLI has been successfully used to monitor infections caused by various microorganisms, in particular bacteria. However, first studies have also been performed on the fungi Candida albicans and Aspergillus fumigatus. Although BLI was, in principle, suitable to study the infection process, some limitations remained. Here, different luciferase systems are introduced, and current approaches are summarised. Finally, suggestions for further improvements of BLI to monitor fungal infections are provided.

  6. Total variation regularization for bioluminescence tomography with the split Bregman method.

    Science.gov (United States)

    Feng, Jinchao; Qin, Chenghu; Jia, Kebin; Zhu, Shouping; Liu, Kai; Han, Dong; Yang, Xin; Gao, Quansheng; Tian, Jie

    2012-07-01

    Regularization methods have been broadly applied to bioluminescence tomography (BLT) to obtain stable solutions, including l2 and l1 regularizations. However, l2 regularization can oversmooth reconstructed images and l1 regularization may sparsify the source distribution, which degrades image quality. In this paper, the use of total variation (TV) regularization in BLT is investigated. Since a nonnegativity constraint can lead to improved image quality, the nonnegative constraint should be considered in BLT. However, TV regularization with a nonnegativity constraint is extremely difficult to solve due to its nondifferentiability and nonlinearity. The aim of this work is to validate the split Bregman method to minimize the TV regularization problem with a nonnegativity constraint for BLT. The performance of split Bregman-resolved TV (SBRTV) based BLT reconstruction algorithm was verified with numerical and in vivo experiments. Experimental results demonstrate that the SBRTV regularization can provide better regularization quality over l2 and l1 regularizations.

  7. Near infrared bioluminescence resonance energy transfer from firefly luciferase—quantum dot bionanoconjugates

    Science.gov (United States)

    Alam, Rabeka; Karam, Liliana M.; Doane, Tennyson L.; Zylstra, Joshua; Fontaine, Danielle M.; Branchini, Bruce R.; Maye, Mathew M.

    2014-12-01

    The bioluminescence resonance energy transfer (BRET) between firefly luciferase enzymes and semiconductive quantum dots (QDs) with near infrared emission is described. The QD were phase transferred to aqueous buffers using a histidine mediated phase transfer route, and incubated with a hexahistidine tagged, green emitting variant of firefly luciferase from Photinus pyralis (PPyGRTS). The PPyGRTS were bound to the QD interface via the hexahistidine tag, which effectively displaces the histidine layer and binds directly to the QD interfaces, allowing for short donor-acceptor distances (˜5.5 nm). Due to this, high BRET efficiency ratios of ˜5 were obtained. These PPyGRTS-QD bio-nano conjugates were characterized by transmission electron microscopy, thermal gravimetric analysis, Fourier transform infrared spectroscopy and BRET emission studies. The final optimized conjugate was easily observable by night vision imaging, demonstrating the potential of these materials in imaging and signaling/sensing applications.

  8. L1/2 regularization based numerical method for effective reconstruction of bioluminescence tomography

    Science.gov (United States)

    Chen, Xueli; Yang, Defu; Zhang, Qitan; Liang, Jimin

    2014-05-01

    Even though bioluminescence tomography (BLT) exhibits significant potential and wide applications in macroscopic imaging of small animals in vivo, the inverse reconstruction is still a tough problem that has plagued researchers in a related area. The ill-posedness of inverse reconstruction arises from insufficient measurements and modeling errors, so that the inverse reconstruction cannot be solved directly. In this study, an l1/2 regularization based numerical method was developed for effective reconstruction of BLT. In the method, the inverse reconstruction of BLT was constrained into an l1/2 regularization problem, and then the weighted interior-point algorithm (WIPA) was applied to solve the problem through transforming it into obtaining the solution of a series of l1 regularizers. The feasibility and effectiveness of the proposed method were demonstrated with numerical simulations on a digital mouse. Stability verification experiments further illustrated the robustness of the proposed method for different levels of Gaussian noise.

  9. Visible light induced ocular delayed bioluminescence as a possible origin of negative afterimage

    CERN Document Server

    Bokkon, I; Wang, C; Dai, J; Salari, V; Grass, F; Antal, I

    2011-01-01

    The delayed luminescence of biological tissues is an ultraweak reemission of absorbed photons after exposure to external monochromatic or white light illumination. Recently, Wang, B\\'okkon, Dai and Antal (Brain Res. 2011) presented the first experimental proof of the existence of spontaneous ultraweak biophoton emission and visible light induced delayed ultraweak photon emission from in vitro freshly isolated rat's whole eye, lens, vitreous humor and retina. Here, we suggest that the photobiophysical source of negative afterimage can also occur within the eye by delayed bioluminescent photons. In other words, when we stare at a colored (or white) image for few seconds, external photons can induce excited electronic states within different parts of the eye that is followed by a delayed reemission of absorbed photons for several seconds. Finally, these reemitted photons can be absorbed by nonbleached photoreceptors that produce a negative afterimage. Although this suggests the photobiophysical source of negativ...

  10. Quantitative bioluminescence imaging--a method for the detection of metabolite distributions in frozen tissues

    Science.gov (United States)

    Mueller-Klieser, Wolfgang; Walenta, Stefan; Schwickert, Georg

    1994-02-01

    A novel technique allows for measurement of metabolite distributions in tissue cryosections at a microscopic level using bioluminescence, single photon imaging, and computerized image analysis. Metabolites, such as ATP, glucose and lactate are registered in absolute concentration units, and the respective images can be correlated with each other and with histological structures by specific algorithms. One striking difference between malignant tumors and normal tissue is the pronounced heterogeneity of metabolite distributions in malignancies contrasted by rather homogeneous patterns obtained in many normal organs. The heterogeneous distribution of metabolites in solid tumors reflects the chaotic organization of the histological architecture and of the microvascular supply in cancerous tissue. Pixel-to-pixel comparison of metabolite distributions measured in cervix cancers of patients revealed a negative linear correlation between glucose and ATP concentrations at identical locations. In contrast, local lactate concentration was positively correlated with ATP.

  11. High Sensitivity Detection of ATP Using Bioluminescence at An Optical Fiber End

    Science.gov (United States)

    Iinuma, Masataka; Ushio, Yasuaki; Kuroda, Akio; Kadoya, Yutaka

    We investigated the sensitivity of ATP detection based on bioluminescence at an optical fiber end where luciferase molecules were immobilized via silica-binding protein molecules. Luminescence was detected by an avalanche photo diode (APD), with coupling optics to make full use of the merit of compactness, high quantum efficiency and low noise of the APD. The core diameter and the numerical aperture of the optical fiber, as well as the design of the coupling optics, were optimized so as to realize high photon-collection efficiency. A detection limit of about 10-10 M was obtained, which corresponds to 10-15 mol of ATP. A rough estimation shows that the photon count rate is still two orders of magnitude lower than that limited by diffusion or reaction processes, implying a possibility of further improvement of the sensitivity.

  12. Rapid identification and antibiotic susceptibility testing of Yersinia pestis using bioluminescent reporter phage

    Science.gov (United States)

    Schofield, David A.; Molineux, Ian J.; Westwater, Caroline

    2012-01-01

    The rapid identification and antibiotic susceptibility testing of Yersinia pestis is paramount for a positive prognosis. We previously engineered a Y. pestis-specific ‘bioluminescent’ reporter phage for the identification of Y. pestis. In this study, we generated an improved reporter phage and evaluated the ability of this phage to provide direct and rapid susceptibility testing. Compared to the first generation reporter, the second generation reporter exhibited a 100-fold increase in signal strength, leading to a 10-fold increase in assay sensitivity. Y. pestis antimicrobial testing in the presence of the reporter elicited bioluminescent signals that were drug concentration-dependent, and produced susceptibility profiles that mirrored the standard CLSI method. The phage-generated susceptibility profiles, however, were obtained within hours in contrast to days with the conventional method. PMID:22579583

  13. Evaluation of On- and Off-Line Bioluminescence Tomography System for Focal Irradiation Guidance.

    Science.gov (United States)

    Zhang, Bin; Wong, John W; Iordachita, Iulian I; Reyes, Juvenal; Nugent, Katriana; Tran, Phuoc T; Tuttle, Stephen W; Koumenis, Constantinos; Wang, Ken Kang-Hsin

    2016-12-01

    In response to the limitations of computed tomography (CT) and cone-beam CT (CBCT) in irradiation guidance, especially for soft-tissue targets without the use of contrast agents, our group developed a solution that implemented bioluminescence tomography (BLT) as the image-guidance modality for preclinical radiation research. However, adding such a system to existing small animal irradiators is no small task. A potential solution is to utilize an off-line BLT system in close proximity to the irradiator, with stable and effective animal transport between the two systems. In this study, we investigated the localization accuracy of an off-line BLT system when used for the small animal radiation research platform (SARRP) and compared the results with those of an on-line system. The CBCT was equipped on both the off-line BLT system and the SARRP, with a distance of 5 m between them. To evaluate the setup error during animal transport between the two systems, the mice underwent CBCT imaging on the SARRP and were then transported to the off-line system for a second CBCT imaging session. The normalized intensity difference of the two images and the corresponding histogram and correlation were computed to evaluate if the transport process perturbed animal positioning. Strong correlation (correlation coefficients >0.95) between the SARRP and the off-line mouse CBCT was observed. The offset of the implanted light source center can be maintained within 0.2 mm during transport. To compare the target localization accuracy using the on-line SARRP BLT and the off-line system, a self-illuminated bioluminescent source was implanted in the abdomen of anesthetized mice. In addition to the application for dose calculation, CBCT imaging was also employed to generate the mesh grid of the imaged mouse for BLT reconstruction. Two scenarios were devised and compared, which involved localization of the luminescence source based on either: 1. on-line SARRP bioluminescence image and CBCT; or 2

  14. Hybrid Multilevel Sparse Reconstruction for a Whole Domain Bioluminescence Tomography Using Adaptive Finite Element

    Directory of Open Access Journals (Sweden)

    Jingjing Yu

    2013-01-01

    Full Text Available Quantitative reconstruction of bioluminescent sources from boundary measurements is a challenging ill-posed inverse problem owing to the high degree of absorption and scattering of light through tissue. We present a hybrid multilevel reconstruction scheme by combining the ability of sparse regularization with the advantage of adaptive finite element method. In view of the characteristics of different discretization levels, two different inversion algorithms are employed on the initial coarse mesh and the succeeding ones to strike a balance between stability and efficiency. Numerical experiment results with a digital mouse model demonstrate that the proposed scheme can accurately localize and quantify source distribution while maintaining reconstruction stability and computational economy. The effectiveness of this hybrid reconstruction scheme is further confirmed with in vivo experiments.

  15. The Drug Excipient Cyclodextrin Interacts With d-Luciferin and Interferes With Bioluminescence Imaging.

    Science.gov (United States)

    Kumar, Jeyan S; Miller Jenkins, Lisa M; Gottesman, Michael M; Hall, Matthew D

    2016-01-01

    Cyclodextrins are well-characterized, barrel-shaped molecules that can solubilize organic small molecules in aqueous solution via host-guest interactions. As such, cyclodextrins are used as excipients for experimental therapeutics in vivo. We observed unanticipated modifications to bioluminescence imaging (BLI) signal intensity when 2-hydroxy-propyl-β-cyclodextrin (HPCD) was coinjected as an excipient. We hypothesized that HPCD bindsd-luciferin and interferes with the BLI signal. Using luciferase-expressing cell lines, we showed that HPCD lowers the BLI signal in a concentration-dependent manner. Flow cytometry revealed that HPCD resulted in reduced cellular accumulation ofd-luciferin, and mass spectrometry revealedd-luciferin HPCD species, confirming a direct interaction. In vivo imaging using a luciferase mouse model demonstrated that HPCD reduced luciferin-mediated BLI compared to luciferin alone. The implications of using HPCD as an excipient in BLI studies are discussed.

  16. Lessons in microcapsule assembly from imaging delivery of a bioluminescent enzyme.

    Science.gov (United States)

    Pavlov, Anton M; Sukhorukov, Gleb B; Gould, David J

    2013-03-11

    Layer-by-layer assembled microcapsules have potential applications as delivery and biosensing systems, which make them attractive tools for use in various aspects of nanomedicine. We examined the effect of microcapsule location on activity of the bioluminescent enzyme luciferase in both intact capsules and following cell uptake. In intact capsules, the rate of reaction of luciferase was greatest for luciferase in the outer layer and least in the core. Following cell uptake, luciferase in the outer layer was rapidly reactive, and a similar rate of reaction and activity was observed for luciferase placed in capsule interior (core). By contrast, there was minimal activity detected when microcapsules with luciferase sandwiched between polyelectrolytes in a middle layer were delivered to cells. This study informs us of the availability of bioactive molecules located in different positions within microcapsules and will enable better microcapsule construction in line with the intended application, particularly delivery of functional proteins to cells.

  17. Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.

    Science.gov (United States)

    Song, Qinxin; Wei, Guijiang; Zhou, Guohua

    2014-07-01

    A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis.

  18. The Flashlight Fish Anomalops katoptron Uses Bioluminescent Light to Detect Prey in the Dark

    Science.gov (United States)

    Hellinger, Jens; Jägers, Peter; Donner, Marcel; Sutt, Franziska; Mark, Melanie D.; Senen, Budiono; Tollrian, Ralph

    2017-01-01

    Bioluminescence is a fascinating phenomenon occurring in numerous animal taxa in the ocean. The reef dwelling splitfin flashlight fish (Anomalops katoptron) can be found in large schools during moonless nights in the shallow water of coral reefs and in the open surrounding water. Anomalops katoptron produce striking blink patterns with symbiotic bacteria in their sub-ocular light organs. We examined the blink frequency in A. katoptron under various laboratory conditions. During the night A. katoptron swims in schools roughly parallel to their conspecifics and display high blink frequencies of approximately 90 blinks/minute with equal on and off times. However, when planktonic prey was detected in the experimental tank, the open time increased compared to open times in the absence of prey and the frequency decreased to 20% compared to blink frequency at night in the absence of planktonic prey. During the day when the school is in a cave in the reef tank the blink frequency decreases to approximately 9 blinks/minute with increasing off-times of the light organ. Surprisingly the non-luminescent A. katoptron with non-functional light organs displayed the same blink frequencies and light organ open/closed times during the night and day as their luminescent conspecifics. In the presence of plankton non-luminescent specimens showed no change in the blink frequency and open/closed times compared to luminescent A. katoptron. Our experiments performed in a coral reef tank show that A. katoptron use bioluminescent illumination to detect planktonic prey and that the blink frequency of A. katoptron light organs follow an exogenous control by the ambient light. PMID:28178297

  19. High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.

    Directory of Open Access Journals (Sweden)

    Michelle Cronin

    Full Text Available The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v. administered to mice bearing subcutaneous (s.c FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

  20. Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice

    Directory of Open Access Journals (Sweden)

    Zagozdzon Agnieszka M

    2012-05-01

    Full Text Available Abstract Background Numerous transgenic models have been generated to study breast cancer. However, despite many advantages, traditional transgenic models for breast cancer are also burdened with difficulties in early detection and longitudinal observation of transgene-induced tumours, which in most cases are randomly located and occur at various time points. Methods such as palpation followed by mechanical measurement of the tumours are of limited value in transgenic models. There is a crucial need for making these previously generated models suitable for modern methods of tumour visualisation and monitoring, e.g. by bioluminescence-based techniques. This approach was successfully used in the current study. Results A new mouse strain (MMTV-Luc2 mice expressing Luc2 luciferase primarily in mammary tissue in females, with low-level background expression in internal organs, was generated and bred to homozygosity. After these mice were intercrossed with MMTV-PyVT mice, all double transgenic females developed mammary tumours by the age of 10 weeks, the localisation and progression of which could be effectively monitored using the luminescence-based in vivo imaging. Luminescence-based readout allowed for early visualisation of the locally overgrown mammary tissue and for longitudinal evaluation of local progression of the tumours. When sampled ex vivo at the age of 10 weeks, all tumours derived from MMTV-Luc2PyVT females displayed robust bioluminescent signal. Conclusions We have created a novel transgenic strain for visualisation and longitudinal monitoring of mammary tumour development in transgenic mice as an addition and/or a new and more advanced alternative to manual methods. Generation of this mouse strain is vital for making many of the existing mammary tumour transgenic models applicable for in vivo imaging techniques.

  1. Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice

    LENUS (Irish Health Repository)

    Zagozdzon, Agnieszka M

    2012-05-30

    AbstractBackgroundNumerous transgenic models have been generated to study breast cancer. However, despite many advantages, traditional transgenic models for breast cancer are also burdened with difficulties in early detection and longitudinal observation of transgene-induced tumours, which in most cases are randomly located and occur at various time points. Methods such as palpation followed by mechanical measurement of the tumours are of limited value in transgenic models. There is a crucial need for making these previously generated models suitable for modern methods of tumour visualisation and monitoring, e.g. by bioluminescence-based techniques. This approach was successfully used in the current study.ResultsA new mouse strain (MMTV-Luc2 mice) expressing Luc2 luciferase primarily in mammary tissue in females, with low-level background expression in internal organs, was generated and bred to homozygosity. After these mice were intercrossed with MMTV-PyVT mice, all double transgenic females developed mammary tumours by the age of 10 weeks, the localisation and progression of which could be effectively monitored using the luminescence-based in vivo imaging. Luminescence-based readout allowed for early visualisation of the locally overgrown mammary tissue and for longitudinal evaluation of local progression of the tumours. When sampled ex vivo at the age of 10 weeks, all tumours derived from MMTV-Luc2PyVT females displayed robust bioluminescent signal.ConclusionsWe have created a novel transgenic strain for visualisation and longitudinal monitoring of mammary tumour development in transgenic mice as an addition and\\/or a new and more advanced alternative to manual methods. Generation of this mouse strain is vital for making many of the existing mammary tumour transgenic models applicable for in vivo imaging techniques.

  2. Multi-projection bioluminescence tomography guided system for small animal radiation research platform (SARRP)

    Science.gov (United States)

    Zhang, Bin; Iordachita, Iulian; Wong, John W.; Wang, Ken Kang-Hsin

    2016-03-01

    Cone beam computed tomography (CBCT) is limited in guiding irradiation for soft tissue targets. As a complementary imaging modality, bioluminescence tomography (BLT) provides strong soft tissue contrast. We developed a dual-use BLT system which consists of an optical assembly, a mobile cart and an independent mouse bed. The system is motorized which can easily dock onto an independent mouse bed operating as a standalone system for longitudinal bioluminescence imaging (BLI)/BLT studies and also dock onto the SARRP for on-line radiation guidance. Our initial tests for the system demonstrate that (i) the imaging depth is 28 mm, (ii) the optical background is sufficiently low and uniform, (iii) the non-uniform response of the optical imaging can be corrected by the flat field correction, and (iv) the imaging acquisition speed was improved by an average of 3.7 times faster than our previous systems. We also presented a geometry calibration procedure to map the planar BLIs acquired at multi-projections onto the surface of the CBCT image. The CBCT is required to generate the mesh for BLT reconstruction and used for treatment planning and radiation delivery. Feasibility study of the geometry calibration was performed on a manual-docking prototype. The mean and maximum mapping accuracy is 0.3 and 0.6 mm. The performance of the proposed motorized dual-use system is expected to be superior to that of the manual-docking prototype because of the mechanism stability. We anticipate the dual-use system as a highly efficient and cost-effective platform to facilitate optical imaging for preclinical radiation research.

  3. Interference of some organic substances and microorganisms adhered to stainless steel in ATP-bioluminescence measurement

    Directory of Open Access Journals (Sweden)

    Erny Marcelo Simm

    2008-06-01

    Full Text Available Adhesion of organic substances and microorganisms on stainless steel surface was evaluated. The log RLU measurement was affected at a lower or higher degree, by type or concentration of the organic substances (casein, lipid, sucrose and their combinations adhered to surface. However, if the samples analyzed were from the food processing surfaces, they would have been considered to be under satisfactory hygienic conditions with log RLU 0.05 on ATP-bioluminescence measurements. However, the surfaces adhered with 2.9x10³ DMC.cm-2 of B. subtilis spores and organic substances showed an increase in the log RLU measurement compared to surfaces containing only the organic substances.A adesão de substâncias orgânicas e de microrganismos em cupons de aço inoxidável ASI 304, #4 foi avaliada. A medida de Unidades Relativas de Luz (RLU foi afetada em menor ou maior nível pelo tipo e concentração das substâncias orgânicas e suas combinações. Entretanto, se as amostras fossem oriundas de superfícies de processamento de alimentos, elas seriam consideradas em boas condições higiênicas pois as medidas de log URL ficaram abaixo de 2,18 log (0.05 na medida de ATP-bioluminescência. Entretanto, superfícies aderidas com 2.9x10³ CDM.cm-2 de esporos de B. subtilis e as substâncias orgânicas mostraram um aumento no log URL comparado com as superfícies contendo somente substâncias orgânicas.

