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Sample records for rapid fluorescent focus

  1. Comparison of a Micro-Neutralization Test with the Rapid Fluorescent Focus Inhibition Test for Measuring Rabies Virus Neutralizing Antibodies

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    Todd G. Smith

    2017-07-01

    Full Text Available The rapid fluorescent focus inhibition test (RFFIT is routinely used in the United States to measure rabies virus neutralizing antibodies (rVNA. RFFIT has a long history of reproducible and reliable results. The test has been modified over the years to use smaller volumes of reagents and samples, but requires a 50 μL minimum volume of test serum. To conduct pathogenesis studies, small laboratory animals such as mice are regularly tested for rVNA, but the minimum volume for a standard RFFIT may be impossible to obtain, particularly in scenarios of repeated sampling. To address this problem, a micro-neutralization test was developed previously. In the current study, the micro-neutralization test was compared to the RFFIT using 129 mouse serum samples from rabies vaccine studies. Using a cut-off value of 0.1 IU/mL, the sensitivity, specificity, and concordance of the micro-neutralization test were 100%, 97.5%, and 98%, respectively. The geometric mean titer of all samples above the cut-off was 2.0 IU/mL using RFFIT and 3.4 IU/mL using the micro-neutralization test, indicating that titers determined using the micro-neutralization test are not equivalent to RFFIT titers. Based on four rVNA-positive hamster serum samples, the intra-assay coefficient of variability was 24% and inter-assay coefficient of variability was 30.4%. These results support continued use of the micro-neutralization test to determine rabies virus neutralizing antibody titers for low-volume serum samples.

  2. Two-focus fluorescence correlation spectroscopy

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    Dertinger, T.

    2007-05-15

    Fluorescence Correlation Spectroscopy (FCS) has been invented more than 30 years ago and experienced a renaissance after stable and affordable laser sources and low-noise single-photon detectors have become available. Its ability to measure diffusion coefficients at nanomolar concentrations of analyte made it a widely used tool in biophysics. However, in recent years it has been shown by many authors that aberrational (e.g. astigmatism) and photophysical effects (e.g. optical saturation) may influence the result of an FCS experiment dramatically, so that a precise and reliable estimation of the diffusion coefficient is no longer possible. In this thesis, we report on the development, implementation, and application of a new and robust modification of FCS that we termed two-focus FCS (2fFCS) and which fulfils two requirements: (i) It introduces an external ruler into the measurement by generating two overlapping laser foci of precisely known and fixed distance. (ii) These two foci and corresponding detection regions are generated in such a way that the corresponding molecule detection functions (MDFs) are sufficiently well described by a simple two-parameter model yielding accurate diffusion coefficients when applied to 2fFCS data analysis. Both these properties enable us to measure absolute values of the diffusion coefficient with an accuracy of a few percent. Moreover, it will turn out that the new technique is robust against refractive index mismatch, coverslide thickness deviations, and optical saturation effects, which so often trouble conventional FCS measurements. This thesis deals mainly with the introduction of the new measurement scheme, 2fFCS, but also presents several applications with far-reaching importance. (orig.)

  3. Screen-printed fluorescent sensors for rapid and sensitive anthrax biomarker detection

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    Lee, Inkyu; Oh, Wan-Kyu; Jang, Jyongsik, E-mail: jsjang@plaza.snu.ac.kr

    2013-05-15

    Highlights: •We fabricated flexible anthrax sensors with a simple screen-printing method. •The sensors selectively detected B. anthracis biomarker. •The sensors provide the visible alarm against anthrax attack. -- Abstract: Since the 2001 anthrax attacks, efforts have focused on the development of an anthrax detector with rapid response and high selectivity and sensitivity. Here, we demonstrate a fluorescence sensor for detecting anthrax biomarker with high sensitivity and selectivity using a screen-printing method. A lanthanide–ethylenediamine tetraacetic acid complex was printed on a flexible polyethersulfone film. Screen-printing deposition of fluorescent detecting moieties produced fluorescent patterns that acted as a visual alarm against anthrax.

  4. Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

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    Xiangdong Kong

    Full Text Available The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR, for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36; trisomy 18 (n = 6; trisomy 13 (n = 4; 45, X (n = 5; 47, XXX (n = 3; 48, XXYY (n = 2; and unaffected controls (n = 40. We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.

  5. Rapid measurement of meat spoilage using fluorescence spectroscopy

    Science.gov (United States)

    Wu, Binlin; Dahlberg, Kevin; Gao, Xin; Smith, Jason; Bailin, Jacob

    2017-02-01

    Food spoilage is mainly caused by microorganisms, such as bacteria. In this study, we measure the autofluorescence in meat samples longitudinally over a week in an attempt to develop a method to rapidly detect meat spoilage using fluorescence spectroscopy. Meat food is a biological tissue, which contains intrinsic fluorophores, such as tryptophan, collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) etc. As meat spoils, it undergoes various morphological and chemical changes. The concentrations of the native fluorophores present in a sample may change. In particular, the changes in NADH and FAD are associated with microbial metabolism, which is the most important process of the bacteria in food spoilage. Such changes may be revealed by fluorescence spectroscopy and used to indicate the status of meat spoilage. Therefore, such native fluorophores may be unique, reliable and nonsubjective indicators for detection of spoiled meat. The results of the study show that the relative concentrations of all above fluorophores change as the meat samples kept in room temperature ( 19° C) spoil. The changes become more rapidly after about two days. For the meat samples kept in a freezer ( -12° C), the changes are much less or even unnoticeable over a-week-long storage.

  6. Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

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    Takahashi, Tadanobu; Agarikuchi, Takashi; Kurebayashi, Yuuki; Shibahara, Nona; Suzuki, Chihiro; Kishikawa, Akiko; Fukushima, Keijo; Takano, Maiko; Suzuki, Fumie; Wada, Hirohisa; Otsubo, Tadamune; Ikeda, Kiyoshi; Minami, Akira; Suzuki, Takashi

    2015-01-01

    Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

  7. Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

    Directory of Open Access Journals (Sweden)

    Tadanobu Takahashi

    Full Text Available Mumps viruses show diverse cytopathic effects (CPEs of infected cells and viral plaque formation (no CPE or no plaque formation in some cases depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac, was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study, even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

  8. Rapid screening test for porphyria diagnosis using fluorescence spectroscopy

    Science.gov (United States)

    Lang, A.; Stepp, H.; Homann, C.; Hennig, G.; Brittenham, G. M.; Vogeser, M.

    2015-07-01

    Porphyrias are rare genetic metabolic disorders, which result from deficiencies of enzymes in the heme biosynthesis pathway. Depending on the enzyme defect, different types of porphyrins and heme precursors accumulate for the different porphyria diseases in erythrocytes, liver, blood plasma, urine and stool. Patients with acute hepatic porphyrias can suffer from acute neuropathic attacks, which can lead to death when undiagnosed, but show only unspecific clinical symptoms such as abdominal pain. Therefore, in addition to chromatographic methods, a rapid screening test is required to allow for immediate identification and treatment of these patients. In this study, fluorescence spectroscopic measurements were conducted on blood plasma and phantom material, mimicking the composition of blood plasma of porphyria patients. Hydrochloric acid was used to differentiate the occurring porphyrins (uroporphyrin-III and coproporphyrin-III) spectroscopically despite their initially overlapping excitation spectra. Plasma phantom mixtures were measured using dual wavelength excitation and the corresponding concentrations of uroporphyrin-III and coproporphyrin-III were determined. Additionally, three plasma samples of porphyria patients were examined and traces of coproporphyrin-III and uroporphyrin-III were identified. This study may therefore help to establish a rapid screening test method with spectroscopic differentiation of the occurring porphyrins, which consequently allows for the distinction of different porphyrias. This may be a valuable tool for clinical porphyria diagnosis and rapid or immediate treatment.

  9. Rapid effects of diverse toxic water pollutants on chlorophyll a fluorescence: variable responses among freshwater microalgae.

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    Choi, Chang Jae; Berges, John A; Young, Erica B

    2012-05-15

    Chlorophyll a fluorescence of microalgae is a compelling indicator of toxicity of dissolved water contaminants, because it is easily measured and responds rapidly. While different chl a fluorescence parameters have been examined, most studies have focused on single species and/or a narrow range of toxins. We assessed the utility of one chl a fluorescence parameter, the maximum quantum yield of PSII (F(v)/F(m)), for detecting effects of nine environmental pollutants from a range of toxin classes on 5 commonly found freshwater algal species, as well as the USEPA model species, Pseudokirchneriella subcapitata. F(v)/F(m) declined rapidly over glyphosate (glyphosate increased exponentially with concentration. F(v)/F(m) provides a sensitive and easily-measured parameter for rapid and cost-effective detection of effects of many dissolved toxins. Field-portable fluorometers will facilitate field testing, however distinct responses between different species may complicate net F(v)/F(m) signal from a community. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Rapid Concentration of Nanoparticles with DC Dielectrophoresis in Focused Electric Fields

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    Chen Dafeng

    2009-01-01

    Full Text Available Abstract We report a microfluidic device for rapid and efficient concentration of micro/nanoparticles with direct current dielectrophoresis (DC DEP. The concentrator is composed of a series of microchannels constructed with PDMS-insulating microstructures for efficiently focusing the electric field in the flow direction to provide high field strength and gradient. The location of the trapped and concentrated particles depends on the strength of the electric field applied. Both ‘streaming DEP’ and ‘trapping DEP’ simultaneously take place within the concentrator at different regions. The former occurs upstream and is responsible for continuous transport of the particles, whereas the latter occurs downstream and rapidly traps the particles delivered from upstream. The performance of the device is demonstrated by successfully concentrating fluorescent nanoparticles. The described microfluidic concentrator can be implemented in applications where rapid concentration of targets is needed such as concentrating cells for sample preparation and concentrating molecular biomarkers for detection.

  11. Focused ion beam lithography for rapid prototyping of metallic films

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    Osswald, Patrick; Kiermaier, Josef; Becherer, Markus; Schmitt-Landsiedel, Doris [Lehrstuhl fuer Technische Elektronik, TU Muenchen, Munich (Germany)

    2010-07-01

    We present FIB-lithography methods for rapid and cost-effective prototyping of metal structures covering the deep-submicron- to the millimeter-range in a single lithography cycle. Focused ion beam (FIB) systems are widely used in semiconductor industry and research facilities for both analytical testing and prototyping. A typical application is to apply electrical contact to micron-sized sensors/particles by FIB induced metal deposition. However, as for E-beam lithography, patterning times for large area bonding pads are unacceptably long, resulting in cost-intensive prototyping. In this work, we optimized FIB lithography processing for negative and positive imaging mode to form metallic structures for large-areas down do the sub-100 nm range. For negative lithography features are defined by implanting Ga{sup +}-ions into a commercial photo resist, without affecting the underlying structures by impinging ions. The structures are highly suitable for following lift-off processing due to the undercut of the resist.Metallic feature size of down to 150 nm are achievable. For positive lithography a PMMA resist is exposed in FIB irradiation. Due to the very low dose (3.10{sup 12} ions/cm{sup 2}) the writing time for an e.g. 100 {mu}m x 100 {mu}m square is approx. 15 seconds. The developed resist is used for subsequent wet chemical etching, obtaining a 100 nm resolution in metal layers.

  12. Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.

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    Sean C Warren

    Full Text Available Fluorescence lifetime imaging (FLIM is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset. This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis

  13. Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.

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    Warren, Sean C; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda; Dunsby, Chris; French, Paul M W

    2013-01-01

    Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell

  14. Upconversion fluorescent strip sensor for rapid determination of Vibrio anguillarum

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    Zhao, Peng; Wu, Yuanyuan; Zhu, Yihua; Yang, Xiaoling; Jiang, Xin; Xiao, Jingfan; Zhang, Yuanxing; Li, Chunzhong

    2014-03-01

    Here, we report a simple and ultrasensitive upconversion fluorescent strip sensor based on NaYF4:Yb,Er nanoparticles (NPs) and the lateral flow immunochromatographic assay (LFIA). Carboxyl-modified β-NaYF4:Yb,Er NPs were successfully synthesized by a facile one-pot solvothermal approach, upon further coupling with monoclonal antibody, the resultant UCNPs-antibody conjugates probes were used in LFIA and served as signal vehicles for the fluorescent reporters. V. anguillarum was used as a model analyte to demonstrate the use of this strip sensor. The limit of the detection for the fluorescent strip was determined as 102 CFU mL-1, which is 100 times lower than those displayed by enzyme-linked immunosorbent assays, while the time needed for the detection was only 15 min. Furthermore, no cross-reaction with other eight pathogens was found, indicating the good specificity of the strip. This developed LFIA would offer the potential as a useful tool for the quantification of pathogens analysis in the future.

  15. Rapid intermittent movement of axonal neurofilaments observed by fluorescence photobleaching

    National Research Council Canada - National Science Library

    Wang, L; Brown, A

    2001-01-01

    Observations on naturally occurring gaps in the axonal neurofilament array of cultured neurons have demonstrated that neurofilament polymers move along axons in a rapid, intermittent, and highly asynchronous manner...

  16. Rapid identification of pathogens in blood cultures with a modified fluorescence in situ hybridization assay

    NARCIS (Netherlands)

    Peters, Remco P. H.; van Agtmael, Michiel A.; Simoons-Smit, Alberdina M.; Danner, Sven A.; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2006-01-01

    We evaluated a modified fluorescence in situ hybridization (FISH) assay for rapid ( <1 h) identification of microorganisms in growth-positive blood cultures. The results were compared to those of the standard FISH technique and conventional culturing. The rapid identification of microorganisms with

  17. Rapid detection of avian influenza A virus by immunochromatographic test using a novel fluorescent dye.

    Science.gov (United States)

    Yeo, Seon-Ju; Cuc, Bui Thi; Kim, Soon-Ai; Kim, Do Thi Hoang; Bao, Duong Tuan; Tien, Trinh Thi Thuy; Anh, Nguyen Thi Viet; Choi, Do-Young; Chong, Chom-Kyu; Kim, Hak Sung; Park, Hyun

    2017-08-15

    Sensitive and rapid diagnostic systems for avian influenza (AI) virus are required to screen large numbers of samples during a disease outbreak and to prevent the spread of infection. In this study, we employed a novel fluorescent dye for the rapid and sensitive recognition of AI virus. The styrylpyridine phosphor derivative was synthesized by adding allyl bromide as a stable linker and covalently immobilizing it on latex beads with antibodies generating the unique Red dye 53-based fluorescent probe. The performance of the innovative rapid fluorescent immnunochromatographic test (FICT) employing Red dye 53 in detecting the AI virus (A/H5N3) was 4-fold and 16-fold higher than that of Europium-based FICT and the rapid diagnostic test (RDT), respectively. In clinical studies, the presence of human nasopharyngeal specimens did not alter the performance of Red dye 53-linked FICT for the detection of H7N1 virus. Furthermore, in influenza A virus-infected human nasopharyngeal specimens, the sensitivity of the Red dye 53-based assay and RDT was 88.89% (8/9) and 55.56% (5/9) relative to rRT-PCR, respectively. The photostability of Red dye 53 was higher than that of fluorescein isothiocyanate (FITC), showing a stronger fluorescent signal persisting up to 8min under UV. The Red dye 53 could therefore be a potential probe for rapid fluorescent diagnostic systems that can recognize AI virus in clinical specimens. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Fusion-fission-fusion fast ignition plasma focus [rapid communication

    Science.gov (United States)

    Winterberg, F.

    2005-03-01

    A crucial advancement in the problem for the controlled release of energy by nuclear fusion appears possible by an autocatalytic fusion-fission-fusion microexplosion, where the deuterium-tritium (DT) fusion reaction of a dense magnetized DT plasma placed inside a thin liner made up of U238, Th232 (perhaps B10) releases a sufficient number of 14 MeV fusion neutrons which by fission reactions in the liner implode the liner on the DT plasma. The liner implosion increases the DT plasma density and with it the neutron output accelerating the fast fission reactions. Following the fast fission assisted ignition, a thermonuclear detonation wave can propagate into unburnt DT to reach a high gain. The simplest way for the realization of this concept appears to be the dense plasma focus configuration, amended with a nested high voltage magnetically insulated transmission line for the heating of the DT. The large magnetic field needed for the α-particle entrapment of the DT fusion reaction is here generated by the thermomagnetic Nernst effect, amplifying the magnetic field of the plasma focus current sheet.

  19. Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

    Science.gov (United States)

    Hemadi, Ahmad; Ekrami, Alireza; Oormazdi, Hormozd; Meamar, Ahmad Reza; Akhlaghi, Lame; Samarbaf-Zadeh, Ali Reza; Razmjou, Elham

    2015-05-01

    Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Rapid identification of Stenotrophomonas maltophilia by peptide nucleic acid fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Knudsen, Nanna Reumert; Rasmussen, A. K. I.; Fiandaca, M. J.

    2014-01-01

    The objective of this study was to develop a novel peptide nucleic acid (PNA) probe for Stenotrophomonas maltophilia identification by fluorescence in situ hybridization (FISH). The probe was evaluated using 33 human and veterinary clinical S. maltophilia isolates and 45 reference strains...... horses. Altogether the study shows that this species-specific PNA FISH probe facilitates rapid detection of S. maltophilia in biological specimens....

  1. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

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    Zhou, Changhua; Mao, Mao [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Yuan, Hang [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Shen, Huaibin [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Wu, Feng; Ma, Lan, E-mail: malan@sz.tsinghua.edu.cn [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Li, Lin Song, E-mail: lsli@henu.edu.cn [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China)

    2013-09-15

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 Degree-Sign C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

  2. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    Science.gov (United States)

    Zhou, Changhua; Mao, Mao; Yuan, Hang; Shen, Huaibin; Wu, Feng; Ma, Lan; Li, Lin Song

    2013-09-01

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 °C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

  3. Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes

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    Yu, F. P.; McFeters, G. A.

    1994-01-01

    Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.