  4. Rapid detection and identification of bacterial pathogens by using an ATP bioluminescence immunoassay.

    Science.gov (United States)

    Hunter, Dawn M; Lim, Daniel V

    2010-04-01

    Rapid identification of viable bacterial contaminants in food products is important because of their potential to cause disease. This study examined a method for microbial detection by using a combined ATP bioluminescence immunoassay. Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium were selected as target organisms because of their implication in foodborne illness. Various matrices containing the target cells were examined, including ground beef homogenate, apple juice, milk, and phosphate-buffered saline. Specific antibodies were immobilized on the surface of 96-well plates, and then the sample matrices containing target cells in the wells were incubated. Sample matrix (no cells) was used to establish background. The plates were washed, and the wells were incubated with BacTiter-Glo reagent in Mueller-Hinton II broth. Bioluminescent output was measured with the GloMax 96 luminometer. Signal-to-noise ratios were calculated, resulting in a limit of detection of 10(4) CFU/ml for both E. coli O157:H7 and Salmonella Typhimurium. The limit of detection for both species was not affected by the presence of nontarget cells. The various sample matrices did not affect signal-to-noise ratios when E. coli O157:H7 was the target. A weak matrix effect was observed when Salmonella Typhimurium was the target. A strong linear correlation was observed between the number of cells and luminescent output over 4 orders of magnitude for both species. This method provides a means of simultaneously detecting and identifying viable pathogens in complex matrices, and could have wider application in food microbiology.

  5. Rapid Detection of Bacillus anthracis in Complex Food Matrices Using Phage-Mediated Bioluminescence.

    Science.gov (United States)

    Sharp, Natasha J; Vandamm, Joshua P; Molineux, Ian J; Schofield, David A

    2015-05-01

    Bacillus anthracis, the causative agent of anthrax, is considered a high-priority agent that may be used in a food-related terrorist attack because it can be contracted by ingestion and it also forms spores with heat and chemical resistance. Thus, novel surveillance methodologies to detect B. anthracis on adulterated foods are important for bioterrorism preparedness. We describe the development of a phage-based bioluminescence assay for the detection of B. anthracis on deliberately contaminated foods. We previously engineered the B. anthracis phage Wβ with genes encoding bacterial luciferase (luxA and luxB) to create a "light-tagged" reporter (Wβ::luxAB) that is able to rapidly detect B. anthracis by transducing a bioluminescent signal response. Here, we investigate the ability of Wβ::luxAB to detect B. anthracis Sterne, an attenuated select agent strain, in inoculated food (ground beef) and milk (2%, baby formula, and half and half) matrices after incubation with spores for 72 h at 4°C as per AOAC testing guidelines. The majority of B. anthracis bacilli remained in spore form, and thus were potentially infectious, within each of the liquid matrices for 14 days. Detection limits were 80 CFU/ml after 7 h of enrichment; sensitivity of detection increased to 8 CFU/ml when enrichment was extended to 16 h. The limit of detection in ground beef was 3.2 × 10(3) CFU/g after 7 h of enrichment, improving to 3.2 × 10(2) CFU/g after 16 h. Because the time to result is rapid and minimal processing is required, and because gastrointestinal anthrax can be fatal, the reporter technology displays promise for the protection of our food supply following a deliberate release of this priority pathogen.

  6. Imaging of bubonic plague dynamics by in vivo tracking of bioluminescent Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Toan Nham

    Full Text Available Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba, followed by a colonization of the draining inguinal lymph node(s, and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.

  7. Phylogenetic and transcriptomic analyses reveal the evolution of bioluminescence and light detection in marine deep-sea shrimps of the family Oplophoridae (Crustacea: Decapoda).

    Science.gov (United States)

    Wong, Juliet M; Pérez-Moreno, Jorge L; Chan, Tin-Yam; Frank, Tamara M; Bracken-Grissom, Heather D

    2015-02-01

    Bioluminescence is essential to the survival of many organisms, particularly in the deep sea where light is limited. Shrimp of the family Oplophoridae exhibit a remarkable mechanism of bioluminescence in the form of a secretion used for predatory defense. Three of the ten genera possess an additional mode of bioluminescence in the form of light-emitting organs called photophores. Phylogenetic analyses can be useful for tracing the evolution of bioluminescence, however, the few studies that have attempted to reconcile the relationships within Oplophoridae have generated trees with low-resolution. We present the most comprehensive phylogeny of Oplophoridae to date, with 90% genera coverage using seven genes (mitochondrial and nuclear) across 30 oplophorid species. We use our resulting topology to trace the evolution of bioluminescence within Oplophoridae. Previous studies have suggested that oplophorid visual systems may be tuned to differentiate the separate modes of bioluminescence. While all oplophorid shrimp possess a visual pigment sensitive to blue-green light, only those bearing photophores have an additional pigment sensitive to near-ultraviolet light. We attempt to characterize opsins, visual pigment proteins essential to light detection, in two photophore-bearing species (Systellaspis debilis and Oplophorus gracilirostris) and make inferences regarding their function and evolutionary significance.

  8. A transcriptional and proteomic survey of Arachnocampa luminosa (Diptera: Keroplatidae) lanterns gives insights into the origin of bioluminescence from the Malpighian tubules in Diptera.

    Science.gov (United States)

    Silva, J R; Amaral, D T; Hastings, J W; Wilson, T; Viviani, V R

    2015-11-01

    Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue-green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera.

  9. The first report of luminescent liver tissue in fishes: evolution and structure of bioluminescent organs in the deep-sea naked barracudinas (Aulopiformes: Lestidiidae).

    Science.gov (United States)

    Ghedotti, Michael J; Barton, Ryan W; Simons, Andrew M; Davis, Matthew P

    2015-03-01

    Bioluminescent organs that provide ventral camouflage are common among fishes in the meso-bathypelagic zones of the deep sea. However, the anatomical structures that have been modified to produce light vary substantially among different groups of fishes. Although the anatomical structure and evolutionary derivation of some of these organs have been well studied, the light organs of the naked barracudinas have received little scientific attention. This study describes the anatomy and evolution of bioluminescent organs in the Lestidiidae (naked barracudinas) in the context of a new phylogeny of barracudinas and closely related alepisauroid fishes. Gross and histological examination of bioluminescent organs or homologous structures from preserved museum specimens indicate that the ventral light organ is derived from hepatopancreatic tissue and that the antorbital spot in Lestrolepis is, in fact, a second dermal light organ. In the context of the phylogeny generated from DNA-sequence data from eight gene fragments (7 nuclear and 1 mitochondrial), a complex liver with a narrow ventral strand running along the ventral midline evolves first in the Lestidiidae. The ventral hepatopancreatic tissue later evolves into a ventral bioluminescent organ in the ancestor of Lestidium and Lestrolepis with the lineage leading to the genus Lestrolepis evolving a dermal antorbital bioluminescent organ, likely for light-intensity matching. This is the first described hepatopancreatic bioluminescent organ in fishes.

  10. Role of certain amino acid residues of the coelenterazine-binding cavity in bioluminescence of light-sensitive Ca(2+)-regulated photoprotein berovin.

    Science.gov (United States)

    Burakova, Ludmila P; Stepanyuk, Galina A; Eremeeva, Elena V; Vysotski, Eugene S

    2016-05-11

    Bright bioluminescence of ctenophores is caused by Ca(2+)-regulated photoproteins. Although these photoproteins are functionally identical to and share many properties of cnidarian photoproteins, like aequorin and obelin, and retain the same spatial architecture, they are extremely sensitive to light, i.e. lose the ability to bioluminesce on exposure to light over the entire absorption spectrum. In addition, the degree of identity of their amino acid sequences with those of cnidarian photoproteins is only 29.4%. This suggests that the residues involved in bioluminescence of ctenophore and cnidarian photoproteins significantly differ. Here we describe the bioluminescent properties of berovin mutants with substitution of the residues located in the photoprotein internal cavity. Since the spatial structure of berovin bound with a substrate is not determined yet, to identify these residues we have modeled it with an accommodated substrate using the structures of some cnidarian Ca(2+)-regulated photoproteins with bound coelenterazine or coelenteramide as templates in order to obtain an adequate sampling and to take into account all possible conformers and variants for ligand-protein docking. Based on the impact of substitutions on the bioluminescent properties and model structures we speculate that within the internal cavity of ctenophore photoproteins, coelenterazine is bound as a 2-peroxy anion adduct which is stabilized owing to Coulomb interaction with a positively charged guanidinium group of Arg41 paired with Tyr204. In this case, the bioluminescence reaction is triggered by only calcium-induced conformational changes leading to the disturbance of charge-charge interaction.

  11. Expression of a Humanized Viral 2A-Mediated lux Operon Efficiently Generates Autonomous Bioluminescence in Human Cells

    Science.gov (United States)

    Xu, Tingting; Ripp, Steven; Sayler, Gary S.; Close, Dan M.

    2014-01-01

    Background Expression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines. Methodology/Principal Findings The lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations. Conclusions/Significance Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines

  12. Global solution of the finite element shape-from-shading model with a bioluminescent molecular imaging application.

    Science.gov (United States)

    Zhong, Jianghong; Tian, Jie; Yang, Xin; Qin, Chenghu

    2010-01-01

    Only a planar bioluminescence image acquired from an ordinary cooled charge-coupled device (CCD) array every time, how to re-establish the three-dimensional small animal shape and light intensity distribution on the surface has become urgent to be solved as a bottleneck of bioluminescence tomography (BLT) reconstruction. In this paper, a finite element algorithm to solve the Dirichlet type problem for the first order Hamilton-Jacobi equation related to the shape-fromshading model is adopted. The algorithm outputting the globally maximal solution of the above problem avoids cumbersome boundary conditions on the interfaces between light and shadows and the use of additional information on the surface. The results of the optimization method are satisfied. It demonstrates the feasibility and potential of the finite element shape-fromshading (FE-SFS) model for reconstructing the small animal surface that lays one of key foundations for a fast low-cost application of the BLT in the next future.

  13. Integrated CMOS photodetectors and signal processing for very low-level chemical sensing with the bioluminescent bioreporter integrated circuit

    Science.gov (United States)

    Bolton, Eric K.; Sayler, Gary S.; Nivens, David E.; Rochelle, James M.; Ripp, Steven; Simpson, Michael L.

    2002-01-01

    We report an integrated CMOS microluminometer optimized for the detection of low-level bioluminescence as part of the bioluminescent bioreporter integrated circuit (BBIC). This microluminometer improves on previous devices through careful management of the sub-femtoampere currents, both signal and leakage, that flow in the front-end processing circuitry. In particular, the photodiode is operated with a reverse bias of only a few mV, requiring special attention to the reset circuitry of the current-to-frequency converter (CFC) that forms the front-end circuit. We report a sub-femtoampere leakage current and a minimum detectable signal (MDS) of 0.15 fA (1510 s integration time) using a room temperature 1.47 mm2 CMOS photodiode. This microluminometer can detect luminescence from as few as 5000 fully induced Pseudomonas fluorescens 5RL bacterial cells. c2002 Elsevier Science B.V. All rights reserved.

  14. Hybrid radiosity-SP3 equation based bioluminescence tomography reconstruction for turbid medium with low- and non-scattering regions

    Science.gov (United States)

    Chen, Xueli; Zhang, Qitan; Yang, Defu; Liang, Jimin

    2014-01-01

    To provide an ideal solution for a specific problem of gastric cancer detection in which low-scattering regions simultaneously existed with both the non- and high-scattering regions, a novel hybrid radiosity-SP3 equation based reconstruction algorithm for bioluminescence tomography was proposed in this paper. In the algorithm, the third-order simplified spherical harmonics approximation (SP3) was combined with the radiosity equation to describe the bioluminescent light propagation in tissues, which provided acceptable accuracy for the turbid medium with both low- and non-scattering regions. The performance of the algorithm was evaluated with digital mouse based simulations and a gastric cancer-bearing mouse based in situ experiment. Primary results demonstrated the feasibility and superiority of the proposed algorithm for the turbid medium with low- and non-scattering regions.

  15. Evaluation of a bioluminescence method, contact angle measurements and topography for testing the cleanability of plastic surfaces under laboratory conditions

    Science.gov (United States)

    Redsven, I.; Kymäläinen, H.-R.; Pesonen-Leinonen, E.; Kuisma, R.; Ojala-Paloposki, T.; Hautala, M.; Sjöberg, A.-M.

    2007-04-01

    Detection of adenosine triphosphate (ATP) by bioluminescence is used, for instance, in the food industry and in hospitals to assess the hygiene status of surfaces. The aim of this laboratory study was to investigate the feasibility of the ATP method for estimating the cleanability of resilient floor coverings from biological soil. The surfaces were worn using a Soiling and Wearing Drum Tester, and soiled and cleaned with an Erichsen Washability and Scrubbing Resistance Tester. In the laboratory test carried out with the bioluminescence method, most of the new and worn floor coverings that were biologically soiled were cleaned efficiently. According to this study, the semiquantitative ATP screening method can be used for hygiene monitoring of flooring materials. No correlation was found between cleanability and contact angles or surface topography measured using a profilometer. However, by revealing local irregularities and damage on surfaces, scanning electron micrographs appeared useful in explaining differences in cleanability.

  16. ATP生物发光测定试剂研究进展%Reserach progress on ATP bioluminescence reagent

    Institute of Scientific and Technical Information of China (English)

    吴慧清; 李程思; 吴清平; 张菊梅

    2012-01-01

    Firefly luciferase is the key component of ATP bioluminescence reagent, gained from firefly lantern throuh extraction and purification or preparation through genetic engineering, the performance of ATP bioluminescence reagent was decided by the vitality and the purity of firefly luciferase.Up to now, many present advanced technology were applied on preparation the reagent such as genetic engineering, ATP amplification device, stabilization technology of luciferase protein and luminescence, and so on.Now research focus on improving detection sensitivity and luminescence performance of the ATP bioluminescence reagents, further raising the adaptability of ATP bioluminescence reagents.%萤火虫荧光素酶是ATP生物发光试剂的关键组成部分,可通过萤火虫尾提取纯化或基因工程技术制备,酶的活力和纯度决定了ATP生物发光试剂的性能.迄今许多先进技术在ATP生物发光试剂的制备中均有应用,包括酶基因工程改造技术、ATP循环的酶法放大技术、荧光素酶蛋白的活力及发光稳定技术,特异的细胞ATP提取技术等.ATP生物发光试剂的研究焦点主要集中在提高发光试剂的检测灵敏度和性能、增加产品的适应性等方面.

  17. Direct Evidence of Mesenchymal Stem Cell Tropism for Tumor and Wounding Microenvironments using In Vivo Bioluminescence Imaging

    Science.gov (United States)

    Kidd, Shannon; Spaeth, Erika; Dembinski, Jennifer L.; Dietrich, Martin; Watson, Keri; Klopp, Ann; Battula, Lokesh; Weil, Micheal; Andreeff, Michael; Marini, Frank C.

    2014-01-01

    Multipotent mesenchymal stromal/stem cells (MSC) have shown potential clinical utility. However, previous assessments of MSC behavior in recipients have relied on visual detection in host tissue following sacrifice, failing to monitor in vivo MSC dispersion in a single animal and limiting the number of variables that can be observed concurrently. In this study, we utilized noninvasive, in vivo bioluminescent imaging to determine conditions under which MSC selectively engraft in sites of inflammation. MSC modified to express firefly luciferase (MSC-ffLuc) were injected into healthy mice or mice bearing inflammatory insults, and MSC localization was followed with bioluminescent imaging. Inflammatory insults investigated included cutaneous needle-stick and surgical incision wounds, as well as xenogeneic and syngeneic tumors. We also compared tumor models in which MSC were intraveneously or intraperitoneally delivered. Our results demonstrate hMSC-ffLuc systemically delivered to non-tumor bearing animals initially reside in the lungs, then egress to the liver and spleen and decrease in signal over time. However, hMSC in wounded mice engraft and remain detectable only in injured sites. Similarly, in syngeneic and xenogeneic breast carcinoma-bearing mice, bioluminescent detection of systemically delivered MSC revealed persistent, specific co-localization with sites of tumor development. This pattern of tropism was also observed in an ovarian tumor model in which MSC were IP injected. In this study we have identified conditions under which MSC tropism and selective engraftment in sites of inflammation can be monitored by bioluminescent imaging over time. Importantly, these consistent findings were independent of tumor type, immunocompetence and route of MSC delivery. PMID:19650040

  18. Direct evidence of mesenchymal stem cell tropism for tumor and wounding microenvironments using in vivo bioluminescent imaging.

    Science.gov (United States)

    Kidd, Shannon; Spaeth, Erika; Dembinski, Jennifer L; Dietrich, Martin; Watson, Keri; Klopp, Ann; Battula, Venkata Lokesh; Weil, Micheal; Andreeff, Michael; Marini, Frank C

    2009-10-01

    Multipotent mesenchymal stromal/stem cells (MSC) have shown potential clinical utility. However, previous assessments of MSC behavior in recipients have relied on visual detection in host tissue following sacrifice, failing to monitor in vivo MSC dispersion in a single animal and limiting the number of variables that can be observed concurrently. In this study, we used noninvasive, in vivo bioluminescent imaging to determine conditions under which MSC selectively engraft in sites of inflammation. MSC modified to express firefly luciferase (ffLuc-MSC) were injected into healthy mice or mice bearing inflammatory insults, and MSC localization was followed with bioluminescent imaging. The inflammatory insults investigated included cutaneous needle-stick and surgical incision wounds, as well as xenogeneic and syngeneic tumors. We also compared tumor models in which MSC were i.v. or i.p. delivered. Our results demonstrate that ffLuc-expressing human MSC (hMSC) systemically delivered to nontumor-bearing animals initially reside in the lungs, then egress to the liver and spleen, and decrease in signal over time. However, hMSC in wounded mice engraft and remain detectable only at injured sites. Similarly, in syngeneic and xenogeneic breast carcinoma-bearing mice, bioluminescent detection of systemically delivered MSC revealed persistent, specific colocalization with sites of tumor development. This pattern of tropism was also observed in an ovarian tumor model in which MSC were i.p. injected. In this study, we identified conditions under which MSC tropism and selective engraftment in sites of inflammation can be monitored by bioluminescent imaging over time. Importantly, these consistent findings were independent of tumor type, immunocompetence, and route of MSC delivery.

  19. How do marine bacteria produce light, why are they luminescent, and can we employ bacterial bioluminescence in aquatic biotechnology?

    OpenAIRE

    Grzegorz Wêgrzyn; Agata Czy¿

    2002-01-01

    Bioluminescence, the phenomenon of light production by living organisms, occurs in forms of life as various as bacteria, fungi and animals. Nevertheless, light-emitting bacteria are the most abundant and widespread of luminescent organisms. Interestingly, most species of such bacteria live in marine environments. In this article, the biochemical mechanism of bacterial luminescence and its genetic regulation are summarized. Although the biochemistry and genetics of light emission by cel...

  20. Dual monitoring using {sup 124}I-FIAU and bioluminescence for HSV1-tk suicide gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, T. S.; Kim, J. H.; Kwon, H. C. [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)] (and others)

    2007-07-01

    Herpes simplex virus type I thymidine kinase (HSV-tk) is the most common reporter gene and is used in cancer gene therapy with a prodrug nucleoside analog, ganciclovir (GCV). The aim of this study is to evaluate therapeutic efficacy of suicide gene therapy with 2'-fluoro-2'-deoxy-1-D-arabinofuranosyl-5-[{sup 124}I] iodouracil ({sup 124}I - FIAU) and bioluminescence in retrovirally HSV -tk and firefly luciferase transduced hepatoma model. The HSV -tk and firefly luciferase (Luc) was retrovirally transduced and expressed in MCA rat Morris hepatoma cells. Nude mice with subcutaneous tumors, MCA and MCA-TK-Luc, were subjected to GCV treatment (50mg/Kg/d intraperitoneally) for 5 day. PET imaging and biodistribution with ({sup 124}I-FIAU) were performed at before and after initiation of therapy with GCV. Bioluminescent signal was also measured during GCV treatment. Before GCV treatment, no significant difference in tumor volume was found in tumors between MCA and MCA-TK-Luc. After GCV treatment, tumor volume of MCA-TK-Luc markedly reduced compared to that of MCA. In biodistribution study, {sup 124}I-FIAU uptake after GCV therapy significantly decreased compared with pretreatment levels (34.8 13.67 %ID/g vs 7.6 2.59 %ID/g) and bioluminescent signal was also significantly decreased compared with pretreatment levels. In small animal PET imaging, {sup 124}I-FIAU selectively localized in HSV -tk expressing tumor and the therapeutic efficacy of GCV treatment was evaluated by {sup 124}I-FIAU PET imaging. {sup 124}I-FIAU PET and bioluminescence imaging in HSV-tk suicide gene therapy were effective to evaluate the therapeutic response. {sup 124}I-FIAU may serve as an efficient and selective agent for monitoring of transduced HSV1-tk gene expression in vivo in clinical trials.

  1. Comparison of a rapid ATP bioluminescence assay and standard plate count methods for assessing microbial contamination of consumers' refrigerators.

    Science.gov (United States)

    Chen, Fur-Chi; Godwin, Sandria L

    2006-10-01

    The feasibility of using an ATP bioluminescence assay for assessing microbial contamination of home refrigerators was evaluated and compared with the standard culture methods. Samples of refrigerator surfaces were collected from 123 households by swabbing an area of 100 cm2 on three locations in the refrigerator with premoisturized sterile swabs. Microbial contaminations were determined by aerobic plate count (APC; incubated at 35 degrees C for 48 h) and psychrotrophic plate count (PPC; incubated at 7 degrees C for 10 days) on plate count agar. The results were compared to the readings from the microbial ATP (mATP) bioluminescence assay. The correlation coefficient (r) between mATP and PPC (r = 0.851) was slightly higher than that between mATP and APC (r = 0.823). Our results indicated a potential discrepancy in the population of mesophilic and psychrotrophic bacteria in the refrigerator samples. Nevertheless, mATP appeared to be a reliable indication of the average of APC and PPC (r = 0.895). The mATP bioluminescence assay would provide a rapid and convenient test for researchers in field studies to assess microbial contamination in refrigerators.

  2. Monitoring Disease Progression and Therapeutic Response in a Disseminated Tumor Model for Non-Hodgkin Lymphoma by Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Margarethe Köberle

    2015-07-01

    Full Text Available Xenograft tumor models are widely studied in cancer research. Our aim was to establish and apply a model for aggressive CD20-positive B-cell non-Hodgkin lymphomas, enabling us to monitor tumor growth and shrinkage in a noninvasive manner. By stably transfecting a luciferase expression vector, we created two bioluminescent human non-Hodgkin lymphoma cell lines, Jeko1(luci and OCI-Ly3(luci, that are CD20 positive, a prerequisite to studying rituximab, a chimeric anti-CD20 antibody. To investigate the therapy response in vivo, we established a disseminated xenograft tumor model injecting these cell lines in NOD/SCID mice. We observed a close correlation of bioluminescence intensity and tumor burden, allowing us to monitor therapy response in the living animal. Cyclophosphamide reduced tumor burden in mice injected with either cell line in a dose-dependent manner. Rituximab alone was effective in OCI-Ly3(luci-injected mice and acted additively in combination with cyclophosphamide. In contrast, it improved the therapeutic outcome of Jeko1(luci-injected mice only in combination with cyclophosphamide. We conclude that well-established bioluminescence imaging is a valuable tool in disseminated xenograft tumor models. Our model can be translated to other cell lines and used to examine new therapeutic agents and schedules.

  3. [Quantitative criteria for the estimation of the effectiveness of bioluminescence expression in natural and transgenic luminescent bacteria].

    Science.gov (United States)

    Gusev, A A; Kargatova, T V; Medvedeva, S E; Popova, L Iu

    2008-01-01

    Computation coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria P. leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in multicopy plasmid are suggested, and their use on the molecular, cell, and population levels was considered. It was shown that, on the population level, all transgenic variants got the better of natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. On the cell level, in the bright and dim variants of the transgenic strain, the effectiveness of bioluminescence expression increases by several orders. On the level of one lux-operon, the effectiveness of expression of the bright variant of transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar, and the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2 /pPHL7 is by two orders lower than that in the dark variant of P. leiognathi 54.

  4. In vivo bioluminescence imaging of neurogenesis - the role of the blood brain barrier in an experimental model of Parkinson's disease.

    Science.gov (United States)

    Fricke, Inga B; Schelhaas, Sonja; Zinnhardt, Bastian; Viel, Thomas; Hermann, Sven; Couillard-Després, Sébastien; Jacobs, Andreas H

    2017-02-13

    Bioluminescence imaging in transgenic mice expressing firefly luciferase in Doublecortin(+) (Dcx) neuroblasts might serve as a powerful tool to study the role of neurogenesis in models of brain injury and neurodegeneration using non-invasive, longitudinal in vivo imaging. Therefore, we aimed to use BLI in B6(Cg)-Tyrc-2J/J Dcx-Luc (Doublecortin-Luciferase, Dcx-Luc) mice to investigate its suitability to assess neurogenesis in a unilateral injection model of Parkinson's disease. We further aimed to assess the blood brain barrier leakage associated with the intranigral 6-OHDA injection to evaluate its impact on substrate delivery and bioluminescence signal intensity. Two weeks after lesion, we observed an increase in bioluminescence signal in the ipsilateral hippocampal region in both, 6-OHDA and vehicle injected Dcx-Luc mice. At the same time, no corresponding increase in Dcx(+) neuroblast numbers could be observed in the dentate gyrus of C57Bl6 mice. Blood brain barrier leakage was observed in the hippocampal region and in the degenerating substantia nigra of C57Bl6 mice in vivo using T1 weighted Magnetic Resonance Imaging with Gadovist(®) and ex vivo using Evans Blue Fluorescence Reflectance Imaging and mouse Immunoglobulin G staining. Our data suggests a BLI signal dependency on blood brain barrier permeability, underlining a major pitfall of substrate/tracer dependent imaging in invasive disease models.

  5. Real-time monitoring of bioaerosols via cell-lysis by air ion and ATP bioluminescence detection.

    Science.gov (United States)

    Park, Chul Woo; Park, Ji-Woon; Lee, Sung Hwa; Hwang, Jungho

    2014-02-15

    In this study, we introduce a methodology for disrupting cell membranes with air ions coupled with ATP bioluminescence detection for real-time monitoring of bioaerosol concentrations. A carbon fiber ionizer was used to extract ATP from bacterial cells for generating ATP bioluminescence. Our methodology was tested using Staphylococcus epidermidis and Escherichia coli, which were aerosolized with an atomizer, and then indoor bioaerosols were also used for testing the methodology. Bioaerosol concentrations were estimated without culturing which requires several days for colony formation. Correlation equations were obtained for results acquired using our methodology (Relative Luminescent Unit (RLU)/m(3)) and a culture-based (Colony Forming Unit (CFU)/m(3)) method; CFU/m(3)=1.8 × measured RLU/m(3) for S. epidermidis and E. coli, and CFU/m(3)=1.1 × measured RLU/m(3) for indoor bioaerosols under the experimental conditions. Our methodology is an affordable solution for rapidly monitoring bioaerosols due to rapid detection time (cell-lysis time: 3 min; bioluminescence detection time: <1 min) and easy operation.

  6. Firefly Luciferase-Based Sequential Bioluminescence Resonance Energy Transfer (BRET)-Fluorescence Resonance Energy Transfer (FRET) Protease Assays.

    Science.gov (United States)

    Branchini, Bruce

    2016-01-01

    We describe here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyzes yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-Infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. This method successfully employs an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence to assay physiologically important protease activities and should be generally applicable to the measurement of any endoprotease lacking accessible cysteine residues.

  7. Effects of properties of metal-contaminated soils on bacterial bioluminescence activity, seed germination, and root and shoot growth.

    Science.gov (United States)

    Kang, Il-Mo; Kong, In Chul

    2016-01-01

    This study examined the effects of several factors (metal contents and soil properties) on bacterial bioluminescence activity, seed germination and root/shoot growth of Lactuca in metal-contaminated soils. Each bioassay showed different sensitivities to extractants of soil samples. Average sensitivities of the bioassay were in the following order: root growth > bioluminescence ≥ shoot growth ≥ seed germination. Both total and weak acid-extracted metal contents showed no observable correlations with the activity of any bioassays (r(2) bioluminescence activity and organics (r(2) = 0.7198) as well as between root growth and CEC (r(2) = 0.6676). Effects of soils were difficult to generalize since they were dependent on many factors, such as soil properties, metal contents, and the organism used in each test. Nonetheless, these results indicated that a battery of bioassays is an effective strategy for assessment of contaminated soils. Furthermore, specific soil factors were shown to more influence on soil toxicity, depending on the type of bioassay.

  8. Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity.

    Science.gov (United States)

    Cevenini, Luca; Calabretta, Maria Maddalena; Lopreside, Antonia; Tarantino, Giuseppe; Tassoni, Annalisa; Ferri, Maura; Roda, Aldo; Michelini, Elisa

    2016-12-01

    The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.

  9. Use of a highly sensitive two-dimensional luminescence imaging system to monitor endogenous bioluminescence in plant leaves

    Directory of Open Access Journals (Sweden)

    Flor-Henry Michel

    2004-11-01

    Full Text Available Abstract Background All living organisms emit spontaneous low-level bioluminescence, which can be increased in response to stress. Methods for imaging this ultra-weak luminescence have previously been limited by the sensitivity of the detection systems used. Results We developed a novel configuration of a cooled charge-coupled device (CCD for 2-dimensional imaging of light emission from biological material. In this study, we imaged photon emission from plant leaves. The equipment allowed short integration times for image acquisition, providing high resolution spatial and temporal information on bioluminescence. We were able to carry out time course imaging of both delayed chlorophyll fluorescence from whole leaves, and of low level wound-induced luminescence that we showed to be localised to sites of tissue damage. We found that wound-induced luminescence was chlorophyll-dependent and was enhanced at higher temperatures. Conclusions The data gathered on plant bioluminescence illustrate that the equipment described here represents an improvement in 2-dimensional luminescence imaging technology. Using this system, we identify chlorophyll as the origin of wound-induced luminescence from leaves.

  10. Visualization of in Vivo Hydrogen Sulfide Production by a Bioluminescence Probe in Cancer Cells and Nude Mice.

    Science.gov (United States)

    Tian, Xiaodong; Li, Zhiyan; Lau, Choiwan; Lu, Jianzhong

    2015-11-17

    Hydrogen sulfide (H2S) has emerged as an exciting endogenous gasotransmitter in addition to nitric oxide and carbon monoxide. However, its precise measurement in living cells and animals remains a challenge. In this study, a novel bioluminescence H2S probe was designed and synthesized by modifying the 6'-amino group of d-aminoluciferin into a 6'-azido group, which was highly selective against other reactive sulfur, nitrogen, and oxygen species. Our H2S probe azidoluciferin sensitively reacted with H2S to release d-aminoluciferin with a strong bioluminescence signal. On the basis of its high selectivity and sensitivity, the H2S probe was used to detect H2S production in live cancer cells and nude mice. The bioluminescence signal decreased in mice treated with propargylglycine, an inhibitor of H2S, suggesting that our H2S probe can detect endogenous H2S in real time, in vivo. Overall, the excellent sensing properties of the probe combined with its bioimaging capability make it a useful tool to study H2S biological roles.

  11. A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.

    Science.gov (United States)

    Mezzanotte, Laura; Blankevoort, Vicky; Löwik, Clemens W G M; Kaijzel, Eric L

    2014-09-01

    Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 10(3) and 5 × 10(4) cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.

  12. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.

    Science.gov (United States)

    Ramanathan, Chidambaram; Khan, Sanjoy K; Kathale, Nimish D; Xu, Haiyan; Liu, Andrew C

    2012-09-27

    In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host

  13. A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations.

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    Amaral, Danilo T; Oliveira, Gabriela; Silva, Jaqueline R; Viviani, Vadim R

    2016-09-31

    Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 μM and 17 μM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation.

  14. Mouse Reporter Strain for Noninvasive Bioluminescent Imaging of Cells that have Undergone Cre-Mediated Recombination

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    Michal Safran

    2003-10-01

    Full Text Available Conditional alleles containing LoxP recombination sites, in conjunction with Cre recombinase delivered by a variety of means, allows for spatial and temporal control of gene expression in mouse models. Here we describe a mouse strain in which a luciferase (Luc cDNA, preceded by a LoxP-stop-LoxP (L-S-L cassette, was introduced into the ubiquitously expressed ROSA26 locus. Mouse embryo fibroblasts derived from this strain expressed luciferase after Cre-mediated recombination in vitro. ROSA26 L-S-L-Luc/+ mice expressed luciferase in a diffuse or liver-restricted pattern, as determined by noninvasive, bioluminescent imaging, when crossed to transgenic mice in which Cre was under the control of a zygotically expressed (EIIA-Cre, or a liver-restricted (albumin-Cre, promoter, respectively. Organ-specific luciferase expression was also seen after intraparenchymal administration of an adenovirus encoding Cre. The ROSA26 L-S-L-Luc/+ strain should be useful for characterizing Cre mouse strains and for following the fate of cells that have undergone Cre-mediated recombination in vivo.

  15. Effect of source-separated urine storage on estrogenic activity detected using bioluminescent yeast Saccharomyces cerevisiae.

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    Jaatinen, Sanna; Kivistö, Anniina; Palmroth, Marja R T; Karp, Matti

    2016-09-01

    The objective was to demonstrate that a microbial whole cell biosensor, bioluminescent yeast, Saccharomyces cerevisiae (BMAEREluc/ERα) can be applied to detect overall estrogenic activity from fresh and stored human urine. The use of source-separated urine in agriculture removes a human originated estrogen source from wastewater influents, subsequently enabling nutrient recycling. Estrogenic activity in urine should be diminished prior to urine usage in agriculture in order to prevent its migration to soil. A storage period of 6 months is required for hygienic reasons; therefore, estrogenic activity monitoring is of interest. The method measured cumulative female hormone-like activity. Calibration curves were prepared for estrone, 17β-estradiol, 17α- ethinylestradiol and estriol. Estrogen concentrations of 0.29-29,640 μg L(-1) were detectable while limit of detection corresponded to 0.28-35 μg L(-1) of estrogens. The yeast sensor responded well to fresh and stored urine and gave high signals corresponding to 0.38-3,804 μg L(-1) of estrogens in different urine samples. Estrogenic activity decreased during storage, but was still higher than in fresh urine implying insufficient storage length. The biosensor was suitable for monitoring hormonal activity in urine and can be used in screening anthropogenic estrogen-like compounds interacting with the receptor.

  16. Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.

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    Mobin A Karimi

    Full Text Available The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform.

  17. Rapid and Quantitative Assessment of Cancer Treatment Response Using In Vivo Bioluminescence Imaging

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    Alnawaz Rehemtulla

    2000-01-01

    Full Text Available Current assessment of orthotopic tumor models in animals utilizes survival as the primary therapeutic end point. In vivo bioluminescence imaging (BLI is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating antineoplastic therapies [1 ]. Using human tumor cell lines constitutively expressing luciferase, the kinetics of tumor growth and response to therapy have been assessed in intraperitoneal [2], subcutaneous, and intravascular [3] cancer models. However, use of this approach for evaluating orthotopic tumor models has not been demonstrated. In this report, the ability of BLI to noninvasively quantitate the growth and therapeuticinduced cell kill of orthotopic rat brain tumors derived from 9L gliosarcoma cells genetically engineered to stably express firefly luciferase (9LLuc was investigated. Intracerebral tumor burden was monitored over time by quantitation of photon emission and tumor volume using a cryogenically cooled CCD camera and magnetic resonance imaging (MRI, respectively. There was excellent correlation (r=0.91 between detected photons and tumor volume. A quantitative comparison of tumor cell kill determined from serial MRI volume measurements and BLI photon counts following 1,3-bis(2-chloroethyl-1-nitrosourea (BCNU treatment revealed that both imaging modalities yielded statistically similar cell kill values (P=.951. These results provide direct validation of BLI imaging as a powerful and quantitative tool for the assessment of antineoplastic therapies in living animals.

  18. L{sub 1/2} regularization based numerical method for effective reconstruction of bioluminescence tomography

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    Chen, Xueli, E-mail: xlchen@xidian.edu.cn, E-mail: jimleung@mail.xidian.edu.cn; Yang, Defu; Zhang, Qitan; Liang, Jimin, E-mail: xlchen@xidian.edu.cn, E-mail: jimleung@mail.xidian.edu.cn [School of Life Science and Technology, Xidian University, Xi' an 710071 (China); Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education (China)

    2014-05-14

    Even though bioluminescence tomography (BLT) exhibits significant potential and wide applications in macroscopic imaging of small animals in vivo, the inverse reconstruction is still a tough problem that has plagued researchers in a related area. The ill-posedness of inverse reconstruction arises from insufficient measurements and modeling errors, so that the inverse reconstruction cannot be solved directly. In this study, an l{sub 1/2} regularization based numerical method was developed for effective reconstruction of BLT. In the method, the inverse reconstruction of BLT was constrained into an l{sub 1/2} regularization problem, and then the weighted interior-point algorithm (WIPA) was applied to solve the problem through transforming it into obtaining the solution of a series of l{sub 1} regularizers. The feasibility and effectiveness of the proposed method were demonstrated with numerical simulations on a digital mouse. Stability verification experiments further illustrated the robustness of the proposed method for different levels of Gaussian noise.

  19. CD4+ T cell effects on CD8+ T cell location defined using bioluminescence.

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    Mitra Azadniv

    Full Text Available T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are "helped" by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell "help" is to program the homing potential of CD8+ T cells.

  20. Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice

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    Liu Zhihui

    2008-08-01

    Full Text Available Abstract Background Interleukin 1 beta (IL-1β plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1β in disease processes and develop an in vivo screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1β gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models. Results In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1β mRNA and pro-IL-1β protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model. Conclusion Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1β gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug in vivo.

  1. Light Emission Requires Exposure to the Atmosphere in Ex Vivo Bioluminescence Imaging

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    Yusuke Inoue

    2006-04-01

    Full Text Available The identification of organs bearing luciferase activity by in vivo bioluminescence imaging (BLI is often difficult, and ex vivo imaging of excised organs plays a complementary role. This study investigated the importance of exposure to the atmosphere in ex vivo BLI. Mice were inoculated with murine pro-B cell line Ba/F3 transduced with firefly luciferase and p190 BCR-ABL. They were killed following in vivo BLI, and whole-body imaging was done after death and then after intraperitoneal air injection. In addition, the right knee was exposed and imaged before and after the adjacent bones were cut. Extensive light signals were seen on in vivo imaging. The luminescence disappeared after the animal was killed, and air injection restored the light emission from the abdomen only, suggesting a critical role of atmospheric oxygen in luminescence after death. Although no substantial light signal at the right knee was seen before bone cutting, light emission was evident after cutting. In conclusion, in ex vivo BLI, light emission requires exposure to the atmosphere. Bone destruction is required to demonstrate luciferase activity in the bone marrow after death.

  2. Incorporating MRI structural information into bioluminescence tomography: system, heterogeneous reconstruction and in vivo quantification.

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    Zhang, Jun; Chen, Duofang; Liang, Jimin; Xue, Huadan; Lei, Jing; Wang, Qin; Chen, Dongmei; Meng, Ming; Jin, Zhengyu; Tian, Jie

    2014-06-01

    Combining two or more imaging modalities to provide complementary information has become commonplace in clinical practice and in preclinical and basic biomedical research. By incorporating the structural information provided by computed tomography (CT) or magnetic resonance imaging (MRI), the ill poseness nature of bioluminescence tomography (BLT) can be reduced significantly, thus improve the accuracies of reconstruction and in vivo quantification. In this paper, we present a small animal imaging system combining multi-view and multi-spectral BLT with MRI. The independent MRI-compatible optical device is placed at the end of the clinical MRI scanner. The small animal is transferred between the light tight chamber of the optical device and the animal coil of MRI via a guide rail during the experiment. After the optical imaging and MRI scanning procedures are finished, the optical images are mapped onto the MRI surface by interactive registration between boundary of optical images and silhouette of MRI. Then, incorporating the MRI structural information, a heterogeneous reconstruction algorithm based on finite element method (FEM) with L 1 normalization is used to reconstruct the position, power and region of the light source. In order to validate the feasibility of the system, we conducted experiments of nude mice model implanted with artificial light source and quantitative analysis of tumor inoculation model with MDA-231-GFP-luc. Preliminary results suggest the feasibility and effectiveness of the prototype system.