  4. Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression

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    Yadetie Fekadu

    2004-06-01

    Full Text Available Abstract Background RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. Results RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. Conclusion RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

  5. Remote temperature measurements in femto-liter volumes using dual-focus-Fluorescence Correlation Spectroscopy.

    Science.gov (United States)

    Müller, Claus B; Weiss, Kerstin; Loman, Anastasia; Enderlein, Jörg; Richtering, Walter

    2009-05-07

    Remote temperature measurements in microfluidic devices with micrometer spatial resolution are important for many applications in biology, biochemistry and chemistry. The most popular methods use the temperature-dependent fluorescence lifetime of Rhodamine B, or the temperature-dependent size of thermosensitive materials such as microgel particles. Here, we use the recently developed method of dual-focus fluorescence correlation spectroscopy (2fFCS) for measuring the absolute diffusion coefficient of small fluorescent molecules at nanomolar concentrations and show how these data can be used for remote temperature measurements on a micrometer scale. We perform comparative temperature measurements using all three methods and show that the accuracy of 2fFCS is comparable or even better than that achievable with Rhodamine B fluorescence lifetime measurements. The temperature dependent microgel swelling leads to an enhanced accuracy within a narrow temperature range around the volume phase transition temperature, but requires the availability of specific microgels, whereas 2fFCS is applicable under very general conditions.

  6. Rapid analysis & design methodologies of High-Frequency LCLC Resonant Inverter as Electrodeless Fluorescent Lamp Ballast

    OpenAIRE

    Ang, Y A; Stone, D A; Bingham, Chris; Foster, M

    2007-01-01

    The papers presents methodologies for the analysis of 4th-order LCLC resonant power converters operating at 2.63 MHz as fluorescent lamp ballasts, where high frequency operation facilitates capacitive discharge into the tube, with near resonance operation at high load quality factor enabling high efficiency. State-variable dynamic descriptions of the converter are employed to rapidly determine the steady-state cyclic behaviour of the ballast during nominal operation. Simulation and experiment...

  7. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    OpenAIRE

    Hoseok Choi; Bomi Choi; Ju Tae Seo; Kyung Jin Lee; Myung Chan Gye; Young-Pil Kim

    2016-01-01

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) p...

  8. Rapid and sensitive detection of early esophageal squamous cell carcinoma with fluorescence probe targeting dipeptidylpeptidase IV

    Science.gov (United States)

    Onoyama, Haruna; Kamiya, Mako; Kuriki, Yugo; Komatsu, Toru; Abe, Hiroyuki; Tsuji, Yosuke; Yagi, Koichi; Yamagata, Yukinori; Aikou, Susumu; Nishida, Masato; Mori, Kazuhiko; Yamashita, Hiroharu; Fujishiro, Mitsuhiro; Nomura, Sachiyo; Shimizu, Nobuyuki; Fukayama, Masashi; Koike, Kazuhiko; Urano, Yasuteru; Seto, Yasuyuki

    2016-01-01

    Early detection of esophageal squamous cell carcinoma (ESCC) is an important prognosticator, but is difficult to achieve by conventional endoscopy. Conventional lugol chromoendoscopy and equipment-based image-enhanced endoscopy, such as narrow-band imaging (NBI), have various practical limitations. Since fluorescence-based visualization is considered a promising approach, we aimed to develop an activatable fluorescence probe to visualize ESCCs. First, based on the fact that various aminopeptidase activities are elevated in cancer, we screened freshly resected specimens from patients with a series of aminopeptidase-activatable fluorescence probes. The results indicated that dipeptidylpeptidase IV (DPP-IV) is specifically activated in ESCCs, and would be a suitable molecular target for detection of esophageal cancer. Therefore, we designed, synthesized and characterized a series of DPP-IV-activatable fluorescence probes. When the selected probe was topically sprayed onto endoscopic submucosal dissection (ESD) or surgical specimens, tumors were visualized within 5 min, and when the probe was sprayed on biopsy samples, the sensitivity, specificity and accuracy reached 96.9%, 85.7% and 90.5%. We believe that DPP-IV-targeted activatable fluorescence probes are practically translatable as convenient tools for clinical application to enable rapid and accurate diagnosis of early esophageal cancer during endoscopic or surgical procedures. PMID:27245876

  9. Rapid diagnosis and intraoperative margin assessment of human lung cancer with fluorescence lifetime imaging microscopy

    Directory of Open Access Journals (Sweden)

    Mengyan Wang

    2017-12-01

    Full Text Available A method of rapidly differentiating lung tumor from healthy tissue is extraordinarily needed for both the diagnosis and the intraoperative margin assessment. We assessed the ability of fluorescence lifetime imaging microscopy (FLIM for differentiating human lung cancer and normal tissues with the autofluorescence, and also elucidated the mechanism in tissue studies and cell studies. A 15-patient testing group was used to compare FLIM results with traditional histopathology diagnosis. Based on the endogenous fluorescence lifetimes of the testing group, a criterion line was proposed to distinguish normal and cancerous tissues. Then by blinded examined 41 sections from the validation group of other 16 patients, the sensitivity and specificity of FLIM were determined. The cellular metabolism was studied with specific perturbations of oxidative phosphorylation and glycolysis in cell studies. The fluorescence lifetime of cancerous lung tissues is consistently lower than normal tissues, and this is due to the both decrease of reduced nicotinamide adenine dinucleotide (NADH and flavin adenine dinucleotide (FAD lifetimes. A criterion line of lifetime at 1920 ps can be given for differentiating human lung cancer and normal tissues.The sensitivity and specificity of FLIM for lung cancer diagnosis were determined as 92.9% and 92.3%. These findings suggest that NADH and FAD can be used to rapidly diagnose lung cancer. FLIM is a rapid, accurate and highly sensitive technique in the judgment during lung cancer surgery and it can be potential in earlier cancer detection.

  10. Rapid diagnosis and intraoperative margin assessment of human lung cancer with fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Wang, Mengyan; Tang, Feng; Pan, Xiaobo; Yao, Longfang; Wang, Xinyi; Jing, Yueyue; Ma, Jiong; Wang, Guifang; Mi, Lan

    2017-12-01

    A method of rapidly differentiating lung tumor from healthy tissue is extraordinarily needed for both the diagnosis and the intraoperative margin assessment. We assessed the ability of fluorescence lifetime imaging microscopy (FLIM) for differentiating human lung cancer and normal tissues with the autofluorescence, and also elucidated the mechanism in tissue studies and cell studies. A 15-patient testing group was used to compare FLIM results with traditional histopathology diagnosis. Based on the endogenous fluorescence lifetimes of the testing group, a criterion line was proposed to distinguish normal and cancerous tissues. Then by blinded examined 41 sections from the validation group of other 16 patients, the sensitivity and specificity of FLIM were determined. The cellular metabolism was studied with specific perturbations of oxidative phosphorylation and glycolysis in cell studies. The fluorescence lifetime of cancerous lung tissues is consistently lower than normal tissues, and this is due to the both decrease of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) lifetimes. A criterion line of lifetime at 1920 ps can be given for differentiating human lung cancer and normal tissues.The sensitivity and specificity of FLIM for lung cancer diagnosis were determined as 92.9% and 92.3%. These findings suggest that NADH and FAD can be used to rapidly diagnose lung cancer. FLIM is a rapid, accurate and highly sensitive technique in the judgment during lung cancer surgery and it can be potential in earlier cancer detection.

  11. Rapid fluorometric determination of perfluorooctanoic acid by its quenching effect on the fluorescence of quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi; Huang, Aizhen; Wang, Nan, E-mail: nwang@hust.edu.cn; Zheng, Guan; Zhu, Lihua

    2015-05-15

    Analysis of perfluorooctanoic acid (PFOA) usually requires a combination of high-performance liquid chromatography and mass spectrometry, which is expensive and time-consuming. In the present work, water-soluble CdS quantum dots (QDs) were employed to develop a simple and rapid fluorometric method for the determination of PFOA. Strongly fluorescent CdS QDs were prepared by using 3-mercaptopropionic acid (MPA) as a stabilizer. It was observed that PFOA strongly quenched the fluorescence emission of the MPA-CdS QDs because PFOA promotes the aggregation of MPA-CdS QDs through a fluorine–fluorine affinity interaction. Under optimum conditions, the fluorescence intensity of MPA-CdS QDs was observed to decrease linearly with an increase in the concentration of PFOA from 0.5 to 40 μmol L{sup −1}, with a limit of detection of 0.3 μmol L{sup −1}. This new method was successfully implemented for the analysis of PFOA-spiked textile samples, with recoveries ranging from 95% to 113%. - Highlights: • PFOA significantly quenched the fluorescence emission of quantum dots (QDs). • A rapid and simple fluorescence sensor was proposed for determining PFOA by QDs. • PFOA determination could be completed within approximately 10 min. • The developed method had a working range of 0.5 to 40 μmol L{sup −1} and a detection limit of 0.3 μmol L{sup −1}.

  12. Rapid biocompatibility analysis of materials via in vivo fluorescence imaging of mouse models.

    Directory of Open Access Journals (Sweden)

    Kaitlin M Bratlie

    Full Text Available BACKGROUND: Many materials are unsuitable for medical use because of poor biocompatibility. Recently, advances in the high throughput synthesis of biomaterials has significantly increased the number of potential biomaterials, however current biocompatibility analysis methods are slow and require histological analysis. METHODOLOGY/PRINCIPAL FINDINGS: Here we develop rapid, non-invasive methods for in vivo quantification of the inflammatory response to implanted biomaterials. Materials were placed subcutaneously in an array format and monitored for host responses as per ISO 10993-6: 2001. Host cell activity in response to these materials was imaged kinetically, in vivo using fluorescent whole animal imaging. Data captured using whole animal imaging displayed similar temporal trends in cellular recruitment of phagocytes to the biomaterials compared to histological analysis. CONCLUSIONS/SIGNIFICANCE: Histological analysis similarity validates this technique as a novel, rapid approach for screening biocompatibility of implanted materials. Through this technique there exists the possibility to rapidly screen large libraries of polymers in vivo.

  13. Fluorescence-based lateral flow assays for rapid oral fluid roadside detection of cannabis use.

    Science.gov (United States)

    Plouffe, Brian D; Murthy, Shashi K

    2017-02-01

    With the recent worldwide changes in the legalization of marijuana, there is a significant need for rapid, roadside screening test for driving under the influence of drugs. A robust, sensitive, lateral flow assay has been developed to detect recent use via oral-fluid testing for Δ 9 -tetrahydrocannabinol (THC). This proof-of-concept assay uses a fluorescent-based immunoassay detection of polymeric beads, conjugated to antibodies against native THC. The fluorescent technique allows for significantly lower limits of detection and higher precision determination of recent marijuana use without the use of urine or blood sampling-thus allowing for roadside identification. Detection levels of 0.01 ng/mL were distinguished from background and the lower limit of quantification was determined to approach 1 ng/mL. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

    Directory of Open Access Journals (Sweden)

    Amar B. T. Ghisaidoobe

    2014-12-01

    Full Text Available F resonance energy transfer (FRET occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (\\(\\uplambda_{\\textsc{ex}}\\sim\\ nm, \\(\\uplambda_{\\textsc{em}}\\sim\\ 350 nm, in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the proteinlocal environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic F resonance energy transfer (iFRET, a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins.

  15. Fluorescent microscope system to monitor real-time interactions between focused ultrasound, echogenic drug delivery vehicles, and live cell membranes.

    Science.gov (United States)

    Ibsen, Stuart; Benchimol, Michael; Esener, Sadik

    2013-01-01

    Rapid development in the field of ultrasound triggered drug delivery has made it essential to study the real-time interaction between the membranes of live cells and the membranes of echogenic delivery vehicles under exposure to focused ultrasound. The objective of this work was to design an analysis system that combined fluorescent imagining, high speed videography, and definable pulse sequences of focused ultrasound to allow for real time observations of both cell and vehicle membranes. Documenting the behavior of the membranes themselves has not previously been possible due to limitations with existing optical systems used to understand the basic physics of microbubble/ultrasound interaction and the basic interaction between microbubbles and cells. The performance of this new system to monitor membrane behavior was demonstrated by documenting the modes of vehicle fragmentation at different ultrasound intensity levels. At 1.5MPa the membranes were shown to completely fragment while at intensities below 1MPa the membranes pop open and slowly unfold. The interaction between these vehicles and cell membranes was also documented by the removal of fluorescent particles from the surfaces of live cells out to 20μm from the microbubble location. The fluid flow created by microstreaming around ensonated microbubbles was documented at video recording speeds from 60 to 18,000 frames per second. This information about membrane behavior allows the chemical and physical properties of the drug delivery vehicle to be designed along with the ultrasound pulse sequence to cause the most efficient drug delivery. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Rapid identification of Stenotrophomonas maltophilia by peptide nucleic acid fluorescence in situ hybridization.

    Science.gov (United States)

    Hansen, N; Rasmussen, A K I; Fiandaca, M J; Kragh, K N; Bjarnsholt, T; Høiby, N; Stender, H; Guardabassi, L

    2014-05-01

    The objective of this study was to develop a novel peptide nucleic acid (PNA) probe for Stenotrophomonas maltophilia identification by fluorescence in situ hybridization (FISH). The probe was evaluated using 33 human and veterinary clinical S. maltophilia isolates and 45 reference strains representing common bacterial species in the respiratory tract. The probe displayed 100% sensitivity and 100% specificity on pure cultures and allowed detection in sputum from cystic fibrosis patients. The detection limit was 10(4) CFU/mL in spiked tracheal aspirate and bronchoalveolar lavage from healthy horses. Altogether the study shows that this species-specific PNA FISH probe facilitates rapid detection of S. maltophilia in biological specimens.

  17. Characterization of protein adsorption onto FePt nanoparticles using dual-focus fluorescence correlation spectroscopy

    Directory of Open Access Journals (Sweden)

    Pauline Maffre

    2011-07-01

    Full Text Available Using dual-focus fluorescence correlation spectroscopy, we have analyzed the adsorption of three human blood serum proteins, namely serum albumin, apolipoprotein A-I and apolipoprotein E4, onto polymer-coated, fluorescently labeled FePt nanoparticles (~12 nm diameter carrying negatively charged carboxyl groups on their surface. For all three proteins, a step-wise increase in hydrodynamic radius with protein concentration was observed, strongly suggesting the formation of protein monolayers that enclose the nanoparticles. Consistent with this interpretation, the absolute increase in hydrodynamic radius can be correlated with the molecular shapes of the proteins known from X-ray crystallography and solution experiments, indicating that the proteins bind on the nanoparticles in specific orientations. The equilibrium dissociation coefficients, measuring the affinity of the proteins to the nanoparticles, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of the electrostatic properties of the proteins. From structure-based calculations of the surface potentials, positively charged patches of different extents can be revealed, through which the proteins interact electrostatically with the negatively charged nanoparticle surfaces.

  18. Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

    Directory of Open Access Journals (Sweden)

    Heinz-Ulrich G. Weier

    2012-12-01

    Full Text Available Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols.

  19. [Rapid selection of recombinant orf virus expression vectors using green fluorescent protein].

    Science.gov (United States)

    Zhang, Jiachun; Guo, Xianfeng; Zhang, Min; Wu, Feifan; Peng, Yongzheng

    2016-01-01

    To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green fluorescent protein (GFP) as the reporter gene. The flanking regions of the ORFV132 of orf virus DNA were amplified by PCR to construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The shuttle plasmid was transfected into OFTu cells and GFP was incorporated into orf virus IA82Delta 121 by homologous recombination. The recombinant IA82Delta121-V was selected by green fluorescent signal. The deletion gene was identified by PCR and sequencing. The effects of ORFV132 knockout were evaluated by virus titration and by observing the proliferation of the infected vascular endothelial cells in vitro. The recombinant orf virus IA82Delta121-V was obtained successfully and quickly, and the deletion of ORFV132 did not affect the replication of the virus in vitro but reduced its virulence. Green fluorescent protein is a selectable marker for rapid, convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82Delta121-V can serve as a new expression vector for exogenous genes.

  20. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.

    Science.gov (United States)

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-04-16

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  1. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  2. Fluorescent immunochromatography for rapid and sensitive typing of seasonal influenza viruses.

    Directory of Open Access Journals (Sweden)

    Akira Sakurai

    Full Text Available Lateral flow tests also known as Immunochromatography (IC is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60% and the limit of detection (LOD is as low as 10³ pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (fluorescent immunochromatography; FLIC for subtyping H5 influenza viruses (FLIC-H5. Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB. This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75, specificity 100% (54/54, Type B: sensitivity 100% (90/90, specificity 98.2% (54/55 in nasal swab samples in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13 h than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.

  3. Rapid and sensitive screening of some acidic micronutrients in infant foods by HPLC with fluorescent detector.

    Science.gov (United States)

    Li, Guoliang; Kong, Weiheng; Fan, Guangsen; Wang, Wenli; Hu, Na; Chen, Guang; Zhao, Xianen; You, Jinmao

    2016-06-01

    Currently, commercially prepared complementary foods have become an important part of the diet of many infants and toddlers. But the method for simultaneous analysis of different types of micronutrient remains poorly investigated, which hinders the rapid and comprehensive quality control of infant foods. In the presented study, we first tried to employ the fluorescence labeling strategy combined with high-performance liquid chromatography-fluorescence detection for simultaneous determination of some acidic micronutrients including biotin, nicotinic acid, linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid and linoleic acid in infant foods. 2-(5-Benzoacridine) ethyl-p-toluenesulfonate was used as the fluorescence labeling reagent for simultaneous labeling of the seven components. The labeling conditions were optimized systematically by response surface methodology. The correlation coefficients for the calibration curves of the tested compounds ranged from 0.9991 to 0.9998. Limits of detection were in the range of 1.99-3.05 nmol L(-1) . Relative standard deviation values of retention time and peak area of seven compounds were less than 0.05% and 0.75%, respectively. The intra- and inter-day precision was in the range of 1.81-3.80% and 3.21-4.30%, respectively. When applied to analysis of several infant foods it showed good applicability. The developed method has been proven to be simple, inexpensive, selective, sensitive, accurate and reliable for analysis of some acidic micronutrients in infant foodstuffs. Furthermore, this developed method also has powerful potential in the analysis of many other complementary foodstuffs. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  4. A FRET-based ratiometric fluorescent aptasensor for rapid and onsite visual detection of ochratoxin A.