  3. Vibrational spectra of chemical and isotopic variants of oxyluciferin, the light emitter of firefly bioluminescence.

    Science.gov (United States)

    Maltsev, Oleg V; Yue, Ling; Rebarz, Mateusz; Hintermann, Lukas; Sliwa, Michel; Ruckebusch, Cyril; Pejov, Ljupčo; Liu, Ya-Jun; Naumov, Panče

    2014-08-18

    The chemical complexity of oxyluciferin (OxyLH2), the light-emitting molecule in the bioluminescence of fireflies, originates from the possibility of keto/enol tautomerism and single or double deprotonation. Herein, we present detailed infrared spectroscopic analysis of OxyLH2 and several of its chemical isomers and isotopomers. To facilitate the future characterization of its biogenic forms, we provide accurate assignments of the solid-state and solution FTIR spectra of OxyLH2 based on comparison to six isotopically labeled variants ([2-(13)C]-OxyLH2, [3-(15)N]-OxyLH2, [4-(13)C]-OxyLH2, [5-(13)C]-OxyLH2, [2'-(13)C]-OxyLH2, [3'-(15)N]-OxyLH2), five closely related structural analogues, and theoretically computed spectra. The computed DFT harmonic vibrational force fields (B3LYP and M06 functionals with basis sets of varying flexibility up to 6-311++G**) reproduce well the observed shifts in the IR spectra of both isotopically labeled and structurally related analogues.

  4. Bioluminescence tomography using eigenvectors expansion and iterative solution for the optimized permissible source region.

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    Naser, Mohamed A; Patterson, Michael S

    2011-11-01

    A reconstruction algorithm for bioluminescence tomography (BLT) has been developed. The algorithm numerically calculates the Green's function at different wavelengths using the diffusion equation and finite element method. The optical properties used in calculating the Green's function are reconstructed using diffuse optical tomography (DOT) and assuming anatomical information is provided by x-ray computed tomography or other methods. A symmetric system of equations is formed using the Green's function and the measured light fluence rate and the resulting eigenvalue problem is solved to get the eigenvectors of this symmetric system of equations. A space can be formed from the eigenvectors obtained and the reconstructed source is written as an expansion of the eigenvectors corresponding to non-zero eigenvalues. The coefficients of the expansion are found to obtain the reconstructed BL source distribution. The problem is solved iteratively by using a permissible source region that is shrunk by removing nodes with low probability to contribute to the source. Throughout this process the permissible region shrinks from the entire object to just a few nodes. The best estimate of the reconstructed source is chosen that which minimizes the difference between the calculated and measured light fluence rates. 3D simulations presented here show that the reconstructed source is in good agreement with the actual source in terms of locations, magnitudes, sizes, and total powers for both localized multiple sources and large inhomogeneous source distributions.

  5. Computationally efficient perturbative forward modeling for 3D multispectral bioluminescence and fluorescence tomography

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    Dutta, Joyita; Ahn, Sangtae; Li, Changqing; Chaudhari, Abhijit J.; Cherry, Simon R.; Leahy, Richard M.

    2008-03-01

    The forward problem of optical bioluminescence and fluorescence tomography seeks to determine, for a given 3D source distribution, the photon density on the surface of an animal. Photon transport through tissues is commonly modeled by the diffusion equation. The challenge, then, is to accurately and efficiently solve the diffusion equation for a realistic animal geometry and heterogeneous tissue types. Fast analytical solvers are available that can be applied to arbitrary geometries but assume homogeneity of tissue optical properties and hence have limited accuracy. The finite element method (FEM) with volume tessellation allows reasonably accurate modeling of both animal geometry and tissue heterogeneity, but this approach is computationally intensive. The computational challenge is heightened when one is working with multispectral data to improve source localization and conditioning of the inverse problem. Here we present a fast forward model based on the Born approximation that falls in between these two approaches. Our model introduces tissue heterogeneity as perturbations in diffusion and absorption coefficients at rectangular grid points inside a mouse atlas. These reflect as a correction term added to the homogeneous forward model. We have tested our model by performing source localization studies first with a biolumnescence simulation setup and then with an experimental setup using a fluorescent source embedded in an inhomogeneous phantom that mimicks tissue optical properties.

  6. In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography

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    Coralie Genevois

    2016-10-01

    Full Text Available Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI using the firefly luciferase (Fluc as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI, fluorescence diffuse optical tomography (fDOT, and fluorescence molecular Imaging (FMT®. A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors.

  7. Hunting in bioluminescent light: Vision in the nocturnal box jellyfish Copula sivickisi

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    Anders eGarm

    2016-03-01

    Full Text Available Cubomedusae all have a similar set of six eyes on each of their four rhopalia. Still, there is a great variation in activity patterns with some species being strictly day active while others are strictly night active. Here we have examined the visual ecology of the medusa of the night active Copula sivickisi from Okinawa using optics, morphology, electrophysiology, and behavioural experiments. We found the lenses of both the upper and the lower lens eyes to be image forming but under-focused, resulting in low spatial resolution in the order of 10 – 15 degrees. The photoreceptor physiology is similar in the two lens eyes and they have a single opsin peaking around 460 nm and low temporal resolution with a flicker fusion frequency (fff of 2.5 Hz indicating adaptions to vision in low light intensities. Further, the outer segments have fluid filled swellings, which may concentrate the light in the photoreceptor membrane by total internal reflections, and thus enhance the signal to noise ratio in the eyes. Finally our behavioural experiments confirmed that the animals use vision when hunting. When they are active at night they seek out high prey-concentration by visual attraction to areas with abundant bioluminescent flashes triggered by their prey.

  8. Evaluation of monkeypox virus infection of prairie dogs (Cynomys ludovicianus) using in vivo bioluminescent imaging

    Science.gov (United States)

    Falendysz, Elizabeth A.; Londoño-Navas, Angela M.; Meteyer, Carol U.; Pussini, Nicola; Lopera, Juan G.; Osorio, Jorge E.; Rocke, Tonie E.

    2014-01-01

    Monkeypox (MPX) is a re-emerging zoonotic disease that is endemic in Central and West Africa, where it can cause a smallpox-like disease in humans. Despite many epidemiologic and field investigations of MPX, no definitive reservoir species has been identified. Using recombinant viruses expressing the firefly luciferase (luc) gene, we previously demonstrated the suitability of in vivo bioluminescent imaging (BLI) to study the pathogenesis of MPX in animal models. Here, we evaluated BLI as a novel approach for tracking MPX virus infection in black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs were affected during a multistate outbreak of MPX in the US in 2003 and have since been used as an animal model of this disease. Our BLI results were compared with PCR and virus isolation from tissues collected postmortem. Virus was easily detected and quantified in skin and superficial tissues by BLI before and during clinical phases, as well as in subclinical secondary cases, but was not reliably detected in deep tissues such as the lung. Although there are limitations to viral detection in larger wild rodent species, BLI can enhance the use of prairie dogs as an animal model of MPX and can be used for the study of infection, disease progression, and transmission in potential wild rodent reservoirs.

  9. The CUBLAS and CULA based GPU acceleration of adaptive finite element framework for bioluminescence tomography.

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    Zhang, Bo; Yang, Xiang; Yang, Fei; Yang, Xin; Qin, Chenghu; Han, Dong; Ma, Xibo; Liu, Kai; Tian, Jie

    2010-09-13

    In molecular imaging (MI), especially the optical molecular imaging, bioluminescence tomography (BLT) emerges as an effective imaging modality for small animal imaging. The finite element methods (FEMs), especially the adaptive finite element (AFE) framework, play an important role in BLT. The processing speed of the FEMs and the AFE framework still needs to be improved, although the multi-thread CPU technology and the multi CPU technology have already been applied. In this paper, we for the first time introduce a new kind of acceleration technology to accelerate the AFE framework for BLT, using the graphics processing unit (GPU). Besides the processing speed, the GPU technology can get a balance between the cost and performance. The CUBLAS and CULA are two main important and powerful libraries for programming on NVIDIA GPUs. With the help of CUBLAS and CULA, it is easy to code on NVIDIA GPU and there is no need to worry about the details about the hardware environment of a specific GPU. The numerical experiments are designed to show the necessity, effect and application of the proposed CUBLAS and CULA based GPU acceleration. From the results of the experiments, we can reach the conclusion that the proposed CUBLAS and CULA based GPU acceleration method can improve the processing speed of the AFE framework very much while getting a balance between cost and performance.

  10. Small animal fluorescence and bioluminescence tomography: a review of approaches, algorithms and technology update

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    Darne, Chinmay; Lu, Yujie; Sevick-Muraca, Eva M.

    2014-01-01

    Emerging fluorescence and bioluminescence tomography approaches have several common, yet several distinct features from established emission tomographies of PET and SPECT. Although both nuclear and optical imaging modalities involve counting of photons, nuclear imaging techniques collect the emitted high energy (100-511 keV) photons after radioactive decay of radionuclides while optical techniques count low-energy (1.5-4.1 eV) photons that are scattered and absorbed by tissues requiring models of light transport for quantitative image reconstruction. Fluorescence imaging has been recently translated into clinic demonstrating high sensitivity, modest tissue penetration depth, and fast, millisecond image acquisition times. As a consequence, the promise of quantitative optical tomography as a complement of small animal PET and SPECT remains high. In this review, we summarize the different instrumentation, methodological approaches and schema for inverse image reconstructions for optical tomography, including luminescence and fluorescence modalities, and comment on limitations and key technological advances needed for further discovery research and translation.

  11. Algorithm for localized adaptive diffuse optical tomography and its application in bioluminescence tomography

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    Naser, Mohamed A.; Patterson, Michael S.; Wong, John W.

    2014-04-01

    A reconstruction algorithm for diffuse optical tomography based on diffusion theory and finite element method is described. The algorithm reconstructs the optical properties in a permissible domain or region-of-interest to reduce the number of unknowns. The algorithm can be used to reconstruct optical properties for a segmented object (where a CT-scan or MRI is available) or a non-segmented object. For the latter, an adaptive segmentation algorithm merges contiguous regions with similar optical properties thereby reducing the number of unknowns. In calculating the Jacobian matrix the algorithm uses an efficient direct method so the required time is comparable to that needed for a single forward calculation. The reconstructed optical properties using segmented, non-segmented, and adaptively segmented 3D mouse anatomy (MOBY) are used to perform bioluminescence tomography (BLT) for two simulated internal sources. The BLT results suggest that the accuracy of reconstruction of total source power obtained without the segmentation provided by an auxiliary imaging method such as x-ray CT is comparable to that obtained when using perfect segmentation.

  12. The uses and abuses of rapid bioluminescence-based ATP assays.

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    Shama, G; Malik, D J

    2013-03-01

    Bioluminescence-based ATP testing of solid surfaces has become well established in the food processing industry as part of general hazard analysis and critical control points (HACCP) measures. The rise in healthcare associated infections (HAIs) at the turn of the century focussed attention on the environment as a potential reservoir of the agents responsible for such infections. In response to the need for objective methods of assessing the efficiency of cleaning in healthcare establishments and for rapid methods for detecting the presence of the pathogens responsible for HAIs, it was proposed that ATP testing of environmental surfaces be introduced. We examine the basis behind the assumptions inherent in these proposals. Intracellular ATP levels are shown to vary between microbial taxa and according to environmental conditions. Good correlations between microbial numbers and ATP levels have been obtained under certain specific conditions, but never within healthcare settings. Notwithstanding, ATP testing may still have a role in providing reassurance that cleaning regimes are being carried out satisfactorily. However, ATP results should not be interpreted as surrogate indicators for the presence of microbial pathogens.

  13. Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics.

    Science.gov (United States)

    Qiu, Jingmin; Zhou, Yun; Chen, Hui; Lin, Jin-Ming

    2009-08-15

    This paper describes an immunomagnetic separation of target bacterial cells from others by using magnetic bead. The surface of bead was coated with antibodies which can capture specific organism. The binding efficiency of immunomagnetic bead (IMB) capturing target bacterial cells was higher than 98% when the concentrations of target and interferent bacterial cells were at the same level. The concentration of bacteria was determined indirectly by detecting adenosine 5'-triphosphate (ATP) employing bioluminescence (BL) reaction of firefly luciferin-ATP. Benzalkonium chloride (BAC) was used as an ATP extractant from living bacterial cells. We found that BAC could enhance the light emission when the concentration of BAC was less than 5.3 x 10(-2)% (w/v) and the BL intensity reached its maximum at the concentration of BAC was 2.7 x 10(-2)%, which was 10-fold stronger than that without BAC. Based on the principle of the IMB, a microfluidic chip combined with immunofluorescence assay for separating and detecting bacteria simultaneously was also developed. The IMBs were magnetically fixed in the bead-beds of chip channels with a 3-mm diameter of NdFeB permanent magnet. The target bacterial cells can be captured magnetically and observed by a fluorescent microscope.

  14. How reliable are ATP bioluminescence meters in assessing decontamination of environmental surfaces in healthcare settings?

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    Navid Omidbakhsh

    Full Text Available BACKGROUND: Meters based on adenosine triphosphate (ATP bioluminescence measurements in relative light units (RLU are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry? OBJECTIVE: This study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals' interference in their readings. METHODS: First, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meter's ATP readings correlated with the actual CFUs. Next, various types of disinfectant chemistries were tested for their potential to interfere with the standard ATP readings. RESULTS: All four ATP meters tested herein demonstrated acceptable linearity and repeatability in their readings. However, there were significant differences in their sensitivity to detect the levels of viable microorganisms on experimentally contaminated surfaces. Further, most disinfectant chemistries tested here quenched the ATP readings variably in different ATP meters evaluated. CONCLUSIONS: Apart from their limited sensitivity in detecting low levels of microbial contamination, the ATP meters tested were also prone to interference by different disinfectant chemistries.

  15. Metabolic imaging in microregions of tumors and normal tissues with bioluminescence and photon counting

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    Mueller-Klieser, W.; Walenta, S.; Paschen, W.; Kallinowski, F.; Vaupel, P.

    1988-08-03

    A method has been developed for metabolic imaging on a microscopic level in tumors, tumor spheroids, and normal tissues. The technique makes it possible to determine the spatial distribution of glucose, lactate, and ATP in absolute terms at similar locations within tissues or cell aggregates. The substrate distributions are registered in serial cryostat sections from tissue cryobiopsies or from frozen spheroids with the use of bioluminescence reactions. The light emission is measured directly by a special imaging photon counting system enabling on-line image analysis. The technique has been applied to human breast cancer xenografts, to spheroids originating from a human colon adenocarcinoma, and to skeletal rat muscle. Preliminary data obtained indicate that heterogeneities in the substrate distributions measured are much more pronounced in tumors than in normal tissue. There was no obvious correlation among the three quantities measured at similar locations within the tissues. The distribution of ATP corresponded well with the histological structure of larger spheroids; values were low in the necrotic center and high in the viable rim of these cell aggregates.

  16. Lead and Cu in contaminated urban soils: extraction with chemical reagents and bioluminescent bacteria and yeast.

    Science.gov (United States)

    Peltola, Pasi; Ivask, Angela; Aström, Mats; Virta, Marko

    2005-11-01

    Twenty urban soil samples, with a wide range of Pb (14-5323 mg/kg) and Cu (8-12987 mg/kg), were used to compare the operational speciation of a five-step sequential leach with the bioavailability determined with bioluminescent Pb (RN4220(pTOO24)) and Cu (MC1061(pSLcueR/pDNPcopAluc)) specific bacterial biosensors and a Cu specific yeast sensor. The bioavailable Pb concentrations were all similar or lower than the first sequential leach step (1M NaOAc). In contrast, in some samples the bioavailable concentrations of Cu clearly exceeded even the second sequential leach step (0.1 M Na4P2O7). With the yeast sensor 12/20 samples were below detection, however, the yeast sensor was capable of detecting all high Cu concentrations. The biosensors used in this study are not capable of detecting the natural soil concentrations of Pb and Cu in the studied area.

  17. Toxicity Testing of Pristine and Aged Silver Nanoparticles in Real Wastewaters Using Bioluminescent Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    Florian Mallevre

    2016-03-01

    Full Text Available Impact of aging on nanoparticle toxicity in real matrices is scarcely investigated due to a lack of suitable methodologies. Herein, the toxicity of pristine and aged silver nanoparticles (Ag NPs to a bioluminescent Pseudomonas putida bioreporter was measured in spiked crude and final wastewater samples (CWs and FWs, respectively collected from four wastewater treatment plants (WWTPs. Results showed lower toxicity of pristine Ag NPs in CWs than in FWs. The effect of the matrix on the eventual Ag NP toxicity was related to multiple physico-chemical parameters (biological oxygen demand (BOD, chemical oxygen demand (COD, total suspended solids (TSS pH, ammonia, sulfide and chloride based on a multivariate analysis. However, no collection site effect was concluded. Aged Ag NPs (up to eight weeks were found less toxic than pristine Ag NPs in CWs; evident increased aggregation and decreased dissolution were associated with aging. However, Ag NPs exhibited consistent toxicity in FWs despite aging; comparable results were obtained in artificial wastewater (AW simulating effluent. The study demonstrates the potency of performing nanoparticle acute toxicity testing in real and complex matrices such as wastewaters using relevant bacterial bioreporters.

  18. Quick, portable toxicity testing of marine or terrigenous fluids, sediments, or chemicals with bioluminescent organism

    Energy Technology Data Exchange (ETDEWEB)

    Sabate, R.W.; Stiffey, A.V.; Dewailly, E.L. [Lumitox Gulf L.C., New Orleans, LA (United States)

    1995-12-31

    A hand-held, battery-operated instrument, which measures bioluminescence inhibition of the microscopic marine dinoflagellate Pyrocystis lunula, is capable of field-testing substances for toxicity. The organism is sensitive to ppb of strong toxicants. It tolerates some solvents in concentrations necessary for testing lipophylic samples. A test consumes only micrograms of sample. This method requires no adjustments for salinity, pH, color, or turbidity. It has been used successfully to test oil-well drilling fluids, brines produced with oil, waters and sediments from streams and lakes and petroleum-plant effluents containing contaminants such as benzene. The test is non-specific; however, if the substance is known, the end-point effects a direct measurement of its concentration. One-hour toxicity screening tests in the field produce results comparable to the standard four-hour laboratory test. Keeping the sample in the dark during incubation and testing, together with shortness of the overall procedure, eliminates anomalies from light-sensitive substances. Day-to-day variation, as well as among test replicates, is less than 10%. This quick method yields results comparable with a quick test that uses Photobacterium phosphoria, and with 96-hour tests that use Mysidopsis bahia, Artemia salina, Gonyaulax polyedra, Pimephales promelas, Ceriodaphnia dubia, and Cyprinodon variegatus.

  19. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter.

    Science.gov (United States)

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei

    2015-04-01

    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

  20. Analysis of Arrestin Recruitment to Chemokine Receptors by Bioluminescence Resonance Energy Transfer.

    Science.gov (United States)

    Bonneterre, J; Montpas, N; Boularan, C; Galés, C; Heveker, N

    2016-01-01

    Chemokine receptors recruit the multifunctional scaffolding protein beta arrestin in response to binding of their chemokine ligands. Given that arrestin recruitment represents a signaling axis that is in part independent from G-protein signaling, it has become a hallmark of G protein-coupled receptor functional selectivity. Therefore, quantification of arrestin recruitment has become a requirement for the delineation of chemokine and drug candidate activity along different signaling axes. Bioluminescence resonance energy transfer (BRET) techniques provide methodology for such quantification that can reveal differences between nonredundant chemokines binding the same receptor, and that can be upscaled for high-throughput testing. We here provide protocols for the careful setup of BRET-based arrestin recruitment assays, and examples for the application of such systems in dose-response or time-course experiments. Suggestions are given for troubleshooting, optimizing test systems, and the interpretation of results obtained with BRET-based assays, which indeed yield an intricate blend of quantitative and qualitative information.

  1. Noninvasive bioluminescence imaging of the dynamics of sanguinarine induced apoptosis via activation of reactive oxygen species.

    Science.gov (United States)

    Wang, Yan; Zhang, Beilei; Liu, Wei; Dai, Yunpeng; Shi, Yaru; Zeng, Qi; Wang, Fu

    2016-04-19

    Most chemotherapeutic drugs exert their anti-tumor effects primarily by triggering a final pathway leading to apoptosis. Noninvasive imaging of apoptotic events in preclinical models would greatly facilitate the development of apoptosis-inducing compounds and evaluation of their therapeutic efficacy. Here we employed a cyclic firefly luciferase (cFluc) reporter to screen potential pro-apoptotic compounds from a number of natural agents. We demonstrated that sanguinarine (SANG) could induce apoptosis in a dose- and time-dependent manner in UM-SCC-22B head and neck cancer cells. Moreover, SANG-induced apoptosis was associated with the generation of reactive oxygen species (ROS) and activation of c-Jun-N-terminal kinase (JNK) and nuclear factor-kappaB (NF-κB) signal pathways. After intravenous administration with SANG in 22B-cFluc xenograft models, a dramatic increase of luminescence signal can be detected as early as 48 h post-treatment, as revealed by longitudinal bioluminescence imaging in vivo. Remarkable apoptotic cells reflected from ex vivo TUNEL staining confirmed the imaging results. Importantly, SANG treatment caused distinct tumor growth retardation in mice compared with the vehicle-treated group. Taken together, our results showed that SANG is a candidate anti-tumor drug and noninvasive imaging of apoptosis using cFluc reporter could provide a valuable tool for drug development and therapeutic efficacy evaluation.

  2. Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases.

    Science.gov (United States)

    Viviani, V R; Arnoldi, F G C; Venkatesh, B; Neto, A J S; Ogawa, F G T; Oehlmeyer, A T L; Ohmiya, Y

    2006-10-01

    The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

  3. Molecular basis for the blue bioluminescence of the Australian glow-worm Arachnocampa richardsae (Diptera: Keroplatidae).

    Science.gov (United States)

    Trowell, Stephen C; Dacres, Helen; Dumancic, Mira M; Leitch, Virginia; Rickards, Rodney W

    2016-09-16

    Bioluminescence is the emission of visible light by living organisms. Here we describe the isolation and characterisation of a cDNA encoding a MW ≈ 59,000 Da luciferase from the Australian glow-worm, Arachnocampa richardsae. The enzyme is a member of the acyl-CoA ligase superfamily and produces blue light on addition of D-luciferin. These results are contrary to earlier reports (Lee, J., Photochem Photobiol 24, 279-285 (1976), Viviani, V. R., Hastings, J. W. & Wilson, T., Photochem Photobiol 75, 22-27 (2002)), which suggested glow-worm luciferase has MW ≈ 36,000 Da and is unreactive with beetle luciferin. There are more than 2000 species of firefly, which all produce emissions from D-luciferin in the green to red regions of the electromagnetic spectrum. Although blue-emitting luciferases are known from marine organisms, they belong to different structural families and use a different substrate. The observation of blue emission from a D-luciferin-using enzyme is therefore unprecedented.

  4. Light and vision in the deep-sea benthos: I. Bioluminescence at 500-1000 m depth in the Bahamian islands.

    Science.gov (United States)

    Johnsen, Sönke; Frank, Tamara M; Haddock, Steven H D; Widder, Edith A; Messing, Charles G

    2012-10-01

    Bioluminescence is common and well studied in mesopelagic species. However, the extent of bioluminescence in benthic sites of similar depths is far less studied, although the relatively large eyes of benthic fish, crustaceans and cephalopods at bathyal depths suggest the presence of significant biogenic light. Using the Johnson-Sea-Link submersible, we collected numerous species of cnidarians, echinoderms, crustaceans, cephalopods and sponges, as well as one annelid from three sites in the northern Bahamas (500-1000 m depth). Using mechanical and chemical stimulation, we tested the collected species for light emission, and photographed and measured the spectra of the emitted light. In addition, in situ intensified video and still photos were taken of different benthic habitats. Surprisingly, bioluminescence in benthic animals at these sites was far less common than in mesopelagic animals from similar depths, with less than 20% of the collected species emitting light. Bioluminescent taxa comprised two species of anemone (Actinaria), a new genus and species of flabellate Parazoanthidae (formerly Gerardia sp.) (Zoanthidea), three sea pens (Pennatulacea), three bamboo corals (Alcyonacea), the chrysogorgiid coral Chrysogorgia desbonni (Alcyonacea), the caridean shrimp Parapandalus sp. and Heterocarpus ensifer (Decapoda), two holothuroids (Elasipodida and Aspidochirota) and the ophiuroid Ophiochiton ternispinus (Ophiurida). Except for the ophiuroid and the two shrimp, which emitted blue light (peak wavelengths 470 and 455 nm), all the species produced greener light than that measured in most mesopelagic taxa, with the emissions of the pennatulaceans being strongly shifted towards longer wavelengths. In situ observations suggested that bioluminescence associated with these sites was due primarily to light emitted by bioluminescent planktonic species as they struck filter feeders that extended into the water column.

  5. Analytical performance issues: comparison of ATP bioluminescence and aerobic bacterial count for evaluating surface cleanliness in an Italian hospital.

    Science.gov (United States)

    Amodio, Emanuele; Cannova, Lucia; Villafrate, Maria Rosaria; Merendino, Anna Maria; Aprea, Luigi; Calamusa, Giuseppe

    2014-01-01

    Contaminated hospital surfaces have been demonstrated to be an important environmental reservoir of microorganisms that can increase the risk of nosocomial infection in exposed patients. As a consequence, cleaning and disinfecting hospital environments play an important role among strategies for preventing healthcare-associated colonization and infections. The aim of the present study was to evaluate whether adenosine triphosphate (ATP) presence, measured by bioluminescence methods, can predict microbiological contamination of hospital surfaces. The study was carried out between September and December 2012 at the University Hospital "P. Giaccone" of Palermo. A total of 193 randomly selected surfaces (tables, lockers, furnishings) were sampled and analyzed in order to assess ATP levels (expressed as relative light units or RLU) and aerobic colony count (ACC) or presence of S. aureus. ACC had median values of 1.85 cfu/cm(2)(interquartile range = 4.16) whereas ATP median was 44.6 RLU/cm(2)(interquartile range = 92.3). Overall, 85 (44.0%) surfaces exceeded the established microbial benchmark: 73 (37.8%) exceeded the 2.5 cfu/cm(2)ACC standard, 5 (2.6%) surfaces were positive for S. aureus and 7 (3.6%) showed both the presence of S. aureus and an ACC of more than 2.5 cfu/cm(2). ACC and bioluminescence showed significant differences in the different surface sites (p < 0.001). A significant correlation was found between ACC and RLU values (p-value < 0.001; R(2)= 0.29) and increasing RLU values were significantly associated with a higher risk of failing the benchmark (p < 0.001). Our data suggest that bioluminescence could help in measuring hygienic quality of hospital surfaces using a quick and sensitive test that can be an useful proxy of microbial contamination; however, further analysis will be necessary to assess the cost-efficacy of this methodology before requiring incorporation in hospital procedures.

  6. Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.

    Directory of Open Access Journals (Sweden)

    Adil Bakayan

    Full Text Available Bioluminescence recording of Ca(2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET to the green fluorescent protein (GFP. This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+ in various cell compartments. In addition, they would also serve to monitor Ca(2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA showed the highest BRET efficiency (largest energy transfer critical distance R(0 and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+ oscillations in single HeLa cells expressing tdTA. Ca(2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+ activity of HeLa cells injected subcutaneously into mice, and Ca(2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+ sensor reported to date and is, therefore, a promising probe to study Ca(2+ dynamics in whole organisms or tissues expressing the transgene.

  7. The detection of food soils and cells on stainless steel using industrial methods: UV illumination and ATP bioluminescence.

    Science.gov (United States)

    Whitehead, Kathryn A; Smith, Lindsay A; Verran, Joanna

    2008-09-30

    Open food contact surfaces were subjected to organic soiling to provide a source for transfer of microbial cells. Rapid industrial methods used for the detection of residual cells and soil e.g. ATP (adenosine triphosphate) bioluminescence and an ultraviolet (UV) light detection method were assessed for their ability to detect organic soils, or organic soil-cell mix on surfaces. A range of soils (complex [meat extract, fish extract, cottage cheese extract]; oils [cholesterol, fish oil, mixed fatty acids]; proteins [bovine serum albumin, fish peptones casein]; carbohydrates [glycogen, starch, lactose]); was used. Under UV, oily soils, mixed fatty acids, cholesterol and casein were detected at low concentrations, with detection levels ranging from 1% to 0.001% for different substances. Glycogen was the most difficult substance to detect at lower concentrations. Using UV wavelength bands (lambda) of 330-380 nm, 510-560 nm and 590-650 nm, wavelength bands of 330-380 nm, illuminated most of the soils well, whilst the wavelength band of 510-560 nm illuminated the fish extract, cholesterol and fatty acids; the 590-650 nm wavelength band illuminated the lactose. Soils at all concentrations were detected by the ATP bioluminescence method; the complex soils gave the highest readings. When complex soils were combined with Listeria monocytogenes Scott A or a non-pathogenic Escherichia coli O157:H7, ATP measurements increased by 1-2 logs. For UV illumination, the L. monocytogenes and cheese combination was the most intensely illuminated, with E. coli and meat the least. UV illumination is a simple well established method for detecting food soil, with little change in findings when microorganisms are included. Performance can be enhanced in certain circumstances by altering the wavelength. ATP bioluminescence is a proven system for hygienic assessment being especially useful in the presence of microorganisms rather than organic soil alone.

  8. Ultra-thin titanium nanolayers for plasmon-assisted enhancement of bioluminescence of chloroplast in biological light emitting devices

    Energy Technology Data Exchange (ETDEWEB)

    Hsun Su, Yen [Department of Materials Science and Engineering, National Cheng Kung University, Tainan 70101, Taiwan (China); Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan 70101, Taiwan (China); Hsu, Chia-Yun; Chang, Chung-Chien [Science and Technology of Accelerator Light Source, Hsinchu 300, Taiwan (China); Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu 300, Taiwan (China); Tu, Sheng-Lung; Shen, Yun-Hwei [Department of Resource Engineering, National Cheng Kung University, Tainan 70101, Taiwan (China)

    2013-08-05

    Ultra-thin titanium films were deposited via ultra-high vacuum ion beam sputter deposition. Since the asymmetric electric field of the metal foil plane matches the B-band absorption of chlorophyll a, the ultra-thin titanium nanolayers were able to generate surface plasmon resonance, thus enhancing the photoluminescence of chlorophyll a. Because the density of the states of plasmon resonance increases, the enhancement of photoluminescence also rises. Due to the biocompatibility and inexpensiveness of titanium, it can be utilized to enhance the bioluminescence of chloroplast in biological light emitting devices, bio-laser, and biophotonics.

  9. Design Optimization and Evaluation of a Bioluminescence Detection Part on a Microfluidic Device for in situ ATP Quantification

    Science.gov (United States)

    Aoki, Yusuke; Fukuba, Tatsuhiro; Yamamoto, Takatoki; Fujii, Teruo

    An integrated in situ analyzer for microbial ATP (IISA-ATP) has been developed with a microfluidic device as its core component to realize a compact and fully integrated system. In the system, a bioluminescence (luciferin—luciferase) reaction is conducted for ATP quantification. The microfluidic device has a coil-shaped microchannel for highly sensitive photo intensity measurement. In this paper, the concept of the IISA-ATP and optimization of the microchannel design to enhance sensitivity are presented. As a result of the optimization, linear correlation of the luminescence intensity with the ATP concentration in the range of 2 to 2 × 104 pM was achieved.

  10. Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

    Science.gov (United States)

    Hattori, Mitsuru; Ozawa, Takeaki

    2016-01-01

    G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and β-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner.

  11. Ultra-thin titanium nanolayers for plasmon-assisted enhancement of bioluminescence of chloroplast in biological light emitting devices

    Science.gov (United States)

    Hsun Su, Yen; Hsu, Chia-Yun; Chang, Chung-Chien; Tu, Sheng-Lung; Shen, Yun-Hwei

    2013-08-01

    Ultra-thin titanium films were deposited via ultra-high vacuum ion beam sputter deposition. Since the asymmetric electric field of the metal foil plane matches the B-band absorption of chlorophyll a, the ultra-thin titanium nanolayers were able to generate surface plasmon resonance, thus enhancing the photoluminescence of chlorophyll a. Because the density of the states of plasmon resonance increases, the enhancement of photoluminescence also rises. Due to the biocompatibility and inexpensiveness of titanium, it can be utilized to enhance the bioluminescence of chloroplast in biological light emitting devices, bio-laser, and biophotonics.

  12. Stably luminescent Staphylococcus aureus clinical strains for use in bioluminescent imaging.

    Directory of Open Access Journals (Sweden)

    Roger D Plaut

    Full Text Available In vivo bioluminescent imaging permits the visualization of bacteria in live animals, allowing researchers to monitor, both temporally and spatially, the progression of infection in each animal. We sought to engineer stably luminescent clinical strains of Staphylococcus aureus, with the goal of using such strains in mouse models. The gram-positive shuttle vector pMAD was used as the backbone for an integration plasmid. A chloramphenicol resistance gene, a modified lux operon from Photorhabdus luminescens, and approximately 650 bp of homology to the chromosome of the USA300 S. aureus strain NRS384 were added, generating plasmid pRP1195. Electroporation into strain RN4220 followed by temperature shift led to integration of pRP1195 into the chromosome. The integrated plasmid was transferred to clinical strains by phage transduction. Luminescent strains displayed no in vitro growth defects. Moreover, luminescence was stable in vitro after three rounds of subculture over 48 hours of growth in the absence of antibiotics. Mice were infected with a luminescent strain of NRS384 in skin and intravenous models. In a mouse skin model, luminescent bacteria were present in lesions that formed and cleared over the course of several days, and in an intravenous model, bacteria inoculated in the mouse tail vein were observed spreading to multiple tissues. No statistically significant difference in virulence was observed between NRS384 and the luminescent strain in either infection model. These preliminary data suggest that this luminescent USA300 strain is suitable for use in mouse models. Similar strains were engineered using other sequenced clinical strains. Because these strains are stably luminescent, they should prove useful in animal models of infection.

  13. Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.

    Directory of Open Access Journals (Sweden)

    Nan Wu

    Full Text Available BACKGROUND: Fluorescence and bioluminescence resonance energy transfer (F/BRET are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now. METHODOLOGY: We used a thrombin bioprobe based on a form of BRET (BRET(H, which uses the BRET(1 substrate, native coelenterazine, with the typical BRET(2 donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRET(H ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay. PRINCIPAL FINDINGS: The BRET(H thrombin bioprobe has a lower limit of detection (LOD than previously reported for the equivalent BRET(1-based version but it is substantially brighter than the BRET(2 version. The normalised BRET(H ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays. CONCLUSIONS: These data demonstrate that BRET based microfluidic assays are feasible and that BRET(H provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.

  14. Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes.

    Science.gov (United States)

    Kamidate, Tamio; Yanashita, Kenji; Tani, Hirofumi; Ishida, Akihiko; Notani, Mizuyo

    2006-01-01

    Liposomes containing phosphatidylcholine (PC) and cholesterol (Chol) were applied to the enhancer for firefly bioluminescence (BL) assay for ATP in the presence of cationic surfactants using as an extractant for the release of ATP from living cells. Benzalkonium chloride (BAC) was used as an ATP extractant. However, BAC seriously inhibited the activity of luciferase, thus resulting in the remarkable decrease in the sensitivity of the BL assay for ATP. On the other hand, we found that BAC was associated with liposomes to form cationic liposomes containing BAC. The association rate of BAC with liposomes was faster than that of BAC with luciferase. As a result, the inhibitory effect of BAC on luciferase was eliminated in the presence of liposomes. In addition, cationic liposomes thus formed enhanced BL emission. BL measurement conditions were optimized in terms of liposome charge type, liposome size, and total concentration of PC and Chol. ATP can be sensitively determined without dilution of analytical samples by using liposomes. The detection limit of ATP with and without liposomes was 100 amol and 25 fmol in aqueous ATP standard solutions containing 0.06% BAC, respectively. The method was applied to the determination of ATP in Escherichia coli extracts. The BL intensity was linear from 4 x 10(4) to 1 x 10(7) cells mL(-1) in the absence of liposomes. On the other hand, the BL intensity was linear from 4 x 10(3) to 4 x 10(6) cells mL(-1) in the presence of liposomes. The detection limit of ATP in E. coli extracts was improved by a factor of 10 via use of liposomes.

  15. Bioluminescent imaging and histopathologic characterization of WEEV neuroinvasion in outbred CD-1 mice.

    Directory of Open Access Journals (Sweden)

    Aaron T Phillips

    Full Text Available Western equine encephalitis virus (WEEV; Alphavirus is a mosquito-borne virus that can cause severe encephalitis in humans and equids. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan (McM strain result in high mortality within 4 days of infection. Here in vivo and ex vivo bioluminescence (BLM imaging was applied on mice intranasally infected with a recombinant McM virus expressing firefly luciferase (FLUC to track viral neuroinvasion by FLUC detection and determine any correlation between BLM and viral titer. Immunological markers of disease (MCP-1 and IP-10 were measured and compared to wild type virus infection. Histopathology was guided by corresponding BLM images, and showed that neuroinvasion occurred primarily through cranial nerves, mainly in the olfactory tract. Olfactory bulb neurons were initially infected with subsequent spread of the infection into different regions of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few infected glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem. The recombinant virus WEEV.McM.FLUC had attenuated replication kinetics and induced a weaker immunological response than WEEV.McM but produced comparable pathologies. Immunohistochemistry staining for FLUC and WEEV antigen showed that transgene expression was present in all areas of the CNS where virus was observed. BLM provides a quantifiable measure of alphaviral neural disease progression and a method for evaluating antiviral strategies.

  16. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  17. An efficient reconstruction method for bioluminescence tomography based on two-step iterative shrinkage approach

    Science.gov (United States)

    Guo, Wei; Jia, Kebin; Tian, Jie; Han, Dong; Liu, Xueyan; Wu, Ping; Feng, Jinchao; Yang, Xin

    2012-03-01

    Among many molecular imaging modalities, Bioluminescence tomography (BLT) is an important optical molecular imaging modality. Due to its unique advantages in specificity, sensitivity, cost-effectiveness and low background noise, BLT is widely studied for live small animal imaging. Since only the photon distribution over the surface is measurable and the photo propagation with biological tissue is highly diffusive, BLT is often an ill-posed problem and may bear multiple solutions and aberrant reconstruction in the presence of measurement noise and optical parameter mismatches. For many BLT practical applications, such as early detection of tumors, the volumes of the light sources are very small compared with the whole body. Therefore, the L1-norm sparsity regularization has been used to take advantage of the sparsity prior knowledge and alleviate the ill-posedness of the problem. Iterative shrinkage (IST) algorithm is an important research achievement in a field of compressed sensing and widely applied in sparse signal reconstruction. However, the convergence rate of IST algorithm depends heavily on the linear operator. When the problem is ill-posed, it becomes very slow. In this paper, we present a sparsity regularization reconstruction method for BLT based on the two-step iterated shrinkage approach. By employing Two-step strategy of iterative reweighted shrinkage (IRS) to improve IST, the proposed method shows faster convergence rate and better adaptability for BLT. The simulation experiments with mouse atlas were conducted to evaluate the performance of proposed method. By contrast, the proposed method can obtain the stable and comparable reconstruction solution with less number of iterations.

  18. ATP-Binding Cassette Transporters Modulate Both Coelenterazine- and D-Luciferin-Based Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Ruimin Huang

    2011-05-01

    Full Text Available Bioluminescence imaging (BLI of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC transporters on BLI readout, we generated click beetle (cLuc, firefly (fLuc, Renilla (rLuc, and Gaussia (gLuc luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2. In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type–independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.

  19. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

    Science.gov (United States)

    Duellman, Sarah J; Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta

    2015-10-01

    Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

  20. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    Science.gov (United States)

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke.

  1. Bright mutants of Vibrio fischeri ES114 reveal conditions and regulators that control bioluminescence and expression of the lux operon.

    Science.gov (United States)

    Lyell, Noreen L; Dunn, Anne K; Bose, Jeffrey L; Stabb, Eric V

    2010-10-01

    Vibrio fischeri ES114, an isolate from the Euprymna scolopes light organ, produces little bioluminescence in culture but is ∼1,000-fold brighter when colonizing the host. Cell-density-dependent regulation alone cannot explain this phenomenon, because cells within colonies on solid medium are much dimmer than symbiotic cells despite their similar cell densities. To better understand this low luminescence in culture, we screened ∼20,000 mini-Tn5 mutants of ES114 for increased luminescence and identified 28 independent "luminescence-up" mutants with insertions in 14 loci. Mutations affecting the Pst phosphate uptake system led to the discovery that luminescence is upregulated under low-phosphate conditions by PhoB, and we also found that ainS, which encodes an autoinducer synthase, mediates repression of luminescence during growth on plates. Other novel luminescence-up mutants had insertions in acnB, topA, tfoY, phoQ, guaB, and two specific tRNA genes. Two loci, hns and lonA, were previously described as repressors of bioluminescence in transgenic Escherichia coli carrying the light-generating lux genes, and mutations in arcA and arcB were consistent with our report that Arc represses lux. Our results reveal a complex regulatory web governing luminescence and show how certain environmental conditions are integrated into regulation of the pheromone-dependent lux system.