    Science.gov (United States)

    Qian, Jing; Wang, Kan; Wang, Chengquan; Hua, Mengjuan; Yang, Zhenting; Liu, Qian; Mao, Hanping; Wang, Kun

    2015-11-07

    A color change observable by the naked eye to indicate the content of an analyte is considered to be the most conceivable way of various sensing protocols. By taking advantage of the Förster resonance energy transfer (FRET) principles, we herein designed a dual-emission ratiometric fluorescent aptasensor for ochratoxin A (OTA) detection via a dual mode of fluorescent sensing and onsite visual screening. Amino group-modified OTA's aptamer was firstly labeled with the green-emitting CdTe quantum dots (gQDs) donor. The red-emitting CdTe QDs (rQDs) which were wrapped in the silica sphere could serve as the reference signal, while the gold nanoparticle (AuNP) acceptors were attached on the silica surface to bind with the thiolated complementary DNA (cDNA). The hybridization reaction between the aptamer and the cDNA brought gQD-AuNP pair close enough, thereby making the FRET occur in the aptasensor fabrication, while the subsequent fluorescence recovery induced by OTA was obtained in the detection procedure. Based on the red background of the wrapped rQDs, the aptasensor in response to increasing OTA displayed a distinguishable color change from red to yellow-green, which could be conveniently readout in solution even by the naked eye. Since the bioconjugations used as the aptasensor can be produced at large scale, this method can be used for in situ, rapid, or high-throughput OTA detection after only an incubation step in a homogeneous mode. We believe that this novel aptasensing strategy provides not only a promising method for OTA detection but also a universal model for detecting diverse targets by changing the corresponding aptamer.

  5. Mapping metals in Parkinson's and normal brain using rapid-scanning x-ray fluorescence

    Science.gov (United States)

    Popescu, Bogdan F. Gh; George, Martin J.; Bergmann, Uwe; Garachtchenko, Alex V.; Kelly, Michael E.; McCrea, Richard P. E.; Lüning, Katharina; Devon, Richard M.; George, Graham N.; Hanson, Akela D.; Harder, Sheri M.; Chapman, L. Dean; Pickering, Ingrid J.; Nichol, Helen

    2009-02-01

    Rapid-scanning x-ray fluorescence (RS-XRF) is a synchrotron technology that maps multiple metals in tissues by employing unique hardware and software to increase scanning speed. RS-XRF was validated by mapping and quantifying iron, zinc and copper in brain slices from Parkinson's disease (PD) and unaffected subjects. Regions and structures in the brain were readily identified by their metal complement and each metal had a unique distribution. Many zinc-rich brain regions were low in iron and vice versa. The location and amount of iron in brain regions known to be affected in PD agreed with analyses using other methods. Sample preparation is simple and standard formalin-fixed autopsy slices are suitable. RS-XRF can simultaneously and non-destructively map and quantify multiple metals and holds great promise to reveal metal pathologies associated with PD and other neurodegenerative diseases as well as diseases of metal metabolism.

  6. In vitro infectivity of oncolytic Newcastle Disease Virus: Correlation between plaque and fluorescent focus assays.

    Science.gov (United States)

    Rush, Benjamin S; Coughlin, Melissa L; Sanyal, Gautam

    2018-01-01

    Newcastle Disease Virus (NDV) is an avian paramyxovirus that has no significant pathogenicity in humans. Cancer cells with impaired immune defense mechanisms are susceptible to infection and lysis by NDV. A recombinant construct of a lentogenic form of NDV (rNDV) containing an insertion of granulocyte macrophage colony stimulating factor (GMCSF) transgene was earlier reported and shown to have acceptably low avian pathogenicity as well as oncolytic potential. Reliable measurement of infectious titer is key to determining the effectiveness of virus preparations to infect and lyse cells. We report here a comparative evaluation of two infectious titer assays as applied to rNDV: plaque assay and fluorescent focus assay (FFA). Optimization of assay conditions for both titer methods has produced concordant results spanning several orders of magnitude. While plaque formation is the gold standard measure of virus titer, FFA provides higher throughput and faster turn-around. FFA has been further evaluated on two different instrument platforms, for automated versus manual foci recognition and counting, with equivalent results. These results point to amenability of FFA to transfer between different laboratories and analysts, without introducing significant subjectivity in data analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  8. Development of a fluorescence-based sensor for rapid diagnosis of cyanide exposure.

    Science.gov (United States)

    Jackson, Randy; Oda, Robert P; Bhandari, Raj K; Mahon, Sari B; Brenner, Matthew; Rockwood, Gary A; Logue, Brian A

    2014-02-04

    Although commonly known as a highly toxic chemical, cyanide is also an essential reagent for many industrial processes in areas such as mining, electroplating, and synthetic fiber production. The "heavy" use of cyanide in these industries, along with its necessary transportation, increases the possibility of human exposure. Because the onset of cyanide toxicity is fast, a rapid, sensitive, and accurate method for the diagnosis of cyanide exposure is necessary. Therefore, a field sensor for the diagnosis of cyanide exposure was developed based on the reaction of naphthalene dialdehyde, taurine, and cyanide, yielding a fluorescent β-isoindole. An integrated cyanide capture "apparatus", consisting of sample and cyanide capture chambers, allowed rapid separation of cyanide from blood samples. Rabbit whole blood was added to the sample chamber, acidified, and the HCN gas evolved was actively transferred through a stainless steel channel to the capture chamber containing a basic solution of naphthalene dialdehyde (NDA) and taurine. The overall analysis time (including the addition of the sample) was cyanide exposure. Most importantly, the sensor was 100% accurate in diagnosing cyanide poisoning for acutely exposed rabbits.

  9. A Rapid Python-Based Methodology for Target-Focused Combinatorial Library Design.

    Science.gov (United States)

    Li, Shiliang; Song, Yuwei; Liu, Xiaofeng; Li, Honglin

    2016-01-01

    The chemical space is so vast that only a small portion of it has been examined. As a complementary approach to systematically probe the chemical space, virtual combinatorial library design has extended enormous impacts on generating novel and diverse structures for drug discovery. Despite the favorable contributions, high attrition rates in drug development that mainly resulted from lack of efficacy and side effects make it increasingly challenging to discover good chemical starting points. In most cases, focused libraries, which are restricted to particular regions of the chemical space, are deftly exploited to maximize hit rate and improve efficiency at the beginning of the drug discovery and drug development pipeline. This paper presented a valid methodology for fast target-focused combinatorial library design in both reaction-based and production-based ways with the library creating rates of approximately 70,000 molecules per second. Simple, quick and convenient operating procedures are the specific features of the method. SHAFTS, a hybrid 3D similarity calculation software, was embedded to help refine the size of the libraries and improve hit rates. Two target-focused (p38-focused and COX2-focused) libraries were constructed efficiently in this study. This rapid library enumeration method is portable and applicable to any other targets for good chemical starting points identification collaborated with either structure-based or ligand-based virtual screening.

  10. Rapid detection of fluorescent and chemiluminescent total coliforms and Escherichia coli on membrane filters.

    Science.gov (United States)

    Van Poucke, S O; Nelis, H J

    2000-11-01

    The detection of fluorescent colonies of Escherichia coli/total coliforms (TC) on a membrane filter is currently carried out using 4-methylumbelliferyl-beta-D-glycosides as enzyme substrates and a UV-lamp for visualization. The most rapid procedures based on this approach for the demonstration of these indicator bacteria in water take 6-7.5 h to complete. As part of efforts to further reduce the detection time, an improved two-step procedure for the fluorescence or chemiluminescence labelling of microcolonies of E. coli/TC on a membrane filter has been developed. Essential features of this approach include a separation of the bacterial propagation and target enzyme induction from the actual enzymatic labelling, the use of improved fluorogenic, i.e., 4-trifluoromethylumbelliferyl-beta-D-glycosides and fluorescein-di-beta-D-glycosides, or chemiluminogenic (i.e., phenylglucuronic- or galactose-substituted adamantyl 1,2-dioxetanes) substrates for beta-glucuronidase/beta-galactosidase, of enzyme inducers, of special membrane filters and of polymyxin B to promote the cellular uptake of the substrate. This labelling procedure has been applied in conjunction with different detection devices including a UV-lamp, CCD-cameras, X-ray film and the ChemScan((R)) RDI. Using the former three, microcolonies of pure cultures could be detected within 5.5-6.5 h, but waterborne E. coli/TC may fail to form microcolonies in this short time period, thus yielding poor sensitivity and a high false-negative rate. In contrast, a quantitative enumeration was feasible in less than 4 h with the ChemScan((R)) RDI, owing to its ability to detect both microcolonies and non-dividing single cells.

  11. Rapid and accurate identification of Xanthomonas citri subspecies citri by fluorescence in situ hybridization.

    Science.gov (United States)

    Waite, D W; Griffin, R; Taylor, R; George, S

    2016-11-01

    Citrus canker is an economically important disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). This organism targets a wide range of citrus plants, including sweet orange, grapefruit, lemon and lime. As Xcc is spread by environmental factors such as wind and rain, it is difficult to control its movement once the disease has established. In order to facilitate monitoring of citrus canker we sought to design a novel diagnostic protocol based on fluorescence in situ hybridization (FISH) for identification of bacterial cells directly from canker pustules without cultivation or DNA extraction. This method was validated for specificity against a range of Xanthomonas species and strains. We show that our assay is extremely rapid (typically requiring between 2 and 3 h), and possesses a similar specificity to existing PCR diagnostic tools. The sensitivity of the assay is comparable to that of an existing PCR-based technique and sufficient for identifying Xcc in symptomatic plant material. The method is easily transferable to diagnosticians without prior experience using FISH. Xanthomonas citri subsp. citri (Xcc) is an aggressive and hardy pathogen of citrus plants worldwide. Outbreaks are difficult and costly to contain and the establishment of citrus canker results in restricted trade. In order to extend the existing toolkit for identification of Xcc we developed a novel diagnostic approach based on fluorescence in situ hybridization. Our approach is of comparable specificity and sensitivity to existing methods but can be performed directly on infected tissue making it significantly faster than existing PCRs, and requiring fewer laboratory resources. © 2016 The Society for Applied Microbiology.

  12. Improved Savitzky-Golay-method-based fluorescence subtraction algorithm for rapid recovery of Raman spectra.

    Science.gov (United States)

    Chen, Kun; Zhang, Hongyuan; Wei, Haoyun; Li, Yan

    2014-08-20

    In this paper, we propose an improved subtraction algorithm for rapid recovery of Raman spectra that can substantially reduce the computation time. This algorithm is based on an improved Savitzky-Golay (SG) iterative smoothing method, which involves two key novel approaches: (a) the use of the Gauss-Seidel method and (b) the introduction of a relaxation factor into the iterative procedure. By applying a novel successive relaxation (SG-SR) iterative method to the relaxation factor, additional improvement in the convergence speed over the standard Savitzky-Golay procedure is realized. The proposed improved algorithm (the RIA-SG-SR algorithm), which uses SG-SR-based iteration instead of Savitzky-Golay iteration, has been optimized and validated with a mathematically simulated Raman spectrum, as well as experimentally measured Raman spectra from non-biological and biological samples. The method results in a significant reduction in computing cost while yielding consistent rejection of fluorescence and noise for spectra with low signal-to-fluorescence ratios and varied baselines. In the simulation, RIA-SG-SR achieved 1 order of magnitude improvement in iteration number and 2 orders of magnitude improvement in computation time compared with the range-independent background-subtraction algorithm (RIA). Furthermore the computation time of the experimentally measured raw Raman spectrum processing from skin tissue decreased from 6.72 to 0.094 s. In general, the processing of the SG-SR method can be conducted within dozens of milliseconds, which can provide a real-time procedure in practical situations.

  13. Rapid and accurate tumor-target bio-imaging through specific in vivo biosynthesis of a fluorescent europium complex.

    Science.gov (United States)

    Ye, Jing; Wang, Jianling; Li, Qiwei; Dong, Xiawei; Ge, Wei; Chen, Yun; Jiang, Xuerui; Liu, Hongde; Jiang, Hui; Wang, Xuemei

    2016-04-01

    A new and facile method for rapidly and accurately achieving tumor targeting fluorescent images has been explored using a specifically biosynthesized europium (Eu) complex in vivo and in vitro. It demonstrated that a fluorescent Eu complex could be bio-synthesized through a spontaneous molecular process in cancerous cells and tumors, but not prepared in normal cells and tissues. In addition, the proteomics analyses show that some biological pathways of metabolism, especially for NADPH production and glutamine metabolism, are remarkably affected during the relevant biosynthesis process, where molecular precursors of europium ions are reduced to fluorescent europium complexes inside cancerous cells or tumor tissues. These results proved that the specific self-biosynthesis of a fluorescent Eu complex by cancer cells or tumor tissues can provide a new strategy for accurate diagnosis and treatment strategies in the early stages of cancers and thus is beneficial for realizing precise surgical intervention based on the relevant cheap and readily available agents.

  14. Imaging inflammation in mouse colon using a rapid stage-scanning confocal fluorescence microscope.

    Science.gov (United States)

    Saldua, Meagan A; Olsovsky, Cory A; Callaway, Evelyn S; Chapkin, Robert S; Maitland, Kristen C

    2012-01-01

    Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333 lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7 mm/sec. A single 1 × 60 mm(2) field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion.

  15. Rapid microwave-assisted synthesis of molecularly imprinted polymers on carbon quantum dots for fluorescent sensing of tetracycline in milk.

    Science.gov (United States)

    Hou, Juan; Li, Huiyu; Wang, Long; Zhang, Ping; Zhou, Tianyu; Ding, Hong; Ding, Lan

    2016-01-01

    In this paper, a novel, selective and eco-friendly sensor for the detection of tetracycline was developed by grafting imprinted polymers onto the surface of carbon quantum dots. A simple microwave-assisted approach was utilized to fabricate the fluorescent imprinted composites rapidly for the first time, which could shorten the polymerization time and simplify the experimental procedure dramatically. The novel composites not only demonstrated excellent fluorescence stability and special binding sites, but also could selectively accumulate target analytes. Under optimal conditions, the relative fluorescence intensity of the composites decreased linearly with increasing the concentration of tetracycline from 20 nM to 14 µM. The detection limit of tetracycline was 5.48 nM. The precision and reproducibility of the proposed sensor were also acceptable. Significantly, the practicality of this ultrasensitive sensor for tetracycline detection in milk was further validated, revealing the advantages of simplicity, sensitivity, selectivity and low cost. This approach combines the high selective adsorption property of molecular imprinted polymers and the sensitivity of fluorescence detection. It is envisioned that the development of fluorescent molecularly imprinted composites will offer a new way of thinking for rapid analysis in complex samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Time-Resolved Fluorescent Immunochromatography of Aflatoxin B1 in Soybean Sauce: A Rapid and Sensitive Quantitative Analysis.

    Science.gov (United States)

    Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Zhang, Wen

    2016-07-14

    Rapid and quantitative sensing of aflatoxin B1 with high sensitivity and specificity has drawn increased attention of studies investigating soybean sauce. A sensitive and rapid quantitative immunochromatographic sensing method was developed for the detection of aflatoxin B1 based on time-resolved fluorescence. It combines the advantages of time-resolved fluorescent sensing and immunochromatography. The dynamic range of a competitive and portable immunoassay was 0.3-10.0 µg·kg(-1), with a limit of detection (LOD) of 0.1 µg·kg(-1) and recoveries of 87.2%-114.3%, within 10 min. The results showed good correlation (R² > 0.99) between time-resolved fluorescent immunochromatographic strip test and high performance liquid chromatography (HPLC). Soybean sauce samples analyzed using time-resolved fluorescent immunochromatographic strip test revealed that 64.2% of samples contained aflatoxin B1 at levels ranging from 0.31 to 12.5 µg·kg(-1). The strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique in food safety analysis.

  17. Rapid Detection of Enterotoxigenic Clostridium perfringens by Real-Time Fluorescence Resonance Energy Transfer PCR

    National Research Council Canada - National Science Library

    dela Cruz, Wilfred P; Gozum, Mary M.A; Lineberry, Sarah F; Stassen, Sarah D; Daughtry, Marianne; Stassen, Nicholas A; Jones, Morris S; Johnson, Oswald L

    2006-01-01

    ...) produced by some strains during sporulation. We developed a quantitative real-time PCR assay based on fluorescence resonance energy transfer hybridization chemistry that targets the C. perfringens...

  18. Development of a fluorescence in situ hybridization (FISH) method for rapid detection of Ulva prolifera

    Science.gov (United States)

    Zhang, Qing-Chun; Liu, Qing; Kang, Zhen-Jun; Yu, Ren-Cheng; Yan, Tian; Zhou, Ming-Jiang

    2015-09-01

    Large-scale green tides have occurred consecutively since 2007 in the Yellow Sea (YS), China. The dominant causative species of the green tides has been identified as Ulva prolifera. The origin of green tides in the YS has been traced back to the Subei Shoal based on the results of remote-sensing, numerical simulations and field investigations. However, it is difficult to study the early development of green tides in the Subei Shoal because of the mixture of multiple green algae and the morphological diversity of U. prolifera when under variable environmental conditions. In this study, a rapid and accurate fluorescence in situ hybridization (FISH) method was developed to detect U. prolifera from the community of green algae targeting the 5S rDNA spacer region of U. prolifera. Two specific probes, 5S-1 and 5S-2, were designed based on the sequences of the 5S rDNA spacer regions of U. prolifera, Ulva linza and Ulva flexuosa. Specificity of the FISH method was tested using the six species of green algae commonly occurring in the Subei Shoal, including U. prolifera, U. linza, U. flexuosa, Ulva compressa, Ulva pertusa and Blidingia sp. The results showed that only U. prolifera could be labeled with both probes. Probe 5S-1, which showed a much higher labeling efficiency on U. prolifera, was ultimately selected as the probe for the FISH detection. The sample preparation method was optimized, particularly for the mature green algae, by the addition of cellulase and proteinase K in the pre-hybridization solution. Labeling efficiency with the probe 5S-1 reached 96% on average under the optimized conditions. The successful development of the FISH method has been applied to qualitative and quantitative analysis of field samples collected from the YS, and the results indicate a potential use in future green algae studies.

  19. Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts.

    Science.gov (United States)

    Ozbay, Baris N; Losacco, Justin T; Cormack, Robert; Weir, Richard; Bright, Victor M; Gopinath, Juliet T; Restrepo, Diego; Gibson, Emily A

    2015-06-01

    We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2  g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ∼12  μm and an axial scan range of ∼80  μm. The lateral field-of-view is 300 μm, and the lateral resolution is 1.8 μm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP).

  20. Rapid and reliable determination of p-nitroaniline in wastewater by molecularly imprinted fluorescent polymeric ionic liquid microspheres.

    Science.gov (United States)

    Lu, Xing; Yang, Yiwen; Zeng, Yanbo; Li, Lei; Wu, Xiaohua

    2018-01-15

    Rapid and efficient detecting trace amount of environmental p-nitroaniline (p-NA) is in urgent need for security concerns and pollution supervision. In this work we report the use of molecularly imprinted polymeric ionic liquid (MIPIL) microspheres to construct recognizable surfaces for detection of p-NA through fluorescence quenching. The p-NA imprinted microspheres are synthesized by precipitation polymerization upon co-polymerization of 3-(anthracen-9-ylmethyl)-1-vinyl-1H-imidazol-3-ium chloride (Fluorescent IL monomer) with ethyleneglycol dimethacrylate (EGDMA). The electron-rich group alkenyl imidazole in IL functional monomer can dramatically improve the emission of anthracene fluorophore and the π-π stacking, electronic, and hydrogen bond between p-NA and MIPIL can efficiently enhance the selective recognition force. The as-synthesized MIPIL microspheres present spherical shape, high fluorescence emission intensity and specific recognition, which showed rapid detection rate (1min), stable reusable property (at least 4 time recycles), wonderful selectivity over several structural analogs, wide linear range (10nM to 10M) with a correlation coefficient of 0.992, and excellent sensitivity (LOD, 9nM). As synthesis and surface functionalization of MIPIL microspheres are well established, the methods reported in this work are facile, rapid and efficient for monitoring p-NA in environmental wastewater. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. A ratiometric fluorescent probe for rapid and sensitive detection of biothiols in fetal bovine serum.

    Science.gov (United States)

    Wang, Fengyang; Feng, Chongchong; Lu, Linlin; Xu, Zhiai; Zhang, Wen

    2017-07-01

    Herein, a ratiometric turn-on fluorescent probe for sensitive detection of biothiols was designed. The probe consisted of two parts: one was rhodamine B serving as a fluorescence reference, and the other was coumarin derivative as the responsive fluorophore with an acrylate group for biothiols recognition. The response was based on the mechanism of Michael addition and intramolecular cyclization reaction, and the probe showed ratiometric and sensitive response to biothiols. Especially, the detection limit of this probe for cysteine was found to be 0.13μΜ. More importantly, the probe showed the advantage of fast response, of which the fluorescence intensity can reach the maximum within 10min. The ratiometric fluorescent probe has been successfully applied for the determination of biothiols in fetal bovine serum samples and the result was in good agreement with that tested by Ellman method. Copyright © 2017. Published by Elsevier B.V.

  2. Development of immunochromatographic strip test using fluorescent, micellar silica nanosensors for rapid detection of B. abortus antibodies in milk samples.

    Science.gov (United States)

    Vyas, Swati S; Jadhav, Sushma V; Majee, Sharmila B; Shastri, Jayanthi S; Patravale, Vandana B

    2015-08-15

    Presence of bacteria such as Brucella spp. in dairy products is an immense risk to public health. Point of care immunoassays are rapid in that they can quickly screen various samples in a relatively short amount of time, are sensitive, specific and offer a great advantage in accurate and fast diagnosis of infectious diseases. We have fabricated a point of care rapid diagnostic assay that employs fluorescent, micellar silica nanosensors capable of specifically detecting Brucella IgG antibodies in milk samples of afflicted animals. Currently, point of care detection assays are not commercially available for field testing of farm animals using milk samples. The nanosensing allows precise detection of antibodies with low sample volumes (50 μl). We demonstrate recognition of B. abortus antibodies through capture by fluorescent silica nanosensors using spiked and raw milk samples validated by ELISA and PCR. The test results are accurate and repeatable with high sensitivity and specificity, and a short assay time of 10 min for antigenic recognition and do not require any sample processing procedures such as isolation and separation. Additionally, well defined antigenic components and surface biomarkers of various disease causing microbes can be broadly incorporated within the purview of this technology for accurate and rapid detection of suspected bovine pathological conditions, and can largely enable rapid field testing that can be implemented in farms and food industry. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Application of fluorescently labelled lectins for the study of polysaccharides in biofilms with a focus on biofouling of nanofiltration membranes

    Directory of Open Access Journals (Sweden)

    Patrick Di Martino

    2016-07-01

    Full Text Available The biofilm state is the dominant microbial lifestyle in nature. A biofilm can be defined as cells organised as microcolonies embedded in an organic polymer matrix of microbial origin living at an interface between two different liquids, air and liquid, or solid and liquid. The biofilm matrix is made of extracellular polymeric substances, polysaccharides being considered as the major structural components of the matrix. Fluorescently labelled lectins have been widely used to stain microbial extracellular glycoconjugates in natural and artificial environments, and to study specific bacterial species or highly complex environments. Biofilm development at the membrane surface conducting to biofouling is one of the major problems encountered during drinking water production by filtration. Biofouling affects the durability and effectiveness of filtration membranes. Biofouling can be reduced by pretreatments in order to control two key parameters of water, the bioavailable organic matter concentration and the concentration of live bacteria. Nanofiltration (NF is a high technology process particularly suited to the treatment of surface waters to produce drinking water that is highly sensitive to biofouling. The development of strategies for fouling prevention and control requires characterizing the fouling material composition and organisation before and after NF membrane cleaning. The aim of this review is to present basics of biofilm analyses after staining with fluorescently labelled lectins and to focus on the use of fluorescent lectins and confocal laser scanning microscopy to analyse NF membrane biofouling.

  4. Nile Red fluorescence spectrum decomposition enables rapid screening of large protein aggregates in complex biopharmaceutical formulations like influenza vaccines.

    Science.gov (United States)

    Sahin, Ziya; Akkoc, Senem; Neeleman, Ronald; Haines, Jonathan; Kayser, Veysel

    2017-05-25

    The extensive presence of large (high molecular weight) protein aggregates in biopharmaceutical formulations is a concern for formulation stability and possibly safety. Tests to screen large aggregate content in such bioformulations are therefore needed for rapid and reliable quality control in industrial settings. Herein, non-commercial seasonal influenza split-virus vaccine samples, produced using various strains and extracted from selected industrial processing steps, were used as model complex bioformulations. Orthogonal characterization through transmission electron microscopy, UV-Vis absorption spectroscopy, fluorescence emission spectroscopy, high-performance liquid chromatography and single-radial immunodiffusion revealed that large, amorphous protein aggregates are formed after virus splitting and their presence is linked mainly, albeit not only, to surfactant (Triton X-100) content in a sample. Importantly, the presence of large virus aggregates in purified whole virus samples and large protein aggregates in vaccine samples was found to correlate with broadening/shouldering in Nile Red fluorescence spectra. Accordingly, decomposition of Nile Red spectra into components allowed the development of a novel, rapid, reliable and user-friendly test with high-throughput potential for screening large aggregate content in influenza split-virus vaccines. The test can be adapted for screening other complex biopharmaceutical formulations, provided relevant controls are done for informed decomposition of fluorescence spectra into their components. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Toxicant induced changes on delayed fluorescence decay kinetics of cyanobacteria and green algae: a rapid and sensitive biotest.

    Directory of Open Access Journals (Sweden)

    Franziska Leunert

    Full Text Available Algal tests have developed into routine tools for testing toxicity of pollutants in aquatic environments. Meanwhile, in addition to algal growth rates, an increasing number of fluorescence based methods are used for rapid and sensitive toxicity measures. The present study stresses the suitability of delayed fluorescence (DF as a promising parameter for biotests. DF is based on the recombination fluorescence at the reaction centre of photosystem II, which is emitted only by photosynthetically active cells. We analyzed the effects of three chemicals (3-(3,4-dichlorophenyl-1,1-dimethylurea (DCMU, 3,5 Dichlorophenol (3,5 DCP and copper on the shape of the DF decay kinetics for potential use in phytoplankton toxicity tests. The short incubation tests were done with four phytoplankton species, with special emphasis on the cyanobacterium Microcystis aeruginosa. All species exhibited a high sensitivity to DCMU, but cyanobacteria were more affected by copper and less by 3,5 DCP than the tested green algae. Analyses of changes in the DF decay curve in response to the added chemicals indicated the feasibility of the DF decay approach as a rapid and sensitive testing tool.

  6. Rectangular coordination polymer nanoplates: large-scale, rapid synthesis and their application as a fluorescent sensing platform for DNA detection.

    Science.gov (United States)

    Zhang, Yingwei; Luo, Yonglan; Tian, Jingqi; Asiri, Abdullah M; Al-Youbi, Abdulrahman O; Sun, Xuping

    2012-01-01

    In this paper, we report on the large-scale, rapid synthesis of uniform rectangular coordination polymer nanoplates (RCPNs) assembled from Cu(II) and 4,4'-bipyridine for the first time. We further demonstrate that such RCPNs can be used as a very effective fluorescent sensing platform for multiple DNA detection with a detection limit as low as 30 pM and a high selectivity down to single-base mismatch. The DNA detection is accomplished by the following two steps: (1) RCPN binds dye-labeled single-stranded DNA (ssDNA) probe, which brings dye and RCPN into close proximity, leading to fluorescence quenching; (2) Specific hybridization of the probe with its target generates a double-stranded DNA (dsDNA) which detaches from RCPN, leading to fluorescence recovery. It suggests that this sensing system can well discriminate complementary and mismatched DNA sequences. The exact mechanism of fluorescence quenching involved is elucidated experimentally and its use in a human blood serum system is also demonstrated successfully.

  7. A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction

    Directory of Open Access Journals (Sweden)

    Jakobsdottir Klara B

    2002-06-01

    Full Text Available Abstract Background The jellyfish green fluorescent protein (GFP can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. Results We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, αs. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. Conclusion This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

  8. PARAFAC modeling of fluorescence excitation-emission spectra of fish bile for rapid en route screening of PAC exposure.

    Science.gov (United States)

    Christensen, Jan H; Tomasi, Giorgio; Strand, Jakob; Andersen, Ole

    2009-06-15

    Polycyclic aromatic compound (PAC) metabolites in fish bile can be used as biomarkers for recent environmental exposure to PACs. Here, a novel method for rapid screening of nonhydrolyzed fish bile is presented. The method is based on excitation-emission fluorescence spectroscopy combined with parallel factor analysis (PARAFAC) and may constitute an alternative to fixed wavelength fluorescence and synchronous fluorescence spectroscopy (SFS). PARAFAC was applied to excitation-emission matrices (EEMs) of bile samples of shorthorn sculpins and European eels collected in Greenland and Denmark. The EEMs were decomposed into a four-factor PARAFAC model. The comparison of the PARAFAC factors with the EEMs of PAC metabolites and amino acids suggests that two factors are related to PAC metabolites and two correspond to fluorescent residues of tryptophan and tyrosine in bile proteins. A new standardization procedure based on the mean of the scores for the biological factors was used to correct for feeding status and sample dilution and, upon such normalization, the score plots of PARAFAC factors showed a clear distinction between exposed and nonexposed fish. A good correlation was found between the factor scores and 1-hydroxypyrene equivalents determined by SFS for high contamination levels, whereas the sensitivity was better for the EEM method.

  9. Rapid fluorescence lifetime estimation with modified phasor approach and Laguerre deconvolution: a comparative study

    Science.gov (United States)

    Fereidouni, Farzad; Gorpas, Dimitris; Ma, Dinglong; Fatakdawala, Hussain; Marcu, Laura

    2017-09-01

    Fluorescence lifetime imaging has been shown to serve as a valuable tool for interrogating and diagnosis of biological tissue at a mesoscopic level. The ability to analyze fluorescence decay curves to extract lifetime values in real-time is crucial for clinical translation and applications such as tumor margin delineation or intracoronary imaging of atherosclerotic plaques. In this work, we compare the performance of two popular non-parametric (fit-free) methods for determining lifetime values from fluorescence decays in real-time—the Phasor approach and Laguerre deconvolution. We demonstrate results from simulated and experimental data to compare the accuracy and speed of both methods and their dependence on noise and model parameters.

  10. Green Fluorescent Protein-Focused Bioinformatics Laboratory Experiment Suitable for Undergraduates in Biochemistry Courses

    Science.gov (United States)

    Rowe, Laura

    2017-01-01

    An introductory bioinformatics laboratory experiment focused on protein analysis has been developed that is suitable for undergraduate students in introductory biochemistry courses. The laboratory experiment is designed to be potentially used as a "stand-alone" activity in which students are introduced to basic bioinformatics tools and…

  11. PARAFAC modeling of fluorescence excitation - Emission spectra of fish bile for rapid en route screening of PAC exposure

    DEFF Research Database (Denmark)

    Christensen, Jan H.; Tomasi, Giorgio; Strand, Jakob

    2009-01-01

    . The EEMs were decomposed into a four-factor PARAFAC model. The comparison of the PARAFAC factors with the EEMs of PAC metabolites and amino acids suggests that two factors are related to PAC metabolites and two correspond to fluorescent residues of tryptophan and tyrosine in bile proteins. A new......Polycyclic aromatic compound (PAC) metabolites in fish bile can be used as biomarkers for recent environmental exposure to PACs. Here, a novel method for rapid screening of nonhydrolyzed fish bile is presented. The method is based on excitation-emission fluorescence spectroscopy combined...... standardization procedure based on the mean of the scores for the biological factors was used to correct for feeding status and sample dilution and, upon such normalization, the score plots of PARAFAC factors showed a clear distinction between exposed and nonexposed fish. A good correlation was found between...

  12. Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o-phthalaldehyde.

    Science.gov (United States)

    Maddukuri, Naveen; Zhang, Qiyang; Zhang, Ning; Gong, Maojun

    2017-02-01

    LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high-throughput analysis with flow-gated CE. Here, we report a fast fluorescein-labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow-gated CE. This scheme was based on the reaction between primary amines and o-phthalaldehyde in the presence of a fluorescent thiol, 2-((5-fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE-SH). The short reaction time (neurotransmitters by coupling in vitro microdialysis with online derivatization and flow-gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on-chip labeling of proteins retained on support in SPE. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. [Rapid Identification of Epicarpium Citri Grandis via Infrared Spectroscopy and Fluorescence Spectrum Imaging Technology Combined with Neural Network].

    Science.gov (United States)

    Pan, Sha-sha; Huang, Fu-rong; Xiao, Chi; Xian, Rui-yi; Ma, Zhi-guo

    2015-10-01

    To explore rapid reliable methods for detection of Epicarpium citri grandis (ECG), the experiment using Fourier Transform Attenuated Total Reflection Infrared Spectroscopy (FTIR/ATR) and Fluorescence Spectrum Imaging Technology combined with Multilayer Perceptron (MLP) Neural Network pattern recognition, for the identification of ECG, and the two methods are compared. Infrared spectra and fluorescence spectral images of 118 samples, 81 ECG and 37 other kinds of ECG, are collected. According to the differences in tspectrum, the spectra data in the 550-1 800 cm(-1) wavenumber range and 400-720 nm wavelength are regarded as the study objects of discriminant analysis. Then principal component analysis (PCA) is applied to reduce the dimension of spectroscopic data of ECG and MLP Neural Network is used in combination to classify them. During the experiment were compared the effects of different methods of data preprocessing on the model: multiplicative scatter correction (MSC), standard normal variable correction (SNV), first-order derivative(FD), second-order derivative(SD) and Savitzky-Golay (SG). The results showed that: after the infrared spectra data via the Savitzky-Golay (SG) pretreatment through the MLP Neural Network with the hidden layer function as sigmoid, we can get the best discrimination of ECG, the correct percent of training set and testing set are both 100%. Using fluorescence spectral imaging technology, corrected by the multiple scattering (MSC) results in the pretreatment is the most ideal. After data preprocessing, the three layers of the MLP Neural Network of the hidden layer function as sigmoid function can get 100% correct percent of training set and 96.7% correct percent of testing set. It was shown that the FTIR/ATR and fluorescent spectral imaging technology combined with MLP Neural Network can be used for the identification study of ECG and has the advantages of rapid, reliable effect.