  2. [Obtaining of bacterial bioluminescent strain Protobacterium phosphoreum B7071 (lux+) for the determination of zinc ion concentration].

    Science.gov (United States)

    Gruzina, T G; Dybkova, S N; Chekhovskaia, T P; Vember, V V; Zadorozhniaia, A M; Ul'berg, Z R

    2006-01-01

    The transconjugate containing hybrid plasmid (Te(R)Zn(R)lux+) was obtained using the conjugation method on Pseudomonasfragi T2(5) (Te(R)ZnR) strain and bioluminescent strain Protobacterium phosphoreum B7071 (lux+). The expression regulation of lux-genes on the obtained plasmid is carried out by promotor-operational area conjointly with zinc resistance genes. The cells of the obtained genetically modified bacteria have the ability to specific induced luminescence, which is a respond to zinc ions' presence in the measuring medium. It was shown that the cells' bioluminescence intensity of trans-conjugate is linearly dependent on zinc ions' concentration within the range of 1-100 microM, that provides the opportunity of using biosensor as a strain for qualitative and quantitative detection of the metal. The low sensitivity limit of this method is 0.5 microM for the metal. Besides having high sensitivity, the developed lux-biosensor is highly specified.

  3. A model system for pathogen detection using a two-component bacteriophage/bioluminescent signal amplification assay

    Science.gov (United States)

    Bright, Nathan G.; Carroll, Richard J.; Applegate, Bruce M.

    2004-03-01

    Microbial contamination has become a mounting concern the last decade due to an increased emphasis of minimally processed food products specifically produce, and the recognition of foodborne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes. This research investigates a detection approach utilizing bacteriophage pathogen specificity coupled with a bacterial bioluminescent bioreporter utilizing the quorum sensing molecule from Vibrio fischeri, N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL). The 3-oxo-C6-HSL molecules diffuse out of the target cell after infection and induce bioluminescence from a population of 3-oxo-C6-HSL bioreporters (ROLux). E. coli phage M13, a well-characterized bacteriophage, offers a model system testing the use of bacteriophage for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains. These assays showed detection limits of 102cfu after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105cfu target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time.

  4. Non-invasive Bioluminescence Imaging of Myoblast-Mediated Hypoxia-Inducible Factor-1 Alpha Gene Transfer

    Science.gov (United States)

    Gheysens, Olivier; Chen, Ian Y.; Rodriguez-Porcel, Martin; Chan, Carmel; Rasooly, Julia; Vaerenberg, Caroline; Paulmurugan, Ramasamy; Willmann, Juergen K.; Deroose, Christophe; Wu, Joseph; Gambhir, Sanjiv S.

    2011-01-01

    Purpose We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1α (HIF-1α-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively. Procedures The plasmid pUbi-hrl-pUbi-HIF-1α-VP2, which expresses both hrl and HIF-1α-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1α-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI). Results Strong correlations existed between the expression of hRL and each of HIF-1α-VP2, VEGF, and PlGF (r2>0.83, r2>0.82, and r2>0.97, respectively). In vivo, both transplanted cells and HIF-1α-VP2 transgene expression were successfully imaged using BLI. Conclusions An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein. PMID:21267661

  5. Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

    Directory of Open Access Journals (Sweden)

    Tetsuya Kadonosono

    Full Text Available An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1 is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg mice that carry HRE/ODD-luciferase (HOL gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.

  6. Yearlong moored bioluminescence and current data at KM3NeT neutrino telescope sites in the deep Ionian Sea

    Science.gov (United States)

    van Haren, Hans; de Jong, Maarten; Kooijman, Paul

    2015-07-01

    Yearlong observations are presented using stand-alone small optical sensors and current meters in the deep Ionian Sea, E-Mediterranean. At two future neutrino telescope sites, off Sicily (I) and off Peloponessos (Gr), we deployed 2500-3000 m long mooring lines with oceanographic instrumentation. At about 150 m above the sea-floor, a glass sphere was mounted to each line holding two 3″-diameter photo-multiplier-tubes 'PMTs' in opposing directions for a first deep-sea test. Due to technical problems the background optical count rate could not be well established. Here, the focus is on the variations with time of bioluminescence bursts and their correlation with currents. Spectral analysis demonstrates that the PMT data best resemble those of horizontal currents (kinetic energy), significantly peaking at near-inertial, sub-inertial mesoscale and (Gr only) at tidal frequencies. Out-of-phase differences between signals from opposing PMTs in the same optical unit indicate impacts of bioluminescent organisms as a function of current direction, rather than a bacterial glow constant with time.

  7. An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications.

    Science.gov (United States)

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Kohrt, Dawn; Talukder, Munya; Michelini, Elisa; Cevenini, Luca; Roda, Aldo; Grossel, Martha J

    2015-09-01

    Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications.

  8. Use of ATP bioluminescence for assessing the cleanliness of hospital surfaces: a review of the published literature (1990-2012).

    Science.gov (United States)

    Amodio, Emanuele; Dino, Claudia

    2014-01-01

    Hospital cleanliness tends to be considered by patients and the public as an important indicator of the general quality of healthcare. Tests for detecting the presence of adenosine triphosphate (ATP) as a proxy of microbial contamination are increasing in popularity, and several studies have been conducted on this topic in the last few decades. The aim of the present study was to review the published literature on this topic and summarize and discuss the available results. The review focused on relevant English-language articles that were identified through searches of two databases [PubMed and Scopus (1990-2012)] by using the keywords "ATP", "bioluminescence", "hospital", and "surfaces". Twelve articles were included and analyzed. ATP measurements showed a wide variation, with values ranging from 0 to >500,000 relative light units (RLU)/s before cleaning and from 3 to 500,000RLU/s after cleaning. ATP benchmarks used by authors ranged from 100 to 500RLU/s. The percentage of surfaces exceeding the chosen cut-off limit showed a failure rate varying from 21.2% to 93.1% before cleaning and from 5.3% to 96.5% after cleaning. Although the use of ATP bioluminescence can be considered a quick and objective method for assessing hospital cleanliness, it appears to be still poorly standardized at both the national and international level.

  9. Comprehensive assessment of host responses to ionizing radiation by nuclear factor-κB bioluminescence imaging-guided transcriptomic analysis.

    Directory of Open Access Journals (Sweden)

    Chung-Ta Chang

    Full Text Available The aim of this study was to analyze the host responses to ionizing radiation by nuclear factor-κB (NF-κB bioluminescence imaging-guided transcriptomic tool. Transgenic mice carrying the NF-κB-driven luciferase gene were exposed to a single dose of 8.5 Gy total-body irradiation. In vivo imaging showed that a maximal NF-κB-dependent bioluminescent intensity was observed at 3 h after irradiation and ex vivo imaging showed that liver, intestine, and brain displayed strong NF-κB activations. Microarray analysis of these organs showed that irradiation altered gene expression signatures in an organ-specific manner and several pathways associated with metabolism and immune system were significantly altered. Additionally, the upregulation of fatty acid binding protein 4, serum amyloid A2, and serum amyloid A3 genes, which participate in both inflammation and lipid metabolism, suggested that irradiation might affect the cross pathways of metabolism and inflammation. Moreover, the alteration of chemokine (CC-motif ligand 5, chemokine (CC-motif ligand 20, and Jagged 1 genes, which are involved in the inflammation and enterocyte proliferation, suggested that these genes might be involved in the radiation enteropathy. In conclusion, this report describes the comprehensive evaluation of host responses to ionizing radiation. Our findings provide the fundamental information about the in vivo NF-κB activity and transcriptomic pattern after irradiation. Moreover, novel targets involved in radiation injury are also suggested.

  10. Evaluation of impaired beta-cell function in nonobese-diabetic (NOD) mouse model using bioluminescence imaging.

    Science.gov (United States)

    Sever, Dror; Eldor, Roy; Sadoun, Gadi; Amior, Livnat; Dubois, Daniele; Boitard, Christian; Aflalo, Claude; Melloul, Danielle

    2011-02-01

    Insulin-producing pancreatic β cells are functionally impaired or destroyed in diabetes mellitus. The onset of type 1 diabetes (T1D) represents the culmination of a prolonged prediabetic phase of immune-mediated β-cell destruction. To assess the in vivo metabolic status of these cells, we used the ATP-sensitive firefly luciferase bioluminescence imaging approach, as a noninvasive probe to monitor pathological alterations in β-cell function in the nonobese-diabetic (NOD) mouse model of T1D. Hence, we generated the ToIβ-NOD transgenic mice in which doxycycline-inducible luciferase gene is selectively expressed in β cells. A sharp reduction in bioluminescence emitted in vivo from β cells at the early stages, preceded by several weeks of a limited reduction in β-cell mass. Since this decline could be due to the ongoing inflammatory process occurring in vivo, we exposed control islets to inflammatory cytokines and observed a dramatic decrease in luciferase luminescence, which appears to be due in part to a decrease in protein levels and a drop in intracellular ATP levels. This is the first evidence that selective expression of the luciferase gene represents a sensitive method for noninvasive in vivo monitoring of early β-cell dysfunction, subtle metabolic changes, such as endogenous ATP levels, indicative of a pathological condition in a tissue at the cellular level.

  11. Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models.

    Science.gov (United States)

    Cosette, Jeremie; Ben Abdelwahed, Rym; Donnou-Triffault, Sabrina; Sautès-Fridman, Catherine; Flaud, Patrice; Fisson, Sylvain

    2016-07-07

    Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

  12. Dual-color bioluminescence imaging assay using green- and red-emitting beetle luciferases at subcellular resolution.

    Science.gov (United States)

    Yasunaga, Mayu; Nakajima, Yoshihiro; Ohmiya, Yoshihiro

    2014-09-01

    Bioluminescence imaging is widely used to monitor cellular events, including gene expression in vivo and in vitro. Moreover, recent advances in luciferase technology have made possible imaging at the single-cell level. To improve the bioluminescence imaging system, we have developed a dual-color imaging system in which the green-emitting luciferase from a Brazilian click beetle (Emerald Luc, ELuc) and the red-emitting luciferase from a railroad worm (Stable Luciferase Red, SLR) were used as reporters, which were localized to the peroxisome and the nucleus, respectively. We clearly captured simultaneously the subcellular localization of ELuc in the peroxisome and SLR in the nucleus of a single cell using a high-magnification objective lens with 3-min exposure time without binning using a combination of optical filters. Furthermore, to apply this system to quantitative time-lapse imaging, the activation of nuclear factor triggered by tumor necrosis factor α was measured using nuclear-targeted SLR and peroxisome-targeted ELuc as the test and internal control reporters, respectively. We successfully quantified the kinetics of activation of nuclear factor κB using nuclear-targeted SLR and the transcriptional change of the internal control promoter using peroxisome-targeted ELuc simultaneously in a single cell, and showed that the activation kinetics, including activation rate and amplitude, differed among cells. The results demonstrated that this imaging system can visualize the subcellular localization of reporters and track the expressions of two genes simultaneously at subcellular resolution.

  13. Construction of mobilizable mini-Tn7 vectors for bioluminescent detection of gram-negative bacteria and single-copy promoter lux reporter analysis.

    Science.gov (United States)

    Damron, F Heath; McKenney, Elizabeth S; Barbier, Mariette; Liechti, George W; Schweizer, Herbert P; Goldberg, Joanna B

    2013-07-01

    We describe the construction of mini-Tn7-based broad-host-range vectors encoding lux genes as bioluminescent reporters. These constructs can be mobilized into the desired host(s) by conjugation for chromosomal mini-Tn7-lux integration and are useful for localization of bacteria during infections or for characterizing regulation of promoters of interest in Gram-negative bacteria.

  14. A Bone Metastasis Nude Mouse Model Created by Ultrasound Guided Intracardiac Injection of Breast Cancer Cells: the Micro-CT, MRI and Bioluminescence Imaging Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Park, Young Jin; Song, Eun Hye; Kim, Seol Hwa; Song, Ho Taek; Suh, Jin Suck [Yonsei University College of Medicine, Seoul (Korea, Republic of); Choi, Sang Hyun [Korean Minjok Leadership Academy, Heongsung (Korea, Republic of)

    2011-01-15

    The purpose of this study was to develop a nude mouse model of bone metastasis by performing intracardiac injection of breast cancer cells under ultrasonography guidance and we wanted to evaluate the development and the distribution of metastasis in vivo using micro-CT, MRI and bioluminescence imaging. Animal experiments were performed in 6-week-old female nude mice. The animals underwent left ventricular injection of 2x105 MDA-MB-231Bo-Luc cells. After injection of the tumor cells, serial bioluminescence imaging was performed for 7 weeks. The findings of micro-CT, MRI and the histology were correlated with the 'hot' lesions seen on the bioluminescence imaging. Metastasis was found in 62.3% of the animals. Two weeks after intracardiac injection, metastasis to the brain, spine and femur was detected with bioluminescence imaging with an increasing intensity by week 7. Micro-CT scan confirmed multiple osteolytic lesions at the femur, spine and skull. MRI and the histology were able to show metastasis in the brain and extraskeletal metastasis around the femur. The intracardiac injection of cancer cells under ultrasonography guidance is a safe and highly reproducible method to produce bone metastasis in nude mice. This bone metastasis nude mouse model will be useful to study the mechanism of bone metastasis and to validate new therapeutics

  15. Green-Fluorescent Protein from the Bioluminescent Jellyfish Clytia gregaria Is an Obligate Dimer and Does Not Form a Stable Complex with the Ca2+-Discharged Photoprotein Clytin.

    NARCIS (Netherlands)

    Malikova, N.P.; Visser, N.V.; Hoek, van A.; Skakun, V.V.; Vysotski, E.S.; Lee, J.; Visser, A.J.W.G.

    2011-01-01

    Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo¨rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at mi

  16. A Bioluminescent Whole-Cell Reporter for Detection of 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol in Soil

    Science.gov (United States)

    Hay, Anthony G.; Rice, James F.; Applegate, Bruce M.; Bright, Nathan G.; Sayler, Gary S.

    2000-01-01

    A bioreporter was made containing a tfdRPDII-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (R2 = 0.9825) in the range of 2.0 μM to 1.1 × 102 μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 102 μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues. PMID:11010925

  17. Ultrafast fluorescence relaxation spectroscopy of 6,7-dimethyl-(8-ribityl)-lumazine and riboflavin, free and bound to antenna proteins from bioluminescent bacteria

    NARCIS (Netherlands)

    Petushkov, V.N.; Stokkum, van I.H.M.; Gobets, B.; Mourik, van F.; Lee, J.; Grondelle, van R.; Visser, A.J.W.G.

    2003-01-01

    The solvation dynamics of interesting bioluminescent chromophores have been determined, using subpicosecond and wavelength-resolved fluorescence spectroscopy, in combination with global analysis of the multidimensional data sets. The systems investigated comprise the free ligands 6,7-dimethyl-(8-rib

  18. Exploiting in vitro and in vivo bioluminescence for the implementation of the three Rs principle (replacement, reduction, and refinement) in drug discovery.

    Science.gov (United States)

    Michelini, Elisa; Cevenini, Luca; Calabretta, Maria Maddalena; Calabria, Donato; Roda, Aldo

    2014-09-01

    Bioluminescence-based analytical tools are suitable for high-throughput and high-content screening assays, finding widespread application in several fields related to the drug discovery process. Cell-based bioluminescence assays, because of their peculiar advantages of predictability, possibility of automation, multiplexing, and miniaturization, seem the most appealing tool for the high demands of the early stages of drug screening. Reporter gene technology and the bioluminescence resonance energy transfer principle are widely used, and receptor binding studies of new agonists/antagonists for a variety of human receptors expressed in different cell lines can be performed. Moreover, bioluminescence can be used for in vitro and in vivo real-time monitoring of pathophysiological processes within living cells and small animals. New luciferases and substrates have recently arrived on the market, further expanding the spectrum of applications. A new generation of probes are also emerging that promise to revolutionize the preclinical imaging market. This formidable toolbox is demonstrated to facilitate the implementation of the three Rs principle in the early drug discovery process, in compliance with ethical and responsible research to reduce cost and improve the reliability and predictability of results.

  19. Noninvasive monitoring of placenta-specific transgene expression by bioluminescence imaging.

    Directory of Open Access Journals (Sweden)

    Xiujun Fan

    Full Text Available BACKGROUND: Placental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts. METHODOLOGY/PRINCIPAL FINDINGS: Zona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3. Animals were examined for Fluc expression by live bioluminescence imaging (BLI at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm(2/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages. CONCLUSIONS/SIGNIFICANCE: These results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early

  20. Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Burakova, Ludmila P.; Natashin, Pavel V.; Markova, Svetlana V.; Eremeeva, Elena V.; Malikova, Natalia P.; Cheng, Chongyun; Liu, Zhi-Jie; Vysotski, Eugene S.

    2016-09-01

    The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.

  1. Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein.

    Science.gov (United States)

    Burakova, Ludmila P; Natashin, Pavel V; Markova, Svetlana V; Eremeeva, Elena V; Malikova, Natalia P; Cheng, Chongyun; Liu, Zhi-Jie; Vysotski, Eugene S

    2016-09-01

    The full-length cDNA genes encoding five new isoforms of Ca(2+)-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473-474nm with no shoulder at 400nm). Fluorescence spectra of Ca(2+)-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca(2+)-discharged aequorin, but different from Ca(2+)-discharged obelins and clytin which fluorescence is red-shifted by 25-30nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.

  2. The cAMP-dependent protein kinase inhibitor H-89 attenuates the bioluminescence signal produced by Renilla Luciferase.