  14. Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images

    NARCIS (Netherlands)

    Fereidouni, F.|info:eu-repo/dai/nl/372641431; Bader, A.N.|info:eu-repo/dai/nl/291137334; Gerritsen, H.C.|info:eu-repo/dai/nl/071548777

    2012-01-01

    A new global analysis algorithm to analyse (hyper-) spectral images is presented. It is based on the phasor representation that has been demonstrated to be very powerful for the analysis of lifetime imaging data. In spectral phasor analysis the fluorescence spectrum of each pixel in the image is

  15. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes

    NARCIS (Netherlands)

    Jansen, GJ; Mooibroek, M; Idema, J; Harmsen, HJM; Welling, GW; Degener, JE

    The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial

  16. Aptamer contained triple-helix molecular switch for rapid fluorescent sensing of acetamiprid.

    Science.gov (United States)

    Liu, Xin; Li, Ying; Liang, Jing; Zhu, Wenyue; Xu, Jingyue; Su, Ruifang; Yuan, Lei; Sun, Chunyan

    2016-11-01

    In this study, an aptamer-based fluorescent sensing platform using triple-helix molecular switch (THMS) was developed for the pesticide screening represented by acetamiprid. The THMS was composed of two tailored DNA probes: a label-free central target specific aptamer sequence flanked by two arm segments acting as a recognition probe; a hairpin-shaped structure oligonucleotide serving as a signal transduction probe (STP), labeled with a fluorophore and a quencher at the 3' and 5'-end, respectively. In the absence of acetamiprid, complementary bindings of two arm segments of the aptamers with the loop sequence of STP enforce the formation of THMS with the "open" configuration of STP, and the fluorescence of THMS is on. In the presence of target acetamiprid, the aptamer-target binding results in the formation of a structured aptamer/target complex, which disassembles the THMS and releases the STP. The free STP is folded to a stem loop structure, and the fluorescence is quenched. The quenched fluorescence intensity was proportional to the concentration of acetamiprid in the range from 100 to 1200nM, with the limit of detection (LOD) as low as 9.12nM. In addition, this THMS-based method has been successfully used to test and quantify acetamiprid in Chinese cabbage with satisfactory recoveries, and the results were in full agreement with those from LC-MS. The aptamer-based THMS presents distinct advantages, including high stability, remarkable sensitivity, and preservation of the affinity and specificity of the original aptamer. Most importantly, this strategy is convenient and generalizable by virtue of altering the aptamer sequence without changing the triple-helix structure. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. A new aggregation-induced emission fluorescent probe for rapid detection of nitroreductase and its application in living cells

    Science.gov (United States)

    Xu, Gaoping; Tang, Yonghe; Ma, Yanyan; Xu, An; Lin, Weiying

    2018-01-01

    The biological activity of nitroreductase (NTR) is closely related to biological hypoxia status in organisms. The development of effective methods for monitoring the activity of NTR is of great significance for medical diagnosis and tumor research. Toward this goal, we have developed a new aggregation-induced emission (AIE) fluorescence NTR probe TPE-HY used the tetraphenylethene as the fluorophore, and used the nitro group as the NTR recognition site. The probe TPE-HY has many excellent properties, including rapid response, AIE characteristics, high sensitivity and selectivity, and low cytotoxicity. Importantly, the probe TPE-HY is successfully applied to monitor endogenous NTR in living HeLa cells.

  18. Optical Aptamer Probes of Fluorescent Imaging to Rapid Monitoring of Circulating Tumor Cell

    Directory of Open Access Journals (Sweden)

    Ji Yeon Hwang

    2016-11-01

    Full Text Available Fluorescence detecting of exogenous EpCAM (epithelial cell adhesion molecule or muc1 (mucin1 expression correlated to cancer metastasis using nanoparticles provides pivotal information on CTC (circulating tumor cell occurrence in a noninvasive tool. In this study, we study a new skill to detect extracellular EpCAM/muc1 using quantum dot-based aptamer beacon (QD-EpCAM/muc1 ALB (aptamer linker beacon. The QD-EpCAM/muc1 ALB was designed using QDs (quantum dots and probe. The EpCAM/muc1-targeting aptamer contains a Ep-CAM/muc1 binding sequence and BHQ1 (black hole quencher 1 or BHQ2 (black hole quencher2. In the absence of target EpCAM/muc1, the QD-EpCAM/muc1 ALB forms a partial duplex loop-like aptamer beacon and remained in quenched state because the BHQ1/2 quenches the fluorescence signal-on of the QD-EpCAM/muc1 ALB. The binding of EpCAM/muc1 of CTC to the EpCAM/muc1 binding aptamer sequence of the EpCAM/muc1-targeting oligonucleotide triggered the dissociation of the BHQ1/2 quencher and subsequent signal-on of a green/red fluorescence signal. Furthermore, acute inflammation was stimulated by trigger such as caerulein in vivo, which resulted in increased fluorescent signal of the cy5.5-EpCAM/muc1 ALB during cancer metastasis due to exogenous expression of EpCAM/muc1 in Panc02-implanted mouse model.

  19. Rapid fluorescence determination of diquat herbicide in food grains using quantum dots as new reducing agent

    Energy Technology Data Exchange (ETDEWEB)

    Carrillo-Carrion, Carolina; Simonet, Bartolome M. [Department of Analytical Chemistry, University of Cordoba, E-14071 Cordoba (Spain); Valcarcel, Miguel, E-mail: qa1meobj@uco.es [Department of Analytical Chemistry, University of Cordoba, E-14071 Cordoba (Spain)

    2011-04-29

    CdSe/ZnS QDs have demonstrated capacity to act as reducing agent in organic media such as acetonitrile and ethanol. By using fluorescence and Raman spectroscopy, it has been demonstrated that QDs reduce diquat herbicide to its monocation radical. The reaction is characterized to present a high reaction rate making possible to perform the reaction by simple filtration of the solution containing the herbicide through a QDs modified filter. The monocation radical presents a high fluorescence emission spectrum which was selected as the analytical signal to quantify the diquat herbicide. The method described here for the analysis of diquat herbicide in oat grains is simple and fast allowing the analysis of trace level of herbicide in only 6 min. The excellent sensitivity and reproducibility of the methods indicate that the reaction is favoured from both thermodynamic and kinetic point of view. The results presented open up the possibility to use QDs as redox agent. The sensitivity of the method expressed as detection limit was only of 0.01 mg kg{sup -1}.The lineal range was between 0.05 and 0.5 mg kg{sup -1}. The time of analysis per sample, including extraction, reaction and fluorescent measurement was only of 6 min.

  20. Rapid near-infrared fluorescence excitation-emission matrix spectroscopy for multifluorophore characterization using an acousto-optic tunable filter technique.

    Science.gov (United States)

    Li, Hao; Zheng, Wei; Huang, Zhiwei

    2010-01-01

    We report on a novel acousto-optic tunable filter (AOTF)-based near-infrared (NIR) fluorescence excitation-emission matrix (EEM) spectroscopy technique for rapid multifluorophore characterization. We implement a unique light filtering module design by using cascaded AOTFs coupled with three orthogonally oriented polarizers to effectively remove the side-ripple artifacts of AOTFs as well as by using a pair of AOTFs coupled with two orthogonally oriented polarizers to improve detection efficiency for high-quality fluorescence EEM acquisitions. NIR fluorescence EEM spectroscopy (41 excitation wavelengths ranging from 550 to 950 nm in 10-nm increments; fluorescence emission from 570 to 1000 nm at 10-nm intervals) can be acquired from fluorescence dyes [e.g., diethylthiatricarbocyanine (DTTC) iodide, oxazine 750, and IR 140] within 10 s or even less, illustrating the potential of the AOTF-based NIR EEM technique developed for rapid multifluorophore analysis and characterization in biochemical and biomedical systems.

  1. Aptamer-based fluorescence-quenching lateral flow strip for rapid detection of mercury (II) ion in water samples.

    Science.gov (United States)

    Wu, Ze; Shen, Haicong; Hu, Junhui; Fu, Qiangqiang; Yao, Cuize; Yu, Shiting; Xiao, Wei; Tang, Yong

    2017-09-01

    Divalent mercury ion (Hg 2+ ) is one of the most common and stable forms of mercury pollution. In this study, a skillfully designed lateral flow strip (LFS) was developed for sensitive detection of Hg 2+ in river water samples. Aptamer, a specific oligonucleotide probe, was used to selectively identify and target Hg 2+ instead of antibody in traditional immunechromatographic strips; and the fluorescence-quenching system was used to generate positive and low background florescence signals in the competitive-likely LFS. The linear detection range of the LFS for Hg 2+ was 0.13 ng mL -1 to 4 ng mL -1 and the limit of detection (LOD) was 0.13 ng mL -1 . This test provided results in 15 min and demonstrated high specificity. For detection of Hg 2+ in river water, the results were consistent with inductively coupled plasma-mass spectrometry measurements. The aptamer-based fluorescence-quenching LFS was shown to provide a reliable, accurate method for rapid detection of mercury contamination. Graphical Abstract The principle of the aptamer-based fluorescence-quenching LFS.

  2. [Fluorescence microscopy and HPLC assay for rapid detection of distribution and content of resveratrol in Polygonum cuspidatum].

    Science.gov (United States)

    Bu, Xiao-Ying; Dong, Ai-Wen; Guan, Qiong-Yu; Wu, Feng

    2012-12-01

    To establish fluorescence microscopy combined with HPLC method for rapid detection the distribution and content of resveratrol tissues in different growth stages of Polygonum cuspidatum. Used sequential experiment to design conditions of frozen and observe of the section by fluorescence microscopy; Resveratrol was extracted by ultrasonic-assisted extraction and its content was detected by HPLC. The results showed that frozen condition for concentration of gum Arabic was from 20% (dipping time was 5 - 6 h) to 40% (2 - 5 min), the freezer temperature was -5 degrees C, and the thickness was 15 microm. Resveratrol in polygonum cuspidatum was mainly accumulated in the organs, tissues and cells of fiber and cellulose, its content in rhizomes declined as the following sequence: spinal cord > xylem > phloem > periderm; Its content declined in organ as the following sequence: buds > rhizomes > ground stem > leaves; The content of resveratrol in root increased with age. The results of fluorescence microscopic observation is in accordance with the HPLC results, indicating that the method is simple, fast and reliable, and provides a fast and reliable detection method for the determination of optimum harvesting period of Polygonum cuspidatum and acquisition of quality.

  3. Bead-based competitive fluorescence immunoassay for sensitive and rapid diagnosis of cyanotoxin risk in drinking water.

    Science.gov (United States)

    Yu, Hye-Weon; Jang, Am; Kim, Lan Hee; Kim, Sung-Jo; Kim, In S

    2011-09-15

    Due to the increased occurrence of cyanobacterial blooms and their toxins in drinking water sources, effective management based on a sensitive and rapid analytical method is in high demand for security of safe water sources and environmental human health. Here, a competitive fluorescence immunoassay of microcystin-LR (MCYST-LR) is developed in an attempt to improve the sensitivity, analysis time, and ease-of-manipulation of analysis. To serve this aim, a bead-based suspension assay was introduced based on two major sensing elements: an antibody-conjugated quantum dot (QD) detection probe and an antigen-immobilized magnetic bead (MB) competitor. The assay was composed of three steps: the competitive immunological reaction of QD detection probes against analytes and MB competitors, magnetic separation and washing, and the optical signal generation of QDs. The fluorescence intensity was found to be inversely proportional to the MCYST-LR concentration. Under optimized conditions, the proposed assay performed well for the identification and quantitative analysis of MCYST-LR (within 30 min in the range of 0.42-25 μg/L, with a limit of detection of 0.03 μg/L). It is thus expected that this enhanced assay can contribute both to the sensitive and rapid diagnosis of cyanotoxin risk in drinking water and effective management procedures.

  4. In vivo effects of focused shock waves on tumor tissue visualized by fluorescence staining techniques.

    Science.gov (United States)

    Lukes, Petr; Zeman, Jan; Horak, Vratislav; Hoffer, Petr; Pouckova, Pavla; Holubova, Monika; Hosseini, S Hamid R; Akiyama, Hidenori; Sunka, Pavel; Benes, Jiri

    2015-06-01

    Shock waves can cause significant cytotoxic effects in tumor cells and tissues both in vitro and in vivo. However, understanding the mechanisms of shock wave interaction with tissues is limited. We have studied in vivo effects of focused shock waves induced in the syngeneic sarcoma tumor model using the TUNEL assay, immunohistochemical detection of caspase-3 and hematoxylin-eosin staining. Shock waves were produced by a multichannel pulsed-electrohydraulic discharge generator with a cylindrical ceramic-coated electrode. In tumors treated with shock waves, a large area of damaged tissue was detected which was clearly differentiated from intact tissue. Localization and a cone-shaped region of tissue damage visualized by TUNEL reaction apparently correlated with the conical shape and direction of shock wave propagation determined by high-speed shadowgraphy. A strong TUNEL reaction of nuclei and nucleus fragments in tissue exposed to shock waves suggested apoptosis in this destroyed tumor area. However, specificity of the TUNEL technique to apoptotic cells is ambiguous and other apoptotic markers (caspase-3) that we used in our study did not confirmed this observation. Thus, the generated fragments of nuclei gave rise to a false TUNEL reaction not associated with apoptosis. Mechanical stress from high overpressure shock wave was likely the dominant pathway of tumor damage. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging.

    Science.gov (United States)

    Afsari, Hamid Samareh; Cardoso Dos Santos, Marcelina; Lindén, Stina; Chen, Ting; Qiu, Xue; van Bergen En Henegouwen, Paul M P; Jennings, Travis L; Susumu, Kimihiro; Medintz, Igor L; Hildebrandt, Niko; Miller, Lawrence W

    2016-06-01

    Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide-mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.

  6. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    National Research Council Canada - National Science Library

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    .... The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  7. Rapid Sedimentation, Overpressure, and Focused Fluid Flow, Gulf of Mexico Continental Margin

    Directory of Open Access Journals (Sweden)

    Cédric M. John

    2006-09-01

    Full Text Available Expedition 308 of the Integrated Ocean Drilling Program (IODP was the fi rst phase of a two-component project dedicated to studying overpressure and fl uid fl ow on the continental slope of the Gulf of Mexico. We examined how sedimentation, overpressure, fl uid fl ow, and deformation are coupled in a passive margin setting and investigated how extremely rapid deposition of fi ne-grained mud might lead to a rapid build-up of pore pressure in excess of hydrostatic (overpressure, underconsolidation, and sedimentary masswasting. Our tests within the Ursa region, where sediment accumulated rapidly in the late Pleistocene, included the first-ever in situ measurements of how physical properties, pressure, temperature,and pore fluid compositions vary within low-permeability mudstones that overlie a permeable, overpressured aquifer, and we documented severe overpressure in the mudstones overlying the aquifer. We also drilled and logged three references sites in the Brazos-Trinity Basin IV and documented hydrostatic pressure conditions and normalconsolidation. Post-expedition studies will address how the generation and timing of overpressure control slope stability, seafl oor seeps, and large-scale crustal fluid fl ow. The operations ofExpedition 308 provide a foundation for future long-term in situ monitoring experiments in the aquifer and bounding mudstones.

  8. Rapid and Inexpensive Screening of Genomic Copy Number Variations Using a Novel Quantitative Fluorescent PCR Method

    Directory of Open Access Journals (Sweden)

    Martin Stofanko

    2013-01-01

    Full Text Available Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations.

  9. Highly sensitive rapid fluorescence detection of protein residues on surgical instruments

    Energy Technology Data Exchange (ETDEWEB)

    Kovalev, Valeri I [School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS (United Kingdom); Bartona, James S [School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS (United Kingdom); Richardson, Patricia R [School of Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ (United Kingdom); Jones, Anita C [School of Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ (United Kingdom)

    2006-07-15

    There is a risk of contamination of surgical instruments by infectious protein residues, in particular, prions which are the agents for Creutzfeldt-Jakob Disease in humans. They are exceptionally resistant to conventional sterilization, therefore it is important to detect their presence as contaminants so that alternative cleaning procedures can be applied. We describe the development of an optimized detection system for fluorescently labelled protein, suitable for in-hospital use. We show that under optimum conditions the technique can detect {approx}10 attomole/cm{sup 2} with a scan speed of {approx}3-10 cm{sup 2}/s of the test instrument's surface. A theoretical analysis and experimental measurements will be discussed.

  10. Rapid imaging, detection and quantification of Giardia lamblia cysts using mobile-phone based fluorescent microscopy and machine learning.

    Science.gov (United States)

    Koydemir, Hatice Ceylan; Gorocs, Zoltan; Tseng, Derek; Cortazar, Bingen; Feng, Steve; Chan, Raymond Yan Lok; Burbano, Jordi; McLeod, Euan; Ozcan, Aydogan

    2015-03-07

    Rapid and sensitive detection of waterborne pathogens in drinkable and recreational water sources is crucial for treating and preventing the spread of water related diseases, especially in resource-limited settings. Here we present a field-portable and cost-effective platform for detection and quantification of Giardia lamblia cysts, one of the most common waterborne parasites, which has a thick cell wall that makes it resistant to most water disinfection techniques including chlorination. The platform consists of a smartphone coupled with an opto-mechanical attachment weighing ~205 g, which utilizes a hand-held fluorescence microscope design aligned with the camera unit of the smartphone to image custom-designed disposable water sample cassettes. Each sample cassette is composed of absorbent pads and mechanical filter membranes; a membrane with 8 μm pore size is used as a porous spacing layer to prevent the backflow of particles to the upper membrane, while the top membrane with 5 μm pore size is used to capture the individual Giardia cysts that are fluorescently labeled. A fluorescence image of the filter surface (field-of-view: ~0.8 cm(2)) is captured and wirelessly transmitted via the mobile-phone to our servers for rapid processing using a machine learning algorithm that is trained on statistical features of Giardia cysts to automatically detect and count the cysts captured on the membrane. The results are then transmitted back to the mobile-phone in less than 2 minutes and are displayed through a smart application running on the phone. This mobile platform, along with our custom-developed sample preparation protocol, enables analysis of large volumes of water (e.g., 10-20 mL) for automated detection and enumeration of Giardia cysts in ~1 hour, including all the steps of sample preparation and analysis. We evaluated the performance of this approach using flow-cytometer-enumerated Giardia-contaminated water samples, demonstrating an average cyst capture

  11. Fluorescent enzyme immunoassay for rapid screening of Salmonella in foods: collaborative study.

    Science.gov (United States)

    Flowers, R S; Klatt, M J; Keelan, S L; Swaminathan, B; Gehle, W D; Chandonnet, H E

    1989-01-01

    A collaborative study was performed in 13 laboratories to validate an enzyme immunoassay (EIA) procedure for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action as a rapid screening method for detection of Salmonella.

  12. Peripheral and integral membrane binding of peptides characterized by time-dependent fluorescence shifts: focus on antimicrobial peptide LAH₄.

    Science.gov (United States)

    Macháň, Radek; Jurkiewicz, Piotr; Olżyńska, Agnieszka; Olšinová, Marie; Cebecauer, Marek; Marquette, Arnaud; Bechinger, Burkhard; Hof, Martin

    2014-06-03

    Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.

  13. Optimization of Molecularly Imprinted Polymer Method for Rapid Screening of 17β-Estradiol in Water by Fluorescence Quenching

    Directory of Open Access Journals (Sweden)

    Yu Yang

    2011-01-01

    Full Text Available A new method was optimized for rapid screening of 17β-estradiol (E2 in water under 10 min. Molecularly imprinted polymer (MIP particles (325 ± 25 nm were added in a water sample at pH 5.5 and 20∘C to form a suspension. Fluorescence emission from E2 nonspecifically bound onto the MIP particles was first quenched by large gold nanoparticles (43 ± 5 nm. The Stern-Volmer plot was linear, with dynamic quenching constants (Ksv of 2.9 ×104 M-1. Fluorescence emission from E2 specifically bound inside the MIP particles was next quenched by small nitrite anions that easily penetrated the imprinted cavities. The Stern-Volmer plot became nonlinear, with Ksv = 2.1 × 102 M-1 and static quenching constant (V below 1.0 M-1. The difference between these two emission intensities varied as the initial E2 concentration in water, generating a Scatchard calibration curve with R2>0.97 from 0.1 to 10 ppb.

  14. DNA Mimics for the Rapid Identification of Microorganisms by Fluorescence in situ Hybridization (FISH

    Directory of Open Access Journals (Sweden)

    Maria J. Vieira

    2008-10-01

    Full Text Available Fluorescence in situ hybridization (FISH is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA. In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD and recognition of individual genes (RING FISH.

  15. Rapid screening of aflatoxin B1 in beer by fluorescence polarization immunoassay.

    Science.gov (United States)

    Beloglazova, N V; Eremin, S A

    2015-09-01

    This manuscript describes the development of a sensitive, fast and easily-performed fluorescence polarization immunoassay (FPIA) for the mycotoxin aflatoxin B1 (AFB1) in various beer samples, both lager and dark. The highest sensitivity was determined for six poly- and monoclonal antibodies selective towards aflatoxins. The sample pretreatment design was emphasized since beer samples are characterized by extremely diverse matrices. Herein, the choice of sorbent for effective removal of matrix interferences prior to analysis was crucial. The samples were diluted with a borate buffer solution containing 1% PEG 6000 and passed through the clean-up column packed with NH2-derivated silica. This sample pretreatment technique was perfectly suitable for the FPIA of lager beer samples, but for dark beer and ale it did not suffice. An artificial matrix was constructed to plot a calibration curve and quantify the results of the latter samples. The developed immunoassay was characterized by a limit of detection of 1 ng mL(-1). Apparent recovery values of 89-114% for lager and 80-125% for dark beer were established. The FPIA data for AFB1 was characterized by elevated linear regression coefficients, 0.9953 for spiked lager and 0.9895 for dark beer samples respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Cerqueira, Laura; Azevedo, Nuno F; Almeida, Carina; Jardim, Tatiana; Keevil, Charles William; Vieira, Maria J

    2008-10-01

    Fluorescence in situ hybridization (FISH) is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA). In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD) and recognition of individual genes (RING) FISH.

  17. Rapid determination of soil quality and earthworm impacts on soil microbial communities using fluorescence-based respirometry

    Science.gov (United States)

    Prendergast-Miller, Miranda T.; Thurston, Josh; Taylor, Joe; Helgason, Thorunn; Ashauer, Roman; Hodson, Mark E.

    2017-04-01

    We applied a fluorescence-based respirometry method currently devised for aquatic ecotoxicology studies to rapidly measure soil microbial oxygen consumption as a function of soil quality. In this study, soil was collected from an arable wheat field and the field margin. These two soil habitats are known to differ in their soil quality due to differences in their use and management as well as plant, microbial and earthworm community. The earthworm Lumbricus terrestris was incubated in arable or margin soil for three weeks. After this initial phase, a transfer experiment was then conducted to test the hypothesis that earthworm 'migration' alters soil microbial community function and diversity. In this transfer experiment, earthworms incubated in margin soil were transferred to arable soil. The converse transfer (i.e. earthworms incubated in arable soil) was also conducted. Soils of each type with no earthworms were also incubated as controls. After a further four week incubation, the impact of earthworm migration on the soil microbial community was tested by measuring oxygen consumption. Replicated soil slurry subsamples were aliquoted into individual respirometer wells (600 μl volume) on a glass 24-well microplate (Loligo Systems, Denmark) fitted with non-invasive, reusable oxygen sensor spots. The sealed microplate was then attached to an oxygen fluorescence sensor (SDR SensorDish Reader, PreSens, Germany). Oxygen consumption was measured in real-time over a 2 hr period following standard operating procedures. Soil microbial activity was measured with and without an added carbon source (glucose or cellulose, 50 mg C L-1). Using this system, we were able to differentiate between soil type, earthworm treatment and C source. Earthworm-driven impacts on soil microbial oxygen consumption were also supported by changes in soil microbial community structure and diversity revealed using DNA-based sequencing techniques. This method provides a simple and rapid system for

  18. Rapid determination of the damage to photosynthesis caused by salt and osmotic stresses using delayed fluorescence of chloroplasts.

    Science.gov (United States)

    Zhang, Lingrui; Xing, Da

    2008-03-01

    Chloroplasts are one of the most susceptible systems to salt and osmotic stresses. Based on quantitative measurements of delayed fluorescence (DF) of the chloroplasts, we have investigated the damage to photosynthesis caused by these two kinds of stresses in Arabidopsis seedlings by using a custom-built multi-channel biosensor. Results showed that the DF intensity and net photosynthesis rate (Pn) decreased in a similar way with increasing NaCl or sorbitol concentration. Incubation of the seedlings in 200 mM NaCl induced a rapid and reversible decline and subsequent slow and irreversible loss in both the DF intensity and Pn. The rapid decline was dominantly related to osmotic stress, whereas the slow declines in the DF intensity and Pn were specific to ionic stress and could be reversed to a similar extent by a Na+-channel blocker. The DF intensity and Pn also exhibited a similar response to irradiation light under NaCl or sorbitol stress. All results indicated that the DF intensity correlated well with Pn under salt and osmotic stresses. We thus conclude that DF is an excellent marker for detecting the damage to photosynthesis caused by these two stresses. The mechanism of the correlation between the DF intensity and Pn under salt and osmotic stresses was also analyzed in theory and investigated with experiments by measuring intercellular CO2 concetration (Ci), stomatal conductance (Gs), chlorophyll fluorescence parameter, and chlorophyll content. This proposed DF technique holds the potential to be a useful means for analyzing the dynamics of salt and osmotic stresses in vivo and elucidating the mechanism by which plants respond to stress.

  19. Rapid intrinsic fluorescence method for direct identification of pathogens in blood cultures.

    Science.gov (United States)

    Walsh, John D; Hyman, Jay M; Borzhemskaya, Larisa; Bowen, Ann; McKellar, Caroline; Ullery, Michael; Mathias, Erin; Ronsick, Christopher; Link, John; Wilson, Mark; Clay, Bradford; Robinson, Ron; Thorpe, Thurman; van Belkum, Alex; Dunne, W Michael

    2013-11-19

    A positive blood culture is a critical result that requires prompt identification of the causative agent. This article describes a simple method to identify microorganisms from positive blood culture broth within the time taken to perform a Gram stain (identification of the etiologic agent may benefit the clinical management of sepsis. Further evaluation is now warranted to determine the performance of the method using clinical blood culture specimens. Physicians often require the identity of the infective agent in order to make life-saving adjustments to empirical therapy or to switch to less expensive and/or more targeted antimicrobials. However, standard identification procedures take up to 2 days after a blood culture is signaled positive, and even most rapid molecular techniques take several hours to provide a result. Other techniques are faster (e.g., matrix-assisted laser desorption ionization-time of flight [MALDI-TOF] mass spectrometry) but require time-consuming manual processing steps and expensive equipment. There remains a clear need for a simple, inexpensive method to rapidly identify microorganisms directly from positive blood cultures. The promising new method described in this research article can identify microorganisms in minutes by optical spectroscopy, thus permitting the lab to simultaneously report the presence of a positive blood culture and the organism's identity.

  20. Rapid fluorescent lateral-flow immunoassay for hepatitis B virus genotyping.

    Science.gov (United States)

    Song, Liu-Wei; Wang, Ying-Bin; Fang, Lin-Lin; Wu, Yong; Yang, Lin; Chen, Jie-Yu; Ge, Sheng-Xiang; Zhang, Jing; Xiong, You-Zheng; Deng, Xiu-Mei; Min, Xiao-Ping; Zhang, Jun; Chen, Pei-Jer; Yuan, Quan; Xia, Ning-Shao

    2015-01-01

    Hepatitis B virus (HBV) genotyping plays an important role in the clinical management of chronic hepatitis B (CHB) patients. However, the current nucleic acid based techniques are expensive, time-consuming, and inconvenient. Here, we developed a novel DNA-independent HBV genotyping tool based on a one-step fluorescent lateral flow immunoassay (LFIA). Epitope-targeting immunization and screening techniques were used to develop HBV genotype specific monoclonal antibodies (mAbs). These mAbs were used to develop a multitest LFIA with a matched scanning luminoscope for HBV genotyping (named the GT-LFIA). The performance of this novel assay was carefully evaluated in well-characterized clinical cohorts. The GT-LFIA, which can specifically differentiate HBV genotypes A, B, C, and D in a pretreatment-free single test, was successfully developed using four genotype specific mAbs. The detection limits of the GT-LFIA for HBV genotypes A, B, C, and D were 2.5-10.0 IU HBV surface antigen/mL, respectively. Among the sera from 456 CHB patients, 439 (96.3%; 95% confidence interval (CI), 94.1-97.8%) were genotype-differentiable by the GT-LFIA and 437 (99.5%; 95% CI, 98.4-99.9%) were consistent with viral genome sequencing. In the 21 patients receiving nucleos(t)ide analogue therapy, for end-of-treatment specimens that were HBV DNA undetectable and were not applicable for DNA-dependent genotyping, the GT-LFIA presented genotyping results that were consistent with those obtained in pretreatment specimens by viral genome sequencing and the GT-LFIA. In conclusion, the novel GT-LFIA is a convenient, fast, and reliable tool for differential HBV genotyping, especially in patients with low or undetectable HBV DNA levels.

  1. Rapid Cancer Fluorescence Imaging Using A γ-Glutamyltranspeptidase-Specific Probe For Primary Lung Cancer

    Directory of Open Access Journals (Sweden)

    Haruaki Hino

    2016-06-01

    Full Text Available BACKGROUND: We set out to examine the activity of γ-glutamyltranspeptidase (GGT in lung cancer and the validity of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG for intraoperative imaging of primary lung cancer. METHODS: GGT activities and mRNA expression levels of GGT1 (one of the GGT subtypes in five human lung cancer cell lines were examined by fluorescence imaging and quantitative reverse transcription polymerase chain reaction. In vivo imaging of an orthotopic A549 xenograft model in nude mice was performed to confirm its applicability to intraoperative imaging. Furthermore, ex vivo imaging of 73 specimens from lung cancer patients were performed and analyzed to calculate the sensitivity/specificity of gGlu-HMRG for lung cancer diagnosis. RESULTS: GGT activities and mRNA expression levels of GGT1 are diverse depending on cell type; A549, H441, and H460 showed relatively high GGT activities and expression levels, whereas H82 and H226 showed lower values. In the in vivo mouse model study, tiny pleural dissemination and hilar/mediastinal lymph node metastasis (less than 1 mm in diameter were clearly detected 15 minutes after topical application of gGlu-HMRG. In the ex vivo study of specimens from patients, the sensitivity and specificity of gGlu-HMRG were calculated to be 43.8% (32/73 and 84.9% (62/73, respectively. When limited to female patients, never smokers, and adenocarcinomas, these values were 78.9% (15/19 and 73.7% (14/19, respectively. CONCLUSIONS: Although GGT activity of lung cancer cells vary, gGlu-HMRG can serve as an intraoperative imaging tool to detect small foci of lung cancer when such cells have sufficient GGT activity.

  2. Wildlife health in a rapidly changing North: focus on avian disease

    Science.gov (United States)

    Van Hemert, Caroline R.; Pearce, John M.; Handel, Colleen M.

    2014-01-01

    Climate-related environmental changes have increasingly been linked to emerging infectious diseases in wildlife. The Arctic is facing a major ecological transition that is expected to substantially affect animal and human health. Changes in phenology or environmental conditions that result from climate warming may promote novel species assemblages as host and pathogen ranges expand to previously unoccupied areas. Recent evidence from the Arctic and subarctic suggests an increase in the spread and prevalence of some wildlife diseases, but baseline data necessary to detect and verify such changes are still lacking. Wild birds are undergoing rapid shifts in distribution and have been implicated in the spread of wildlife and zoonotic diseases. Here, we review evidence of current and projected changes in the abundance and distribution of avian diseases and outline strategies for future research. We discuss relevant climatic and environmental factors, emerging host–pathogen contact zones, the relationship between host condition and immune function, and potential wildlife and human health outcomes in northern regions.

  3. Fluorescence time-lapse imaging of single cells targeted with a focused scanning charged-particle microbeam

    Energy Technology Data Exchange (ETDEWEB)

    Bourret, Stéphane [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Vianna, François [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); IRSN, BP 3, F-13115 Saint-Paul Lez Durance (France); Devès, Guillaume [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Atallah, Vincent [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Department of Radiation Oncology, Institut Bergonié, Bordeaux (France); Univ. Victor Segalen, Bordeaux (France); Moretto, Philippe; Seznec, Hervé [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Barberet, Philippe, E-mail: barberet@cenbg.in2p3.fr [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France)

    2014-04-01

    Charged particle microbeams provide unique features to study targeted and non-targeted radiation response and have recently emerged as a powerful tool to investigate radiation-induced DNA damage and repair. We have developed a charged particle microbeam delivering protons and alpha particles in the MeV energy range equipped with online time-lapse imaging capabilities. The beam is focused to a sub-micrometer beam spot under vacuum by means of a triplet of magnetic quadrupoles and extracted in air through a 200 nm Si{sub 3}N{sub 4} window. The end-station is equipped with an automated fluorescence microscope used for single cell targeting and online time-lapse imaging. Cells are kept in their medium during the irradiation procedure and the sample temperature is regulated to 37 °C. An overall targeting accuracy of 2.0 ± 0.7 μm has been measured by tracking the re-localization of the XRCC1 protein. First measurements of this re-localization shows the ability of our system to follow online the radiation-induced re-localization of proteins in the first minutes after irradiation.

  4. Rapid determination of gizzerosine in fish meals using microchip capillary electrophoresis with laser-induced fluorescence detection.

    Science.gov (United States)

    Xiao, Meng-Wei; Bai, Xiao-Lin; Xu, Pei-Li; Zhao, Yan; Yang, Li; Liu, Yi-Ming; Liao, Xun

    2017-05-01

    Sensitive detection of gizzerosine, a causative agent for deadly gizzard erosion in chicken feeds, is very important to the poultry industry. In this work, a new method was developed based on microchip capillary electrophoresis (MCE) with laser-induced fluorescence (LIF) detection for rapid analysis of gizzerosine, a biogenic amine in fish meals. The MCE separation was performed on a glass microchip using sodium dodecyl sulfate (SDS) as dynamic coating modifier. Separation conditions, including running buffer pH and concentration, SDS concentration, and the separation voltage were investigated to achieve fast and sensitive quantification of gizzerosine. The assay proposed was very quick and could be completed within 65 s. A linear calibration curve was obtained in the range from 0.04 to 1.8 μg ml-1 gizzerosine. The detection limit was 0.025 μg ml-1 (0.025 mg kg-1), which was far more sensitive than those previously reported. Gizzerosine was well separated from other endogenous components in fish meal samples. Recovery of gizzerosine from this sample matrix (n = 3) was determined to be 97.2-102.8%. The results from analysing fish meal samples indicated that the present MCE-LIF method might hold the potential for rapid detection of gizzerosine in poultry feeds.

  5. A novel method for rapid and non-invasive detection of plants senescence using delayed fluorescence technique

    Science.gov (United States)

    Zhang, Lingrui; Xing, Da; Wang, Junsheng; Zeng, Lizhang; Li, Qiang

    2007-05-01

    Plants senescence is a phase of plants ontogeny marked by declining photosynthetic activity that is paralleled by a decline in chloroplast function. The photosystem II ( PSII ) in a plant is considered the primary site where light-induced delayed fluorescence (DF) is produced. With the leaves of Catharanthus roseus (Catharanthus roseus (L.) G.Don) as testing models, we have studied the effects of plants senescence induced by dark and/or exogenous hormones treatments on characteristics of DF by using a home-made portable DF detection system, which can enable various DF parameters, such as DF decay kinetic curve and DF intensity, to be rapidly produced for the plants in a short time. The results show that the changes in DF intensity of green plants can truly reflect the changes in photosynthetic capacity and chlorophyll content. Therefore, DF may be used an important means of evaluating in vivo plants senescence physiology. The changes in DF intensity may provide a new approach for the rapid and early detection of plants senescence caused by age or other senescence-related factors. DF technique could be potential useful for high throughput screening and less time-consuming and automated identifying the interesting mutants with genetic modifications that change plants senescence progress.

  6. PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

    Science.gov (United States)

    LaDuc, Myron T.; Venkateswaran, Kasthuri; Mohapatra, Bidyut

    2012-01-01

    The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

  7. A combination of positive dielectrophoresis driven on-line enrichment and aptamer-fluorescent silica nanoparticle label for rapid and sensitive detection of Staphylococcus aureus.