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    Katie J Herbst

    Full Text Available BACKGROUND: Investigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells. These cell-based assays are often utilized to test the involvement of PKA-dependent processes by using H-89, a reversible competitive inhibitor of PKA. PRINCIPAL FINDINGS: We present here data to show that H-89, in addition to being a competitive PKA inhibitor, attenuates the bioluminescence signal produced by Renilla luciferase (RLuc variants in a population of cells and also in single cells. Using 10 microM of luciferase substrate and 10 microM H-89, we observed that the signal from RLuc and RLuc8, an eight-point mutation variant of RLuc, in cells was reduced to 50% (+/-15% and 54% (+/-14% of controls exposed to the vehicle alone, respectively. In vitro, we showed that H-89 decreased the RLuc8 bioluminescence signal but did not compete with coelenterazine-h for the RLuc8 active site, and also did not affect the activity of Firefly luciferase. By contrast, another competitive inhibitor of PKA, KT5720, did not affect the activity of RLuc8. SIGNIFICANCE: The identification and characterization of the adverse effect of H-89 on RLuc signal will help deconvolute data previously generated from RLuc-based assays looking at the functional effects of PKA signaling. In addition, for the current application and future development of bioluminscence assays, KT5720 is identified as a more suitable PKA inhibitor to be used in conjunction with RLuc-based assays. These principal findings also provide an important lesson to fully consider all of the potential

  3. LuxCDABE—Transformed Constitutively Bioluminescent Escherichia coli for Toxicity Screening: Comparison with Naturally Luminous Vibrio fischeri

    Directory of Open Access Journals (Sweden)

    Anne Kahru

    2011-08-01

    Full Text Available We show that in vitro toxicity assay based on inhibition of the bioluminescence of recombinant Escherichia coli encoding thermostable luciferase from Photorhabdus luminescens is a versatile alternative to Vibrio fischeri MicrotoxTM test. Performance of two luxCDABE-transformed E. coli MC1061 constructs (pDNlux and (pSLlux otherwise identical, but having 100-fold different background luminescence was compared with the performance of V. fischeri. The microplate luminometer and a kinetic Flash-Assay test format was used that differently from Microtox test is also applicable for high throughput analysis. Toxic effects (30-s till 30-min EC50 of four heavy metals (Zn, Cd, Hg, Cu and three organic chemicals (aniline, 3,5-dichloroaniline and 3,5-dichlorophenol were studied. Both E. coli strains had comparable sensitivity and the respective 30-min EC50 values highly correlated (log-log R2 = 0.99; p < 0.01 showing that the sensitivity of the recombinant bacteria towards chemicals analyzed did not depend on the bioluminescence level of the recombinant cells. The most toxic chemical for all used bacterial strains (E. coli, V. fischeri was mercury whereas the lowest EC50 values for Hg (0.04–0.05 mg/L and highest EC50 values for aniline (1,300–1,700 mg/L were observed for E. coli strains. Despite of that, toxicity results obtained with both E. coli strains (pSLlux and pDNlux significantly correlated with V. fischeri results (log-log R2 = 0.70/0.75; p < 0.05/0.01. The use of amino acids (0.25% and glucose (0.05%-supplemented M9 medium instead of leucine-supplemented saline significantly (p < 0.05 reduced the apparent toxicity of heavy metals to both E. coli strains up to three orders of magnitude, but had little or no complexing effect on organic compounds. Thus, P. luminescens luxCDABE-transformed E. coli strains can be successfully used for the acute toxicity screening of various types of organic chemicals and heavy metals and can replace V. fischeri in

  4. Avaliação da qualidade microbiológica de bebida láctea e creme de leite UAT por ATP-Bioluminescência Evaluation of microbiological quality of UHT milk drink and UHT milk cream by ATP-Bioluminescence

    Directory of Open Access Journals (Sweden)

    A.F. Cunha

    2013-04-01

    Full Text Available Embora métodos tradicionais sejam utilizados na avaliação microbiológica de produtos UAT, metodologias rápidas, baseadas em ATP-Bioluminescência, têm sido desenvolvidas. Os resultados da aplicação dessa técnica em 54 amostras de bebida láctea UAT achocolatada e 12 de creme de leite UAT foram comparados com os resultados de métodos microbiológicos, utilizando-se diferentes meios de cultura e tempos de incubação das referidas amostras. A técnica de ATP-Bioluminescência foi aplicada por meio do sistema MLS, e os resultados foram expressos em unidades relativas de luz (RLU. Em todos os tempos de incubação - 48, 72 e 168 horas - , as amostras apresentaram contagens baixas de microrganismos mesófilos e psicrotróficos aeróbios quando analisadas em meio PCA, BHI, PetrifilmTM AC e por ATP-Bioluminescência (Although traditional methods are used for the microbiological evaluation of UHT products, rapid methodologies based on ATP-Bioluminescence have been developed. The results of applying this technique in 54 samples of chocolate UHT milk drink and 12 of UHT milk cream were compared with the results of microbiological methods, using different culture media and incubation times for the referred samples. The ATP-Bioluminescence technique was applied through the MLS system and the results were expressed as relative light units (RLU. In all incubation times - 48, 72, and 168 hours - , the samples showed lower counts of mesophilic and psychrotrophic aerobic microorganisms when analyzed using PCA, BHI, PetrifilmTM AC and ATP-Bioluminescence (<150RLU, demonstrating the technique's high specificity. Only one sample of UHT milk cream showed a mesophilic aerobic count above the standard established by Brazilian legislation (<100CFU/mL when analyzed in PCA (260 CFU/mL and PetrifilmTM AC (108CFU/mL at 168 hours. This high count of aerobic mesophilic microorganisms was also detected by the ATP-Bioluminescence (416 RLU technique. The results of

  5. Joint effects of heavy metal binary mixtures on seed germination, root and shoot growth, bacterial bioluminescence, and gene mutation

    Institute of Scientific and Technical Information of China (English)

    In Chul Kong

    2013-01-01

    This investigation was to assess the joint effects of metal binary mixtures on seed germination,root and shoot growth,bacterial bioluminescence,and gene mutation based on the one toxic unit (1 TU) approach.Different sensitivities and orders of toxicity of metal mixtures were observed among the bioassays.In general,mostly additive or antagonistic effects were observed,while almost no synergistic effects by the binary metal mixtures in all bioassays.Therefore,the combined effects of heavy metals in the different bioassays were difficult to generalize since they were dependent on both chemical type and the organism used in each bioassay.However,these results indicate that a battery of bioassays with mixture chemicals as opposed to just a single assay with single metal is a better strategy for the bioassessment of environmental pollutants.

  6. Microbial availability of mercury: effective detection and organic ligand effect using a whole-cell bioluminescent bioreporter.

    Science.gov (United States)

    Xu, Xianghua; Oliff, Kathryn; Xu, Tingting; Ripp, Steven; Sayler, Gary; Zhuang, Jie

    2015-12-01

    A luxCDABE-based genetically engineered bacterial bioreporter (Escherichia coli ARL1) was used to detect bioavailable ionic mercury (Hg(II)) and investigate the effects of humic acids and ethylenediaminetetraacetic acid (EDTA) on the bioavailability of mercury in E. c oli. Results showed that the E. c oli ARL1 bioreporter was sensitive to mercury, with a detection limit of Hg(II) of 0.5 µg/L and a linear dose/response relationship up to 2000 µg Hg(II)/L. Humic acids and EDTA decreased the Hg(II)-induced bioluminescent response of strain ARL1, suggesting that the two organic ligands reduced the bioavailability of Hg(II) via complexation with Hg(II). Compared with traditional chemical methods, the use of E. c oli ARL1 is a cost-effective, rapid, and reliable approach for measuring aqueous mercury at very low concentrations and thus has potential for applications in field in situ monitoring.

  7. On-line monitoring of aerobic bioremediation with bioluminescent reporter microbes. Final report, July 1991--December 1994

    Energy Technology Data Exchange (ETDEWEB)

    Sayler, G.S.

    1995-03-01

    A critical issue in the biological characterization of contaminated sites and in the evaluation of relative bioremediation treatment efficiencies is the development of appropriate monitoring methods for the assessment of pollutant bioavailability and microbial in situ activity potential. In nature, pollutants are found dispersed among the solid, liquid and gaseous phases of the complex environments rendering the analytical estimation of their bioavailability and degradation more difficult and irrelevant. Ex situ and extractive analytical techniques have only been misrepresentative of the natural conditions and often resulted in inaccurate estimates of pollutants mass transfer. In this project, the bioluminescent bioreporter bacterium P. Fluorescens HK44 was integrated to an optical device, capable of conducting emitted light, and used as an online biosensor of naphthalene and salicylate. The physiological requirements of the bacteria and the physical limitations of the biosensor were also determined.

  8. The Influence of Hypoxia and pH on Bioluminescence Imaging of Luciferase-Transfected Tumor Cells and Xenografts

    Directory of Open Access Journals (Sweden)

    Ashraf A. Khalil

    2013-01-01

    Full Text Available Bioluminescence imaging (BLI is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment.

  9. SU-E-T-20: A Novel Hybrid CBCT, Bioluminescence and Fluorescence Tomography System for Preclinical Radiation Research

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, B; Eslami, S; Iordachita, I [Johns Hopkins University, Baltimore, Maryland (United States); Yang, Y [University of Miami School of Medicine, Miami, FL (United States); Patterson, M [Hamilton Regional Cancer Ctr., Hamilton, ON (Canada); Wong, J [Johns Hopkins University, Baltimore, MD (United States); Wang, K [Johns Hopkins Hospital, Baltimore, MD (United States)

    2014-06-01

    Purpose: A novel standalone bioluminescence and fluorescence tomography (BLT and FT) system equipped with high resolution CBCT has been built in our group. In this work, we present the system calibration method and validate our system in both phantom and in vivo environment. Methods: The CBCT is acquired by rotating the animal stage while keeping the x-ray source and detector panel static. The optical signal is reflected by the 3-mirror system to a multispectral filter set and then delivered to the CCD camera with f/1.4 lens mounted. Nine fibers passing through the stage and in contact with the mouse skin serve as the light sources for diffuse optical tomography (DOT) and FT. The anatomical information and optical properties acquired from the CBCT and DOT, respectively, are used as the priori information to improve the BLT/FT reconstruction accuracy. Flat field correction for the optical system was acquired at multiple wavelengths. A home-built phantom is used to register the optical and CBCT coordinates. An absolute calibration relating the CCD photon counts rate to the light fluence rate emitted at animal surface was developed to quantify the bioluminescence power or fluorophore concentration. Results: An optical inhomogeneous phantom with 2 light sources (3mm separation) imbedded is used to test the system. The optical signal is mapped onto the mesh generated from CBCT for optical reconstruction. Our preliminary results show that the center of mass can be reconstructed within 2.8mm accuracy. A live mouse with the light source imbedded is also used to validate our system. Liver or lung metastatic luminescence tumor model will be used for further testing. Conclusion: This hybrid system transforms preclinical research to a level that even sub-palpable volume of cells can be imaged rapidly and non-invasively, which largely extends the scope of radiobiological research. The research is supported by the NCI grant R01CA158100-01.

  10. Quantitative imaging of D-2-hydroxyglutarate (D2HG in selected histological tissue areas by a novel bioluminescence technique

    Directory of Open Access Journals (Sweden)

    Nadine Fabienne Voelxen

    2016-03-01

    Full Text Available AbstractPatients with malignant gliomas have a poor prognosis with average survival of less than one year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG, a metabolite, which was discovered first in this tumor entity. D2HG is generated in large amounts due to various gain-of–function mutations in the isocitrate dehydrogenases IDH-1 and IDH-2. Meanwhile, D2HG has been detected in several other tumor entities including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (< 0.1 mM, but its concentration increases up to 35 mM in malignant tumor tissues. Consequently, the oncometabolite D2HG has gained increasing interest in the field of tumor metabolism. To facilitate its quantitative measurement without loss of spatial resolution at a microscopical level, we have developed a novel bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography / mass spectrometry (LC/MS. The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0 – 10 µmol/g tissue (wet weight. In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension.

  11. Bioluminescence in the deep sea: Free-fall lander observations in the Atlantic Ocean off Cape Verde

    Science.gov (United States)

    Priede, I. G.; Bagley, P. M.; Way, S.; Herring, P. J.; Partridge, J. C.

    2006-07-01

    A novel autonomous free-fall lander vehicle, with a capability down to 6000 m, was deployed off Cape Verde for studies on bioluminescence in the deep sea. The system was equipped with a high-sensitivity Intensified Silicon Intensified Target (ISIT) video camera, a programmable control-recording unit and an acoustic current meter with depth and temperature sensors. The ISIT lander was used in three modes: (1) free falling at 34 m min -1, with the camera looking downwards at a mesh screen, recording impacts of luminescent organisms to obtain a vertical profile down to the abyssal sea floor, sampling at >100 l s -1; (2) rotating, with the lander on the sea floor and the camera orienting to the bottom current using a servo-controlled turntable, impacts of luminescent organisms carried by the bottom current onto a mesh screen mounted 0.5 m in front of the camera were recorded to estimate abundance in the benthic boundary layer; (3) baited, with the camera focused on a bait placed on the sea floor. Profiles recorded abundance of luminescent organisms as 26.7 m -3 at 500-999 m depth, decreasing to 1.6 m -3 at 2000-2499 m and 0.5 m -3 between 2500 m and the sea floor at 4046 m, with no further detectable significant change with depth. Rotator measurements at a 0.5 m height above the sea floor gave a mean abundance of 0.47 m -3 in the benthic boundary layer at 4046 m and of 2.04 m -3 at 3200 m. Thirty five minutes after the bait was placed on the sea floor at 3200 m, bioluminescent fauna apparently arrived at the bait and produced luminescent displays at a rate of 2 min -1. Moving, flashing light sources were observed and luminescent material was released into the bottom current.

  12. 5-Fluorouracil induced intestinal mucositis via nuclear factor-κB activation by transcriptomic analysis and in vivo bioluminescence imaging.

    Directory of Open Access Journals (Sweden)

    Chung-Ta Chang

    Full Text Available 5-Fluorouracil (5-FU is a commonly used drug for the treatment of malignant cancers. However, approximately 80% of patients undergoing 5-FU treatment suffer from gastrointestinal mucositis. The aim of this report was to identify the drug target for the 5-FU-induced intestinal mucositis. 5-FU-induced intestinal mucositis was established by intraperitoneally administering mice with 100 mg/kg 5-FU. Network analysis of gene expression profile and bioluminescent imaging were applied to identify the critical molecule associated with 5-FU-induced mucositis. Our data showed that 5-FU induced inflammation in the small intestine, characterized by the increased intestinal wall thickness and crypt length, the decreased villus height, and the increased myeloperoxidase activity in tissues and proinflammatory cytokine production in sera. Network analysis of 5-FU-affected genes by transcriptomic tool showed that the expression of genes was regulated by nuclear factor-κB (NF-κB, and NF-κB was the central molecule in the 5-FU-regulated biological network. NF-κB activity was activated by 5-FU in the intestine, which was judged by in vivo bioluminescence imaging and immunohistochemical staining. However, 5-aminosalicylic acid (5-ASA inhibited 5-FU-induced NF-κB activation and proinflammatory cytokine production. Moreover, 5-FU-induced histological changes were improved by 5-ASA. In conclusion, our findings suggested that NF-κB was the critical molecule associated with the pathogenesis of 5-FU-induced mucositis, and inhibition of NF-κB activity ameliorated the mucosal damage caused by 5-FU.

  13. A biocompatible in vivo ligation reaction and its application for noninvasive bioluminescent imaging of protease activity in living mice.

    Science.gov (United States)

    Godinat, Aurélien; Park, Hyo Min; Miller, Stephen C; Cheng, Ke; Hanahan, Douglas; Sanman, Laura E; Bogyo, Matthew; Yu, Allen; Nikitin, Gennady F; Stahl, Andreas; Dubikovskaya, Elena A

    2013-05-17

    The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. The success of this "split luciferin" ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a "caged" luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH(2)-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting in vivo.

  14. Hybrid radiosity-SP{sub 3} equation based bioluminescence tomography reconstruction for turbid medium with low- and non-scattering regions

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xueli, E-mail: xlchen@xidian.edu.cn, E-mail: jimleung@mail.xidian.edu.cn; Zhang, Qitan; Yang, Defu; Liang, Jimin, E-mail: xlchen@xidian.edu.cn, E-mail: jimleung@mail.xidian.edu.cn [School of Life Science and Technology, Xidian University, Xi' an, Shaanxi 710071 (China)

    2014-01-14

    To provide an ideal solution for a specific problem of gastric cancer detection in which low-scattering regions simultaneously existed with both the non- and high-scattering regions, a novel hybrid radiosity-SP{sub 3} equation based reconstruction algorithm for bioluminescence tomography was proposed in this paper. In the algorithm, the third-order simplified spherical harmonics approximation (SP{sub 3}) was combined with the radiosity equation to describe the bioluminescent light propagation in tissues, which provided acceptable accuracy for the turbid medium with both low- and non-scattering regions. The performance of the algorithm was evaluated with digital mouse based simulations and a gastric cancer-bearing mouse based in situ experiment. Primary results demonstrated the feasibility and superiority of the proposed algorithm for the turbid medium with low- and non-scattering regions.

  15. Sensitive Dual Color in vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

    Science.gov (United States)

    2011-04-01

    expression of the bacterial luciferase gene cassette ( lux ) in a mammalian cell line. PLoS One 5: e12441. 6. Cheong WF, Prahl SA, Welch AJ (1990) A...Chemistry, Connecticut College, New London, Connecticut, United States of America Abstract Background: Despite a plethora of bioluminescent reporter genes ...challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color- coupled

  16. 一种生物在体荧光成像的自适应分割算法%Adaptive Segmentation Algorithm of Bioluminescent Image

    Institute of Scientific and Technical Information of China (English)

    常志军; 杨鑫

    2011-01-01

    Bioluminescence imaging is regarded as an imaging modality with a high performance, low cost and good prospect in molecular imaging technique. This paper proposes a new adaptive segmentation algorithm, which is based on the characteristics and the application requirements of the bioluminescent images. The adaptive segmentation is realized by performing the normalized processing, connectivity operation and the interested regions distinguishing on bioluminescent images. Experimental results show that this algorithm can get better segmentation results in the ease of weak signal, low signal-to-noise ratio and multiple light sources, so it is a kind of effective segmentation algorithm of bioluminescent images.%生物在体荧光成像是新兴分子影像技术中性能高、费用低、前景好的一种成像模态.针对生物在体荧光图像的特点和应用需求,提出一种全新的自适应图像分割算法.通过对荧光图像的归一化处理、连通性操作、感兴趣区域区分实现自适应分割.实验结果表明,该算法能够在弱信号、低信噪比、多光源的情况下得到较理想的分割结果,是一种有效的荧光图像分割算法.

  17. A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part II: technical development and proof of concept of the biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Charrier, Thomas; Thouand, Gerald [UMR CNRS 6144 GEPEA, CBAC, Nantes University, PRES UNAM, Campus de la Courtaisiere-IUT, La Roche-sur-Yon cedex (France); Chapeau, Cyrille [Biolumine, Biokar Diagnostic, Rue des Quarante Mines ZAC de Ther-Allonne, Beauvais Cedex (France); Bendria, Loubna; Daniel, Philippe [UMR CNRS 6087 LPEC, Universite du Maine, Av Olivier Messiaen, Le Mans cedex 9 (France); Picart, Pascal [UMR CNRS 6613 IAM-LAUM, Ecole Nationale des Ingenieurs du Mans, Universite du Maine, Le Mans Cedex 9 (France)

    2011-05-15

    This research study deals with the on-line detection of heavy metals and toxicity within the context of environmental pollution monitoring. It describes the construction and the proof of concept of a multi-channel bioluminescent bacterial biosensor in immobilized phase: Lumisens3. This new versatile device, designed for the non-stop analysis of water pollution, enables the insertion of any bioluminescent strains (inducible or constitutive), immobilized in a multi-well removable card. The technical design of Lumisens3 has benefited from both a classical and a robust approach and includes four main parts: (1) a dedicated removable card contains 64 wells, 3 mm in depth, arranged in eight grooves within which bacteria are immobilized, (2) this card is incubated on a Pelletier block with a CCD cooled camera on top for bioluminescence monitoring, (3) a fluidic network feeds the card with the sample to be analyzed and finally (4) a dedicated computer interface, BIOLUX 1.0, controls all the elements of the biosensor, allowing it to operate autonomously. The proof of concept of this biosensor was performed using a set of four bioluminescent bacteria (Escherichia coli DH1 pBzntlux, pBarslux, pBcoplux, and E. coli XL1 pBfiluxCDABE) in the on-line detection of CdCl{sub 2} 0.5 {mu}M and As{sub 2}O{sub 3} 5 {mu}M from an influent. When considering metals individually, the ''fingerprints'' from the biosensor were as expected. However, when metals were mixed together, cross reaction and synergistic effects were detected. This biosensor allowed us to demonstrate the simultaneous on-line cross detection of one or several heavy metals as well as the measurement of the overall toxicity of the sample. (orig.)

  18. In vivo visualization and monitoring of viable neural stem cells using noninvasive bioluminescence imaging in the 6-hydroxydopamine-induced mouse model of Parkinson disease.

    Science.gov (United States)

    Im, Hyung-Jun; Hwang, Do Won; Lee, Han Kyu; Jang, Jaeho; Lee, Song; Youn, Hyewon; Jin, Yeona; Kim, Seung U; Kim, E Edmund; Kim, Yong Sik; Lee, Dong Soo

    2013-06-01

    Transplantation of neural stem cells (NSCs) has been proposed as a treatment for Parkinson disease (PD). The aim of this study was to monitor the viability of transplanted NSCs expressing the enhanced luciferase gene in a mouse model of PD in vivo. The PD animal model was induced by unilateral injection of 6-hydroxydopamine (6-OHDA). The behavioral test using apomorphine-induced rotation and positron emission tomography with [18F]N-(3-fluoropropyl)-2'-carbomethoxy-3'-(4-iodophenyl)nortropane ([18F]FP-CIT) were conducted. HB1.F3 cells transduced with an enhanced firefly luciferase retroviral vector (F3-effLuc cells) were transplanted into the right striatum. In vivo bioluminescence imaging was repeated for 2 weeks. Four weeks after transplantation, [18F]FP-CIT PET and the rotation test were repeated. All 6-OHDA-injected mice showed markedly decreased [18F]FP-CIT uptake in the right striatum. Transplanted F3-effLuc cells were visualized on the right side of the brain in all mice by bioluminescence imaging. The bioluminescence intensity of the transplanted F3-effLuc cells gradually decreased until it was undetectable by 10 days. The behavioral test showed that stem cell transplantation attenuated the motor symptoms of PD. No significant change was found in [18F]FP-CIT imaging after cell transplantation. We successfully established an in vivo bioluminescence imaging system for the detection of transplanted NSCs in a mouse model of PD. NSC transplantation induced behavioral improvement in PD model mice.

  19. Silicon photomultiplier (SPM) detection of low-level bioluminescence for the development of deployable whole-cell biosensors: Possibilities and limitations

    OpenAIRE

    Li, Huaqing; Lopes, Nicholas; Moser, Scott; Sayler, Gary; Ripp, Steven

    2012-01-01

    Whole-cell bacterial bioreporters await miniaturized photon counting modules with high sensitivity and robust compatible hardware to fulfill their promise of versatile, on-site biosensor functionality. In this study, we explore the photon counting readout properties of the silicon photomultiplier (SPM) with a thermoelectric cooler and the possibilities of detecting low-level bioluminescent signals. Detection performance was evaluated through a simulated LED light source and the bioluminescenc...