    Science.gov (United States)

    Shangguan, Jingfang; Li, Yuhong; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Zou, Zhen; Shi, Hui

    2015-07-07

    Staphylococcus aureus (S. aureus) is an important human pathogen that causes several diseases ranging from superficial skin infections to life-threatening diseases. Here, a method combining positive dielectrophoresis (pDEP) driven on-line enrichment and aptamer-fluorescent silica nanoparticle label has been developed for the rapid and sensitive detection of S. aureus in microfluidic channels. An aptamer, having high affinity to S. aureus, is used as the molecular recognition tool and immobilized onto chloropropyl functionalized fluorescent silica nanoparticles through a click chemistry approach to obtain S. aureus aptamer-nanoparticle bioconjugates (Apt(S.aureus)/FNPs). The pDEP driven on-line enrichment technology was used for accumulating the Apt(S.aureus)/FNP labeled S. aureus. After incubating with S. aureus, the mixture of Apt(S.aureus)/FNP labeled S. aureus and Apt(S.aureus)/FNPs was directly introduced into the pDEP-based microfluidic system. By applying an AC voltage in a pDEP frequency region, the Apt(S.aureus)/FNP labelled S. aureus moved to the electrodes and accumulated in the electrode gap, while the free Apt(S.aureus)/FNPs flowed away. The signal that came from the Apt(S.aureus)/FNP labelled S. aureus in the focused detection areas was then detected. Profiting from the specificity of aptamer, signal amplification of FNP label and pDEP on-line enrichment, this assay can detect as low as 93 and 270 cfu mL(-1)S. aureus in deionized water and spiked water samples, respectively, with higher sensitivities than our previously reported Apt(S.aureus)/FNP based flow cytometry. Moreover, without the need for separation and washing steps usually required for FNP label involved bioassays, the total assay time including sample pretreatment was within 2 h.

  8. Rapid analysis and quantification of fluorescent brighteners in wheat flour by Tri-step infrared spectroscopy and computer vision technology

    Science.gov (United States)

    Guo, Xiao-Xi; Hu, Wei; Liu, Yuan; Gu, Dong-Chen; Sun, Su-Qin; Xu, Chang-Hua; Wang, Xi-Chang

    2015-11-01

    Fluorescent brightener, industrial whitening agent, has been illegally used to whitening wheat flour. In this article, computer vision technology (E-eyes) and colorimetry were employed to investigate color difference among different concentrations of fluorescent brightener in wheat flour using DMS as an example. Tri-step infrared spectroscopy (Fourier transform-infrared spectroscopy coupled with second derivative infrared spectroscopy (SD-IR) and two dimensional correlation infrared spectroscopy (2DCOS-IR)) was used to identify and quantitate DMS in wheat flour. According to color analysis, the whitening effect was significant when added with less than 30 mg/g DMS but when more than 100 mg/g, the flour began greenish. Thus it was speculated that the concentration of DMS should be below 100 mg/g in real flour adulterant with DMS. With the increase of the concentration, the spectral similarity of wheat flour with DMS to DMS standard was increasing. SD-IR peaks at 1153 cm-1, 1141 cm-1, 1112 cm-1, 1085 cm-1 and 1025 cm-1 attributed to DMS were regularly enhanced. Furthermore, it could be differentiated by 2DOS-IR between DMS standard and wheat flour added with DMS low to 0.05 mg/g and the bands in the range of 1000-1500 cm-1 could be an exclusive range to identify whether wheat flour contained DMS. Finally, a quantitative prediction model based on IR spectra was established successfully by Partial least squares (PLS) with a concentration range from 1 mg/g to 100 mg/g. The calibration set gave a determination coefficient of 0.9884 with a standard error (RMSEC) of 5.56 and the validation set presented a determination coefficient of 0.9881 with a standard error of 5.73. It was demonstrated that computer vision technology and colorimetry were effective to estimate the content of DMS in wheat flour and the Tri-step infrared macro-fingerprinting combined with PLS was applicable for rapid and nondestructive fluorescent brightener identification and quantitation.

  9. Assessment of impact of peptide nucleic acid fluorescence in situ hybridization for rapid identification of coagulase-negative staphylococci in the absence of antimicrobial stewardship intervention.

    Science.gov (United States)

    Holtzman, Carol; Whitney, Dana; Barlam, Tamar; Miller, Nancy S

    2011-04-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) was instituted at Boston Medical Center for the rapid identification of coagulase-negative staphylococci (CoNS). Without active notification or antimicrobial stewardship intervention, a pre- and postimpact analysis showed no benefit of this assay with respect to the length of hospital stay or vancomycin use.

  10. Rapid peptide based diagnosis: peptide-based Fluorescence Resonance Energy Transfer (FRET) protease substrates for the detection and diagnosis of bacillus spp

    NARCIS (Netherlands)

    Bikker, F.J.; Kaman, W.E.

    2014-01-01

    We describe the development of a highly specific protease-based Fluorescence Resonance Energy Transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp, including Bacillus anthracis. Synthetic substrates for B. anthracis proteases were designed and exposed to

  11. Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds on both receptor types

    NARCIS (Netherlands)

    Bovee, T.F.H.; Helsdingen, J.R.; Rietjens, I.M.C.M.; Keijer, J.; Hoogenboom, L.A.P.

    2004-01-01

    Previously, we described the construction of a rapid yeast bioassay stably expressing human estrogen receptor (hER) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, the properties of this assay were further studied by testing a series of estrogenic

  12. Rapid Focused Ion Beam Milling Based Fabrication of Plasmonic Nanoparticles and Assemblies via "Sketch and Peel" Strategy.

    Science.gov (United States)

    Chen, Yiqin; Bi, Kaixi; Wang, Qianjin; Zheng, Mengjie; Liu, Qing; Han, Yunxin; Yang, Junbo; Chang, Shengli; Zhang, Guanhua; Duan, Huigao

    2016-12-27

    Focused ion beam (FIB) milling is a versatile maskless and resistless patterning technique and has been widely used for the fabrication of inverse plasmonic structures such as nanoholes and nanoslits for various applications. However, due to its subtractive milling nature, it is an impractical method to fabricate isolated plasmonic nanoparticles and assemblies which are more commonly adopted in applications. In this work, we propose and demonstrate an approach to reliably and rapidly define plasmonic nanoparticles and their assemblies using FIB milling via a simple "sketch and peel" strategy. Systematic experimental investigations and mechanism studies reveal that the high reliability of this fabrication approach is enabled by a conformally formed sidewall coating due to the ion-milling-induced redeposition. Particularly, we demonstrated that this strategy is also applicable to the state-of-the-art helium ion beam milling technology, with which high-fidelity plasmonic dimers with tiny gaps could be directly and rapidly prototyped. Because the proposed approach enables rapid and reliable patterning of arbitrary plasmonic nanostructures that are not feasible to fabricate via conventional FIB milling process, our work provides the FIB milling technology an additional nanopatterning capability and thus could greatly increase its popularity for utilization in fundamental research and device prototyping.

  13. A Rapid and Sensitive HPLC-Fluorescence Method for Determination of Mirtazapine and Its two Major Metabolites in Human Plasma.

    Science.gov (United States)

    Lavasani, Hoda; Giorgi, Mario; Sheikholeslami, Behjat; Hedayati, Mohammadhasan; Rouini, Mohammad Reza

    2014-01-01

    A rapid and sensitive HPLC method has been developed for the quantification of mirtazapine (MRZ), a noradrenergic and specific serotonergic inhibitor antidepressant (NaSSA) and its two major metabolites N-desmethyl mirtazapine (NDM) and 8-hydroxymirtazapine (8-OHM) in human plasma. The separation was achieved using Chromolith C18 column and a mobile phase of acetonitrile: phosphate buffer (pH = 3, 20:80, v/v) in isocratic mode at a flow rate of 2 mL/min. A fluorescence detector was set at 290 and 350 nm for excitation and emission, respectively. Zolpidem was used as the internal standard. Liquid-liquid extraction was applied for sample clean up. All analytes were eluted in less than 5 minutes with LOQ of 1 ng/mL for MRZ and 2 ng/mL for both NDM and 8-OHM. The developed method was successfully applied to quantify MRZ and its metabolites in plasma of a healthy volunteer.

  14. Rapid and accurate analyses of silicon and phosphorus in plants using a portable X-ray fluorescence spectrometer.

    Science.gov (United States)

    Reidinger, Stefan; Ramsey, Michael H; Hartley, Susan E

    2012-08-01

    The elemental analysis of plant material is a frequently employed tool across biological disciplines, yet accurate, convenient and economical methods for the determination of some important elements are currently lacking. For instance, digestion-based techniques are often hazardous and time-consuming and, particularly in the case of silicon (Si), can suffer from low accuracy due to incomplete solubilization and potential volatilization, whilst other methods may require large, expensive and specialised equipment. Here, we present a rapid, safe and accurate procedure for the simultaneous, nonconsumptive analysis of Si and phosphorus (P) in as little as 0.1 g dried and ground plant material using a portable X-ray fluorescence spectrometer (P-XRF). We used certified reference materials from different plant species to test the analytical performance of P-XRF and show that the analysis suffers from very little bias and that the repeatability precision of the measurements is as good as or better than that of other methods. Using this technique we were able to process and analyse 200 ground samples a day, so P-XRF could provide a particularly valuable tool for plant biologists requiring the simultaneous nonconsumptive analysis of multiple elements, including those known to be difficult to measure such as Si, in large numbers of samples. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  15. Cardiac Light-Sheet Fluorescent Microscopy for Multi-Scale and Rapid Imaging of Architecture and Function

    Science.gov (United States)

    Fei, Peng; Lee, Juhyun; Packard, René R. Sevag; Sereti, Konstantina-Ioanna; Xu, Hao; Ma, Jianguo; Ding, Yichen; Kang, Hanul; Chen, Harrison; Sung, Kevin; Kulkarni, Rajan; Ardehali, Reza; Kuo, C.-C. Jay; Xu, Xiaolei; Ho, Chih-Ming; Hsiai, Tzung K.

    2016-03-01

    Light Sheet Fluorescence Microscopy (LSFM) enables multi-dimensional and multi-scale imaging via illuminating specimens with a separate thin sheet of laser. It allows rapid plane illumination for reduced photo-damage and superior axial resolution and contrast. We hereby demonstrate cardiac LSFM (c-LSFM) imaging to assess the functional architecture of zebrafish embryos with a retrospective cardiac synchronization algorithm for four-dimensional reconstruction (3-D space + time). By combining our approach with tissue clearing techniques, we reveal the entire cardiac structures and hypertrabeculation of adult zebrafish hearts in response to doxorubicin treatment. By integrating the resolution enhancement technique with c-LSFM to increase the resolving power under a large field-of-view, we demonstrate the use of low power objective to resolve the entire architecture of large-scale neonatal mouse hearts, revealing the helical orientation of individual myocardial fibers. Therefore, our c-LSFM imaging approach provides multi-scale visualization of architecture and function to drive cardiovascular research with translational implication in congenital heart diseases.

  16. Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Worley, K.C.; Lindsay, E.A.; McCabe, E.R.B. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-07-17

    Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring time-consuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosome deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. 17 refs., 2 figs.

  17. Europium Nanospheres-Based Time-Resolved Fluorescence for Rapid and Ultrasensitive Determination of Total Aflatoxin in Feed.

    Science.gov (United States)

    Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Ding, Xiaoxia; Zhang, Wen

    2015-12-02

    Immunochromatographic (IC) assays are considered suitable diagnostic tools for the determination of mycotoxins. A europium nanospheres-based time-resolved fluorescence immunoassay (Eu-Nano-TRFIA), based on a monoclonal antibody and a portable TRFIA reader, was developed to determine total aflatoxin (including aflatoxins B1, B2, G1, and G2) levels in feed samples. Under optimized conditions, the Eu-Nano-TRFIA method detected total aflatoxin within 12 min. It showed good linearity (R(2) > 0.985), LOD of 0.16 μg/kg, a wide dynamic range of 0.48-30.0 μg/kg, recovery rates of 83.9-113.9%, and coefficients of variation (CVs) of 3.5-8.8%. In the 397 samples from company and livestock farms throughout China, the detection rate was 78.3%, concentrations were 0.50-145.30 μg/kg, the highest total aflatoxin content was found in cottonseed meal, and corn was found to be the most commonly contaminated feed. This method could be a powerful alternative for the rapid and ultrasensitive determination of total aflatoxin in quality control and meet the required Chinese maximum residue limits.

  18. Synthesis of molecular imprinted dye-silica nanocomposites with high selectivity and sensitivity: Fluorescent imprinted sensor for rapid and efficient detection of τ-fluvalinate in vodka.

    Science.gov (United States)

    Wang, Yunyun; Wang, Jixiang; Cheng, Rujia; Sun, Lin; Dai, Xiaohui; Yan, Yongsheng

    2018-02-01

    An imprinted fluorescent sensor was fabricated based on SiO2 nanoparticles encapsulated with molecular imprinted polymer containing allyl fluorescein. High fluorine cypermethirin as template molecules, methyl methacrylate as functional monomer, and allyl fluorescein as optical materials synthesized a core-shell fluorescent molecular imprinted sensor, which showed a high and rapid sensitivity and selectivity for the detection of τ-fluvalinate. The sensor presented appreciable sensitivity with a limit of 13.251 nM, rapid detection that reached to equilibrium within 3 min, great linear relationship in the relevant concentration range from 0 to 150 nM and excellent selectivity over structural analogues. In addition, the fluorescent sensor demonstrated desirable regeneration ability (eight cycling operation). The molecular imprinted polymers ensured specificity, while the fluorescent dyes provided the stabile sensitivity. Finally, an effective application of the sensor was implemented by the detection of τ-fluvalinate in real samples from vodka. The molecular imprinted fluorescent sensor showed a promising potential in environmental monitoring and food safety. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  19. Fluorescence spectroscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2016-01-01

    Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses...... the foundations of the fluorescence phenomenon, introduces some general methodologies and provides selected examples on applications focused to disentangle structural and dynamical aspects of biological processes....

  20. Validation of the rapid fluorescent focus inhibition test for rabies virus-neutralizing antibodies in clinical samples

    NARCIS (Netherlands)

    Kostense, Stefan; Moore, Susan; Companjen, Arjen; Bakker, Alexander B. H.; Marissen, Wilfred E.; von Eyben, Rie; Weverling, Gerrit Jan; Hanlon, Cathleen; Goudsmit, Jaap

    2012-01-01

    Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two

  1. Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

    Directory of Open Access Journals (Sweden)

    Xingmei Xie

    Full Text Available Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR. Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY, five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377, one X/Y-common STR (X22, and two autosomal STRs (D13S305 and D21S11. Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

  2. Evaluation of X-ray fluorescence spectroscopy as a method for the rapid and direct determination of sodium in cheese.

    Science.gov (United States)

    Stankey, J A; Akbulut, C; Romero, J E; Govindasamy-Lucey, S

    2015-08-01

    Cheese manufacturers indirectly determine Na in cheese by analysis of Cl using the Volhard method, assuming that all Cl came from NaCl. This method overestimates the actual Na content in cheeses when Na replacers (e.g., KCl) are used. A direct and rapid method for Na detection is needed. X-ray fluorescence spectroscopy (XRF), a mineral analysis technique used in the mining industry, was investigated as an alternative method of Na detection in cheese. An XRF method for the detection of Na in cheese was developed and compared with inductively coupled plasma optical emission spectroscopy (ICP-OES; the reference method for Na in cheese) and Cl analyzer. Sodium quantification was performed by multi-point calibration with cheese standards spiked with NaCl ranging from 0 to 4% Na (wt/wt). The Na concentration of each of the cheese standards (discs: 30mm×7mm) was quantified by the 3 methods. A single laboratory method validation was performed; linearity, precision, limit of detection, and limit of quantification were determined. An additional calibration graph was created using cheese standards made from natural or process cheeses manufactured with different ratios of Na:K. Both Na and K calibration curves were linear for the cheese standards. Sodium was quantified in a variety of commercial cheese samples. The Na data obtained by XRF were in agreement with those from ICP-OES and Cl analyzer for most commercial natural cheeses. The XRF method did not accurately determine Na concentration for several process cheese samples, compared with ICP-OES, likely due to the use of unknown types of Na-based emulsifying salts (ES). When a calibration curve was created for process cheese with the specific types of ES used for this cheese, Na content was successfully predicted in the samples. For natural cheeses, the limit of detection and limit of quantification for Na that can be determined with an acceptable level of repeatability, precision, and trueness was 82 and 246mg/100g of

  3. Rapid and ultrasensitive detection of microRNA by target-assisted isothermal exponential amplification coupled with poly (thymine)-templated fluorescent copper nanoparticles

    Science.gov (United States)

    Park, Kwan Woo; Batule, Bhagwan S.; Kang, Kyoung Suk; Park, Ki Soo; Park, Hyun Gyu

    2016-10-01

    We devised a novel method for rapid and ultrasensitive detection of target microRNA (miRNA) by employing target-assisted isothermal exponential amplification (TAIEA) combined with poly (thymine)-templated fluorescent copper nanoparticles (CuNPs) as signaling probes. The target miRNA hybridizes to the unimolecular template DNA and works as a primer for the extension reaction to form double-stranded product, which consequently generates two nicking endonuclease recognition sites. By simultaneous nicking and displacement reactions, exponential amplification generates many poly (thymine) strands as final products, which are employed for the synthesis of fluorescent CuNPs. Based on the fluorescent signal from CuNPs, target miRNA is detected as low as 0.27 fM around 1 h of total analysis time. The diagnostic capability of this system has been successfully demonstrated by reliably detecting target miRNA from different cell lysates, showing its great potential towards real clinical applications.

  4. A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

    Science.gov (United States)

    Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

    2017-03-01

    A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

  5. Rapid identification of Burkholderia pseudomallei and Burkholderia mallei by fluorescence in situ hybridization (FISH) from culture and paraffin-embedded tissue samples.

    Science.gov (United States)

    Hagen, Ralf M; Frickmann, Hagen; Elschner, Mandy; Melzer, Falk; Neubauer, Heinrich; Gauthier, Yves P; Racz, Paul; Poppert, Sven

    2011-11-01

    We evaluated newly developed probes for rapid identification of Burkholderia (B.) pseudomallei and B. mallei and differentiation from B. thailandensis by fluorescence in situ hybridization (FISH). FISH correctly identified 100% of the tested B. pseudomallei (11), B. mallei (11), and B. thailandensis (1) strains, excluded 100% of all tested negative controls (61), and allowed demonstration of B. pseudomallei infection in a paraffin-embedded spleen tissue sample of an experimentally infected mouse. Copyright © 2011 Elsevier GmbH. All rights reserved.