  20. Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins.

    Science.gov (United States)

    Natashin, Pavel V; Markova, Svetlana V; Lee, John; Vysotski, Eugene S; Liu, Zhi-Jie

    2014-03-01

    Ca(2+) -regulated photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan photoproteins are a single-chain polypeptide to which 2-hydroperoxycoelenterazine is tightly but non-covalently bound. Bioluminescence results from oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant photoproteins vary in the range 462-495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008-1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6-(p-hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild-type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca(2+) discharge, compared to the conformation states of wild-type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6-(p-hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan photoproteins.

  1. [Photoreactivating Activity of Bioluminescence: Repair of UV-damaged DNA of Escherichia coli Occurs with Assistance of lux-Genes of Marine Bacteria].

    Science.gov (United States)

    Zavilgelsky, G B; Melkina, O E; Kotova, V Yu; Konopleva, M N; Manukhov, I V; Pustovoit, K Ss

    2015-01-01

    The UV resistance of luminescent bacteria Escherichia coli AB1886 uvrA6 (pLeo1) containing the plasmid with luxCDABE genes of marine bacteria Photobacterium leiognathi is approximately two times higher than the UV resistance of non-luminous bacteria E. coli AB1886 uvrA6. Introduction of phr::kan(r) mutations (a defect in the functional activity of photolyase) into the genome of E. coli AB1886 uvrA6 (pLeo1) completely removes the high UV resistance of the cells. Therefore, photoreactivation that involves bacterial photolyase contributes mainly to the bioluminescence-induced DNA repair. It is shown that photoreactivating activity of bioluminescence of P. leiognathi is about 2.5 times lower compared with that one induced by a light source with λ > 385 nm. It is also shown that an increase in the bioluminescence intensity, induced by UV radiation in E. coli bacterial cells with a plasmid containing the luxCD ABE genes under RecA-LexA-regulated promoters, occurs only 25-30 min later after UV irradiation of cells and does not contribute to DNA repair. A quorum sensing regulatory system is not involved in the DNA repair by photolyase.

  2. 有趣的发光生物与奇妙的发光蛋白%Interesting Bioluminescent Creatures and Amazing Fluorescent Proteins

    Institute of Scientific and Technical Information of China (English)

    林济民; 王江云

    2011-01-01

    Although bioluminescence seems to be mysterious, it is common in nature with important ecological functions and evolutionary meanings. From using bioluminescent plant as light, to applying green fluorescent proteins as protein expression markers in cellsi from researches on the mechanism of bioluminescence. To the developments of new types of fluorescent proteins- the secrets of life is lighting up.%“生物发光”现象一直以来都吸引着科学家的好奇心,它看似神奇,但在自然界十分普遍.“生物发光”现象有着独特的生态功能和进化意义.在漫漫历史长河中,人类对生物发光现象的探索从未停止过.从用发光生物作为最原始的照明工具,到利用绿色荧光蛋白标记细胞内组分;从对生物发光机理的探究,到荧光蛋白种类的开发,科学的脚步在一步步前进,生命的秘密也在一点点被点亮.

  3. ATP Bioluminescence Method for Rapidly Determining Colony Counts in Drinking Water%ATP发光技术快速检测饮用水中菌落总数

    Institute of Scientific and Technical Information of China (English)

    邱颖; 邱贺民; 李玉婵; 刘国良

    2011-01-01

    Objective To explore the feasibility of ATP bioluminescence method for rapidly determining colony counts. Methods Colony counts were detected by ATP bioluminescence method and plate count method, and the results of the two detection methods were compared. Results The correlation research indicated that they were positively relative in proper range. Conclusions ATP bioluminescence method is simple and has the advantage of wide application. It can be used for detecting the total number of colony in water rapidly.%目的 探讨三磷酸腺苷(ATP)发光技术快速检测水质菌落总数的可行性.方法 比较ATP生物发光值与平板计数法对细菌总数进行检测.结果 检测结果表明,两者在一定范围内呈正相关.结论 ATP生物发光法操作简便,应用范围广,可应用于饮用水中菌落总数的快速检测.

  4. Molecular insights on the evolution of the lateral and head lantern luciferases and bioluminescence colors in Mastinocerini railroad-worms (Coleoptera: Phengodidae).

    Science.gov (United States)

    Arnoldi, Frederico G C; da Silva Neto, Antonio Joaquim; Viviani, Vadim R

    2010-01-01

    Among bioluminescent beetles of Elateroidea superfamily, railroad-worms (Phengodidae) produce the widest range of colors, from green to red, using the same luciferin-luciferase system. Members of the Mastinocerini tribe display additional unique cephalic organs that emit red-shifted light, with Phrixothrix railroad-worms being the most dramatic cases with head lanterns emitting red light. Although the luciferases from the head lanterns of Phrixothrix hirtus and from the lateral lanterns of P. vivianii were previously cloned, the luciferases from both lanterns of the same species were not cloned yet. Therefore the origin and evolution of head and lateral lanterns luciferases in Phengodidae remains unknown. In the present work, we cloned by PCR the cDNA for lateral lantern luciferases of three Mastinocerini species: Phrixothrix hirtus, Brasilocerus sp(3). and Taximastioncerus sp. The results suggest that the head and lateral lanterns luciferases in Mastinocerini are coded by paralogous genes, and that the ancestral luciferase in the Phengodinae subfamily produced green bioluminescence. The evolutionary history of bioluminescence colors within Phengodinae is discussed.

  5. An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer.

    Science.gov (United States)

    Fourrage, Cécile; Swann, Karl; Gonzalez Garcia, Jose Raul; Campbell, Anthony K; Houliston, Evelyn

    2014-04-09

    Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

  6. 基于受激发光实验的生物光尾流特性%Characteristics of bioluminescent wake based on stimulated luminescence laboratory experiments

    Institute of Scientific and Technical Information of China (English)

    曹静; 王江安; 何友; 吴荣华

    2011-01-01

    Abstract: Based on the relative characteristics of stimulated bioluminescence and hydrodynamic flow, the experimental plat-form of stimulated bioluminescence in Couette flow was built. The hydrodynamic elements stimulated bioluminescence were analy-sised. The threshold of shear stress for stimulated bioluminescence of Lingulodinium polyedrum is 0. 1 N/m2. And biolumines-cence emission is the function of shear stress and increases with it when the shear stress exceeds the threshold. The shear stress distributions of submarine were simulated by FLUENT software. The results of calculation illustrate that the shear stress decrea-ses with the increased length of wake of submarine and it can be decreased as the oscillation of sine wave in the near area. The shear stress of wake decreases rapidly and widely with the higher speed of submarine, when the speed of submarine is above 4. 1 m/s, the shear stress of wake in 2 000 m length is still over the threshold.%基于生物受激发光与流场机械刺激间的相关性,构建了库埃特流场刺激下的生物受激发光实验平台,分析了影响生物发光的水动力因素剪应力.实验结果显示:引起多边膝沟藻受激发光的剪应力阈值为0.1 N/m2;当流场中的剪应力大于发光阈值后,生物发光强度随着剪应力的增大而增强.采用FLUENT软件模拟计算了不同航速下某潜艇尾流场中的剪应力,计算结果表明:剪应力随着尾流长度的增加而减小,在近场区域剪应力出现了类似正弦波振荡的减小,且航速越大减幅越大,减速越快;当潜艇航速大于4.1 m/s航行时,2 000 m处尾流的剪应力仍然大于引起生物发光的剪应力阈值.

  7. Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

    Science.gov (United States)

    Viviani, V R; Simões, A; Bevilaqua, V R; Gabriel, G V M; Arnoldi, F G C; Hirano, T

    2016-08-30

    Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also

  8. In Vivo Tracking of Systemically Administered Allogeneic Bone Marrow Mesenchymal Stem Cells in Normal Rats through Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Juan Cao

    2016-01-01

    Full Text Available Recently, mesenchymal stem cells (MSCs are increasingly used as a panacea for multiple types of disease short of effective treatment. Dozens of clinical trials published demonstrated strikingly positive therapeutic effects of MSCs. However, as a specific agent, little research has focused on the dynamic distribution of MSCs after in vivo administration. In this study, we track systemically transplanted allogeneic bone marrow mesenchymal stem cells (BMSCs in normal rats through bioluminescence imaging (BLI in real time. Ex vivo organ imaging, immunohistochemistry (IHC, and RT-PCR were conducted to verify the histological distribution of BMSCs. Our results showed that BMSCs home to the dorsal skin apart from the lungs and kidneys after tail vein injection and could not be detected 14 days later. Allogeneic BMSCs mainly appeared not at the parenchymatous organs but at the subepidermal connective tissue and adipose tissue in healthy rats. There were no significant MSCs-related adverse effects except for transient decrease in neutrophils. These findings will provide experimental evidences for a better understanding of the biocharacteristics of BMSCs.

  9. Evaluation of bioluminescent imaging for noninvasive monitoring of colorectal cancer progression in the liver and its response to immunogene therapy

    Directory of Open Access Journals (Sweden)

    Gonzalez-Aparicio Manuela

    2009-01-01

    Full Text Available Abstract Background Bioluminescent imaging (BLI is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies. Results A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1 retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US, BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12. Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival. Conclusion We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.

  10. Simulation research on bioluminescence tomography%生物发光断层成像的仿真研究

    Institute of Scientific and Technical Information of China (English)

    刘勇; 齐树波; 方勇军; 陈锋

    2014-01-01

    目的:验证生物发光断层成像技术(bioluminescence tomography,BLT)成像的可行性并克服其成像的病态性.方法:采用BLT的前向数学模型和仿真实验结果进行分析.结果:仿真实验结果表明,基于多角度、非接触BLT成像系统(multi-view non-contact BLT imaging system),自发光源在成像体内的分布能够获得较为精确的解析,定位误差小于1 mm.结论:基于多角度、非接触的BLT成像系统,结合稀疏重建算法,能够在较少(6个)的角度下,获取较好的重建结果,有望为疾病早期诊断及药物研发等研究提供有力工具.

  11. Susceptibility of the wild-derived inbred CAST/Ei mouse to infection by orthopoxviruses analyzed by live bioluminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Americo, Jeffrey L.; Sood, Cindy L.; Cotter, Catherine A.; Vogel, Jodi L.; Kristie, Thomas M.; Moss, Bernard, E-mail: bmoss@nih.gov; Earl, Patricia L., E-mail: pearl@nih.gov

    2014-01-20

    Classical inbred mice are extensively used for virus research. However, we recently found that some wild-derived inbred mouse strains are more susceptible than classical strains to monkeypox virus. Experiments described here indicated that the 50% lethal dose of vaccinia virus (VACV) and cowpox virus (CPXV) were two logs lower in wild-derived inbred CAST/Ei mice than classical inbred BALB/c mice, whereas there was little difference in the susceptibility of the mouse strains to herpes simplex virus. Live bioluminescence imaging was used to follow spread of pathogenic and attenuated VACV strains and CPXV virus from nasal passages to organs in the chest and abdomen of CAST/Ei mice. Luminescence increased first in the head and then simultaneously in the chest and abdomen in a dose-dependent manner. The spreading kinetics was more rapid with VACV than CPXV although the peak photon flux was similar. These data suggest advantages of CAST/Ei mice for orthopoxvirus studies. - Highlights: • Wild-derived inbred CAST/Ei mice are susceptible to vaccinia virus and cowpox virus. • Morbidity and mortality from orthopoxviruses are greater in CAST/Ei than BALB/c mice. • Morbidity and mortality from herpes simplex virus type 1 are similar in both mice. • Imaging shows virus spread from nose to lungs, abdominal organs and brain. • Vaccinia virus spreads more rapidly than cowpox virus.

  12. Physicochemical characterization and in vivo bioluminescence imaging of nanostructured lipid carriers for targeting the brain: apomorphine as a model drug

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Shu-Hui [Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan (China); Wen, Chih-Jen; Yen, Tzu-Chen [Animal Molecular Imaging Center, Chang Gung Memorial Hospital, Kweishan, Taoyuan 333, Taiwan (China); Al-Suwayeh, S A; Fang, Jia-You [Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh (Saudi Arabia); Chang, Hui-Wen, E-mail: fajy@mail.cgu.edu.tw [Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan 333, Taiwan (China)

    2010-10-08

    Nanostructured lipid carriers (NLCs) were prepared to investigate whether the duration of brain targeting and accumulation of drugs in the brain can be improved by intravenous delivery. NLCs were developed using cetyl palmitate as the lipid matrix, squalene as the cationic surfactant, and Pluronic F68, polysorbate 80 and polyethylene glycol as the interfacial additives. Solid lipid nanoparticles (SLNs) and lipid emulsions (LEs) were also prepared for comparison. An anti-Parkinson's drug, apomorphine, was used as the model drug. Nuclear magnetic resonance and differential scanning calorimetry showed possible interactions between the solid and liquid lipids in the inner core. The lipid nanoparticles with different compositions were characterized by mean size, zeta potential, apomorphine encapsulation and in vitro drug release. NLCs were 370-430 nm in size, which was between the sizes of the SLNs and LEs. A cationic surfactant was used to produce a positive surface charge of 42-50 mV. The base form of apomorphine was successfully entrapped by NLCs with an entrapment percentage of > 60%. The loading of apomorphine in nanoparticles resulted in a slower release behavior compared to the aqueous solution, with LEs showing the lowest release. In vivo real-time bioluminescence imaging of the rat brain revealed that NLCs could be targeted, through certain vessels, to selected brain regions. This effect was further confirmed by imaging the entire brain and brain slices. The results indicated that NLCs with moderate additives are a promising controlled-release and drug-targeting system.

  13. Bio-luminescent imaging and characterization of organ-specific metastasis of human cancer in NOD/SCID mice

    Science.gov (United States)

    Chun, Nicole A. L.; Murakami, Takashi

    2010-02-01

    Many clinical evidences demonstrate that the sites of distant metastasis are not random and certain malignant tumors show a tendency to develop metastases in specific organs (e.g., brain, liver, and lungs). However, an appropriate animal model to characterize the metastatic nature of transplantable human cancer cell lines has not been reported well. Recent advances in bio-luminescent imaging (BLI) technologies have facilitated the quantitative analysis of various cellular processes in vivo. To visualize the fate of tumor progression in the living mice, we are constructing a luciferaseexpressing human cancer cell library (including melanoma, colon, breast, and prostate cancer). Herein we demonstrate that the BLI technology in couple with a fine ultrasonic guidance realizes cancer cell-type dependent metastasis to the specific organs. For example, some melanoma cell lines showed frequent metastasis to brain, lungs, and lymph nodes in the mouse model. Notably, reflecting the clinical features of melanoma, breast, and prostate cancer, some of the cell lines showed preferential metastasis to the brain. Moreover, these cellular resources for BLI allow a high throughput screening for potential anti-cancer drugs. Thus, this BLI-mediated additional strategy with the luciferase-expressing cancer cell resources should promote many translational studies for human cancer therapy.

  14. Mathematical Models for Quantitative Assessment of Bioluminescence Resonance Energy Transfer (BRET: Application to Seven Transmembrane Receptors (7TMRs Oligomerization

    Directory of Open Access Journals (Sweden)

    Luka eDrinovec

    2012-08-01

    Full Text Available The idea that seven transmembrane receptors (7TMRs; also designated G-protein coupled receptors (GPCRs might form dimers or higher order oligomeric complexes was formulated more than 20 years ago and has been intensively studied since then. In the last decade, bioluminescence resonance energy transfer (BRET has been one of the most frequently used biophysical methods for studying 7TMRs oligomerization. This technique enables monitoring physical interactions between protein partners in living cells fused to donor and acceptor moieties. It relies on non-radiative transfer of energy between donor and acceptor, depending on their intermolecular distance (1–10 nm and relative orientation. Results derived from BRET-based techniques are very persuasive; however, they need appropriate controls and critical interpretation. To overcome concerns about the specificity of BRET-derived results, a set of experiments has been proposed, including negative control with a non-interacting receptor or protein, BRET dilution, saturation and competition assays. This article presents the theoretical background behind BRET assays, then outlines mathematical models for quantitative interpretation of BRET saturation and competition assay results, gives examples of their utilization and discusses the possibilities of quantitative analysis of data generated with other RET-based techniques.

  15. Detection of urinary tract infections on lab-on-chip device by measuring photons emitted from ATP bioluminescence.

    Science.gov (United States)

    Feng, Shilun; Dong, Tao; Yang, Zhaochu

    2014-01-01

    A microfluidic Lab-on-chip (LOC) platform for in vitro detecting Urinary Tract Infections (UTI) for clinical diagnostic applications has been built. Based on one commercial adenosine 5'-triphosphate (ATP) assay kit, one chip designed before was applied to detect UTI with the help of photomultiplier tube (PMT) and quantitative determination was made by measuring the photons of light emitted in the bioluminescent reaction of ATP with the enzyme luciferase. The chip had been tested and materials had been well prepared before testing the PMT detecting system. The data from PMT were visualized by the Labview™, appearing good linearity between voltage values and the concentration of the ATP ranging from 2×10(-12) M to 2×10(-8) M. Fresh urine sample with different amounts of Escherichia coli had been measured by the system, appearing good linearity trend between the voltage values and number of the E.coli. This study successfully expressed the concept of measuring ATP directly in the urine to quickly and accurately detect UTI on a microfluidic chip.

  16. Development of bioluminescent chick chorioallantoic membrane (CAM) models for primary pancreatic cancer cells: a platform for drug testing

    Science.gov (United States)

    Rovithi, Maria; Avan, Amir; Funel, Niccola; Leon, Leticia G.; Gomez, Valentina E.; Wurdinger, Thomas; Griffioen, Arjan W.; Verheul, Henk M. W.; Giovannetti, Elisa

    2017-01-01

    The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations. PMID:28304379

  17. In Vivo Analysis of Protein-Protein Interactions with Bioluminescence Resonance Energy Transfer (BRET): Progress and Prospects.

    Science.gov (United States)

    Sun, Sihuai; Yang, Xiaobing; Wang, Yao; Shen, Xihui

    2016-10-11

    Proteins are the elementary machinery of life, and their functions are carried out mostly by molecular interactions. Among those interactions, protein-protein interactions (PPIs) are the most important as they participate in or mediate all essential biological processes. However, many common methods for PPI investigations are slightly unreliable and suffer from various limitations, especially in the studies of dynamic PPIs. To solve this problem, a method called Bioluminescence Resonance Energy Transfer (BRET) was developed about seventeen years ago. Since then, BRET has evolved into a whole class of methods that can be used to survey virtually any kinds of PPIs. Compared to many traditional methods, BRET is highly sensitive, reliable, easy to perform, and relatively inexpensive. However, most importantly, it can be done in vivo and allows the real-time monitoring of dynamic PPIs with the easily detectable light signal, which is extremely valuable for the PPI functional research. This review will take a comprehensive look at this powerful technique, including its principles, comparisons with other methods, experimental approaches, classifications, applications, early developments, recent progress, and prospects.

  18. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System

    Directory of Open Access Journals (Sweden)

    Hyuck Joon Kwon

    2015-01-01

    Full Text Available Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

  19. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System.

    Science.gov (United States)

    Kwon, Hyuck Joon; Han, Youngbae

    2015-01-01

    Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

  20. Strengths and weaknesses in the determination of Saccharomyces cerevisiae cell viability by ATP-based bioluminescence assay.

    Science.gov (United States)

    Paciello, Lucia; Falco, Francesco Cristino; Landi, Carmine; Parascandola, Palma

    2013-03-05

    Due to its sensitivity and speed of execution, detection of ATP by luciferin-luciferase reaction is a widely spread system to highlight cell viability. The paper describes the methodology followed to successfully run the assay in the presence of yeast cells of two strains of the yeast Saccharomyces cerevisiae, BY4741 and CEN.PK2-1C and emphasizes the importance of correctly determining the contact time between the lysing agent and the yeast cells. Once this was established, luciferin-luciferase reaction was exploited to determine the maximum specific rate of growth, as well as cell viability in a series of routine tests. The results obtained in this preliminary study highlighted that using luciferin-luciferase can imply an over-estimation of maximum specific growth rate with respect to that determined by optical density and/or viable count. On the contrary, the bioluminescence assay gave the possibility to highlight, if employed together with viable count, physiological changes occurring in yeast cells as response to stressful environmental conditions such as those deriving from exposure of yeast cells to high temperature or those depending on the operative conditions applied during fed-batch operations.