  6. Usefulness of light emitting diode (LED) fluorescent microscopy as a tool for rapid and effective method for the diagnosis of pulmonary tuberculosis.

    Science.gov (United States)

    Khatun, Z; Kamal, M; Roy, C K; Sultana, T; Rahman, M Q; Azad, M B A S; Ahmed, A N N

    2011-04-01

    Tuberculosis remains world's leading cause of death from a single infectious agent. Fluorescence microscopy offers well-described benefits, comparing with brightfield microscopy, for the evaluation sputum smear samples for tuberculosis. We evaluated the diagnostic performance of fluorescence microscopy, using novel Light Emitting Diode (LED) technology as an alternative to the conventional fluorescence microscopy by Auramine stain as well as brightfield microscopy by Ziehl-Neelsen (ZN) stain. The objective of the study was to see the usefulness of LED fluorescent microscopy in the diagnosis of pulmonary tuberculosis. This is a prospective study consisted of 150 sputum samples from the patients of NIDCH, Mohakhali. All samples were stained by auramine and ZN stain at BSMMU and culture was done in Lowenstein-Jensen (L-J) media as gold standard at NTRL, Mohakhali. In this study total 66 (44%) out of 150 sputum specimens were positive for Mycobacterium Tuberculosis by culture. Sensitivity and specificity documented for the different modalities were 95.38% and 94.11%, respectively, for the LED assessment; 68.18% and 90.47%, respectively, for the CFM assessment; and 56.06% and 97.61%, respectively, for brightfield microscopy by ZN stain. The difference in their case detection rate was statistically significant (chi2=119.38, p<0.001). Fluorescence Microscopy (FM) is more sensitive than ZN for diagnosis of pulmonary tuberculosis. However, since FM is more sensitive and rapid, using this method (LED) in clinical laboratories with large specimen numbers is recommended.

  7. A rapid equity focused health impact assessment of a policy implementation plan: An Australian case study and impact evaluation

    Science.gov (United States)

    2011-01-01

    Background Equity focused health impact assessments (EFHIAs), or health equity impact assessments, are being increasingly promoted internationally as a mechanism for enhancing the consideration of health equity in the development of policies, programs and projects. Despite this there are relatively few examples of examples of completed EFHIAs available. This paper presents a case study of a rapid EFHIA that was conducted in Australia on a health promotion policy implementation plan. It briefly describes the process and findings of the EFHIA and evaluates the impact on decision-making and implementation. Methods The rapid EFHIA was undertaken in four days, drawing on an expert panel and limited review of the literature. A process evaluation was undertaken by email one month after the EFHIA was completed. An impact evaluation was undertaken two years later based on five semi-structured interviews with members of the EFHIA working group and policy officers and managers responsible for implementing the plan. A cost estimation was conducted by the EFHIA working group. Findings The EFHIA made both general and specific recommendations about how the health equity impacts of the policy implementation plan could be improved. The impact evaluation identified changes to development and implementation that occurred as a result of the EFHIA, though there was disagreement about the extent to which changes could be attributed solely to the EFHIA. Those responsible considered the recommendations of the EFHIA in the next versions of their ABHI implementation plans. Factors that influenced the impact of the EFHIA included consolidating understandings of equity, enabling discussion of alternatives, and differing understandings of the purpose of the EFHIA. The EFHIA cost US$4,036 to undertake. Conclusions This EFHIA was conducted in a short timeframe using relatively few resources. It had some reported impacts on the development of the implementation plan and enhanced overall

  8. Rapid and Sensitive Detection of Protein Biomarker Using a Portable Fluorescence Biosensor based on Quantum Dots and a Lateral Flow Test Strip

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhaohui; Wang, Ying; Wang, Jun; Tang, Zhiwen; Pounds, Joel G.; Lin, Yuehe

    2010-08-15

    A portable fluorescence biosensor with rapid and ultrasensitive response for trace protein has been built up with quantum dots and lateral flow test strip. The superior signal brightness and high photostability of quantum dots are combined with the promising advantages of lateral flow test strip and resulted in high sensitivity, selectivity and speedy for protein detection. Nitrated ceruloplasmin, a significant biomarker for cardiovascular disease, lung cancer and stress response to smoking, was used as model protein to demonstrate the good performances of this proposed Qdot-based lateral flow test strip. Quantitative detection of nitrated ceruloplasmin was realized by recording the fluorescence intensity of quantum dots captured on the test line. Under optimal conditions, this portable fluorescence biosensor displays rapid responses for nitrated ceruloplasmin in wide dynamic range with a detection limit of 0.1ng/mL (S/N=3). Furthermore, the biosensor was successfully utilized for spiked human plasma sample detection with the concentration as low as 1ng/mL. The results demonstrate that the quantum dot-based lateral flow test strip is capable for rapid, sensitive, and quantitative detection of nitrated ceruloplasmin and hold a great promise for point-of-care and in field analysis of other protein biomarkers.

  9. A rapid total reflection X-ray fluorescence protocol for micro analyses of ion profiles in Arabidopsis thaliana

    Science.gov (United States)

    Höhner, Ricarda; Tabatabaei, Samaneh; Kunz, Hans-Henning; Fittschen, Ursula

    2016-11-01

    The ion homeostasis of macro and micronutrients in plant cells and tissues is a fundamental requirement for vital biochemical pathways including photosynthesis. In nature, ion homeostasis is affected mainly by three processes: 1. Environmental stress factors, 2. Developmental effects, and 3. Loss or gain-of-function mutations in the plant genome. Here we present a rapid total reflection X-ray fluorescence (TXRF) protocol that allows for simultaneous quantification of several elements such as potassium (K), calcium (Ca), sulfur (S), manganese (Mn) and strontium (Sr) in Arabidopsis thaliana leaf specimens. Our procedure is cost-efficient and enables precise, robust and highly reproducible measurements on tissue samples as small as 0.3 mg dry weight. As shown here, we apply the TXRF procedure to detect accurately the early replacement of K by Na ions in leaves of plants exposed to soil salinity, a globally increasing abiotic stress factor. Furthermore, we were able to prove the existence of a leaf development-dependent ion gradient for K, Ca, and other divalent ions in A. thaliana; i.e. old leaves contain significantly lower K but higher Ca than young leaves. Lastly, we show that our procedure can be readily applied to reveal subtle differences in tissue-specific ion contents of plant mutants. We employed independent A. thaliana kea1kea2 loss-of-function mutants that lack KEA1 and KEA2, two highly active chloroplast K exchange proteins. We found significantly increased K levels specifically in kea1kea2 mutants, i.e. 55 mg ∗ g- 1 dry weight, compared to 40 mg ∗ g- 1 dry weight in wild type plants. The TXRF procedure can be supplemented with Flame atomic absorption (FAAS) and emission spectrometry (FAES) to expand the detection range to sodium (Na) and magnesium (Mg). Because of the small sample amounts required, this method is especially suited to probe individual leaves in single plants or even specific leaf areas. Therefore, TXRF represents a powerful method to

  10. Rapid assessment of different oxygenic phototrophs and single-cell photosynthesis with multicolour variable chlorophyll fluorescence imaging

    DEFF Research Database (Denmark)

    Trampe, Erik Christian Løvbjerg; Kolbowski, J.; Schreiber, U.

    2011-01-01

    , red or white light. Automated sequential exposure of microscopic samples to the three excitation colours enables subsequent deconvolution of the resulting fluorescence signals and colour marking of cells with different photopigmentation, i.e., cyanobacteria, green algae, red algae and diatoms......We present a new system for microscopic multicolour variable chlorophyll fluorescence imaging of aquatic phototrophs. The system is compact and portable and enables microscopic imaging of photosynthetic performance of individual cells and chloroplasts using different combinations of blue, green...

  11. Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV-based fluorescent imaging system.

    Science.gov (United States)

    Suzuki, Yoshio; Takagi, Nobuyuki; Sano, Takuma; Chimuro, Tomoyuki

    2013-09-01

    A new fluorescent molecular probe, methyl 3-(3,5-bis((bis(pyridin-2-ylmethyl)amino)-methyl)-4-hydroxyphenyl)-2-(5-(dimethylamino)naphthalene-1-sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl-1-Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high-throughput electrophoresis by using a UV-based detection system. Dansyl-1-Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in-gel SDS-PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl-1-Zn(II) allowed 1-step protein staining (SDS-PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312-nm or 365-nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl-1-Zn(II) was successfully applied to protein identification by MS via in-gel tryptic digestion, Western blotting, and Native-PAGE. Accordingly, Dansyl-1-Zn(II) may facilitate highly sensitive and high-throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

    Science.gov (United States)

    Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

    2017-01-01

    A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy. PMID:28300146

  13. Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol.

    Science.gov (United States)

    Mogollon, Catherin Marin; van Pul, Fiona J A; Imai, Takashi; Ramesar, Jai; Chevalley-Maurel, Séverine; de Roo, Guido M; Veld, Sabrina A J; Kroeze, Hans; Franke-Fayard, Blandine M D; Janse, Chris J; Khan, Shahid M

    2016-01-01

    The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.

  14. Profuse color-evolution-based fluorescent test paper sensor for rapid and visual monitoring of endogenous Cu(2+) in human urine.

    Science.gov (United States)

    Cai, Yueqing; You, Junhui; You, Zhengyi; Dong, Fang; Du, Shuhu; Zhang, Liying

    2018-01-15

    The fluorescent paper for colorimetric detection of metal ions has been widely fabricated using various sensing probes, but it still remains an elusive task to design a test paper with multicolor variation with target dosages for accurate determination. Herein, we report a profuse color-evolution-based fluorescent test paper sensor for rapid and visual monitoring of Cu(2+) in human urine by printing tricolor probe onto filter paper. The tricolor probe consists of blue-emission carbon dots (bCDs), green-emission quantum dots (gQDs) and red-emission quantum dots (rQDs), which is based on the principle that the fluorescence of gQDs and rQDs are simultaneously quenched by Cu(2+), whereas the bCDs as the photostable internal standard is insensitive to Cu(2+). Upon the addition of different amounts of Cu(2+), the ratiometric fluorescence intensity of the tricolor probe continuously varied, leading to color changes from shallow pink to blue with a detection limit of 1.3nM. When the tricolor probe solution was printed onto a sheet of filter paper, as-obtained test paper displayed a more profuse color evolution from shallow pink to light salmon to dark orange to olive drab to dark olive green to slate blue to royal blue and to final dark blue with the increase of Cu(2+) concentration compared with dual-color probe-based test paper, and dosage scale as low as 6.0nM was clearly discriminated. The sensing test paper is simple, rapid and inexpensive, and serves as a visual platform for ultrasensitive monitoring of endogenous Cu(2+) in human urine. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Fluorescence-Free Biosensor Methods in Detection of Food Pathogens with a Special Focus on Listeria monocytogenes.

    Science.gov (United States)

    Radhakrishnan, Rajeswaran; Poltronieri, Palmiro

    2017-12-20

    Food pathogens contaminate food products that allow their growth on the shelf and also under refrigerated conditions. Therefore, it is of utmost importance to lower the limit of detection (LOD) of the method used and to obtain the results within hours to few days. Biosensor methods exploit the available technologies to individuate and provide an approximate quantification of the bacteria present in a sample. The main bottleneck of these methods depends on the aspecific binding to the surfaces and on a change in sensitivity when bacteria are in a complex food matrix with respect to bacteria in a liquid food sample. In this review, we introduce surface plasmon resonance (SPR), new advancements in SPR techniques, and electrochemical impedance spectroscopy (EIS), as fluorescence-free biosensing technologies for detection of L. monocytogenes in foods. The application of the two methods has facilitated L. monocytogenes detection with LOD of 1 log CFU/mL. Further advancements are envisaged through the combination of biosensor methods with immunoseparation of bacteria from larger volumes, application of lab-on-chip technologies, and EIS sensing methods for multiplex pathogen detection. Validation efforts are being conducted to demonstrate the robustness of detection, reproducibility and variability in multi-site installations.

  16. Rapid detection of t(15;17)(q24;q21) in acute promyelocytic leukaemia by microwave-assisted fluorescence in situ hybridization.

    Science.gov (United States)

    Soriani, Silvia; Mura, Cinzia; Panico, Anna Rita; Scarpa, Anna Maria; Recchimuzzo, Patrizia; Dadati, Raffaella; Farioli, Renata; De Canal, Gabriella; Mura, Maria Angela; Cesana, Clara

    2017-03-01

    Acute promyelocytic leukaemia (APL) is a hematologic malignancy characterized by the rearrangement of the PML and RARα genes, mostly due to a reciprocal chromosomal translocation t(15;17)(q24;q21). A quick APL diagnosis is essential for starting a prompt suitable therapy. We describe a new rapid diagnostic laboratory approach to detect the PML-RARα rearrangement, which gives clear genetic results within 30 min of hybridization. It combines quick cell harvesting, fluorescence in situ hybridization performed with commercial DNA probe and microwave beams supplied by a domestic microwave oven. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    Directory of Open Access Journals (Sweden)

    Jordan S. Leyton-Mange

    2014-02-01

    Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

  18. Rapid and accurate simultaneous determination of abamectin and ivermectin in bovine milk by high performance liquid chromatography with fluorescence detection

    OpenAIRE

    Kolberg, D. I. S.; Presta, M.A.; Wickert, C.; Adaime,M. B.; R. Zanella

    2009-01-01

    An analytical method using high performance liquid chromatography with fluorescence detection for the simultaneous determination of abamectin and ivermectin in bovine milk was developed and validated. The best recovery results were achieved by using acetonitrile for extraction of the compounds followed by solid phase extraction in cartridges containing C18 for the purification of the extract. Pre-column derivatization was accomplished with N-methylimidazole and trifluoroacetic anhydride. The ...

  19. Ultrasonic-assisted Kabachnik-Fields reaction for rapid fabrication of AIE-active fluorescent organic nanoparticles.

    Science.gov (United States)

    Long, Zi; Liu, Meiying; Jiang, Ruming; Zeng, Guangjiang; Wan, Qing; Huang, Hongye; Deng, Fengjie; Wan, Yiqun; Zhang, Xiaoyong; Wei, Yen

    2017-03-01

    Aggregation-induced emission (AIE)-active fluorescent organic nanoparticles (FNPs) have been extensively explored for fluorescence "turn-on" bio-imaging applications with the unique advantages over conventional FNPs. Transformation of AIE-active molecules into FNPs can greatly expand their biomedical application potential. Here we reported a novel "one-pot" strategy for fabricating AIE-active FNPs through an ultrasonic-assisted, catalysts-free and solvent-free Kabachnik-Fields (KF) reaction for the first time. The KF reaction can be completed within 10min to generate AIE-active PTH-CHO-PEI-DEP FNPs through mixing polyethylenimine and aldehyde group containing AIE dyes and diethyl phosphate. These PTH-CHO-PEI-DEP FNPs were confirmed by proton nuclear magnetic resonance ((1)H NMR) spectroscopy, transmission electron microscopy (TEM) and fluorescence spectroscopy etc. The cell uptake behavior as well as cell viability of PTH-CHO-PEI-DEP FNPs was examined to evaluate their potential for biomedical application. We demonstrated that the amphiphilic α-aminophosphonate polymers could self-assemble into PTH-CHO-PEI-DEP FNPs in aqueous solution and showed excellent water dispersibility. TEM image shows the size of PTH-CHO-PEI-DEP FNPs is 100-200nm. More importantly, the PTH-CHO-PEI-DEP FNPs emit strong green fluorescence and desirable biocompatibility, making them very suitable for biomedical applications. Finally, thus smart FNPs design together with their excellent performance will open a new avenue in the development of FNPs for following biological processes such as carcinogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Confocal fluorescence microscopy for rapid evaluation of invasive tumor cellularity of inflammatory breast carcinoma core needle biopsies.

    Science.gov (United States)

    Dobbs, Jessica; Krishnamurthy, Savitri; Kyrish, Matthew; Benveniste, Ana Paula; Yang, Wei; Richards-Kortum, Rebecca

    2015-01-01

    Tissue sampling is a problematic issue for inflammatory breast carcinoma, and immediate evaluation following core needle biopsy is needed to evaluate specimen adequacy. We sought to determine if confocal fluorescence microscopy provides sufficient resolution to evaluate specimen adequacy by comparing invasive tumor cellularity estimated from standard histologic images to invasive tumor cellularity estimated from confocal images of breast core needle biopsy specimens. Grayscale confocal fluorescence images of breast core needle biopsy specimens were acquired following proflavine application. A breast-dedicated pathologist evaluated invasive tumor cellularity in histologic images with hematoxylin and eosin staining and in grayscale and false-colored confocal images of cores. Agreement between cellularity estimates was quantified using a kappa coefficient. 23 cores from 23 patients with suspected inflammatory breast carcinoma were imaged. Confocal images were acquired in an average of less than 2 min per core. Invasive tumor cellularity estimated from histologic and grayscale confocal images showed moderate agreement by kappa coefficient: κ = 0.48 ± 0.09 (p fluorescence microscopy can be performed immediately following specimen acquisition and could indicate the need for additional biopsies at the initial visit.

  1. Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein.

    Science.gov (United States)

    Piscitelli, Alessandra; Pennacchio, Anna; Cicatiello, Paola; Politi, Jane; De Stefano, Luca; Giardina, Paola

    2017-01-15

    A fusion protein designed in order to combine the fluorescence emission of the Green Fluorescent Protein (GFP) with the adhesion ability of the class I hydrophobin Vmh2 was heterologously produced in the yeast Pichia pastoris. The Vmh2-GFP fusion protein has proven to be a smart and effective tool for the study of Vmh2 self-assembling. Since the two proteins were linked by the specific cutting site of the thrombin, the fusion protein was used as the active biological element in the realization of a thrombin biosensor. When the thrombin present in the target solution specifically hydrolyzed its cleavage sequence, a consequent decrease in the fluorescence intensity of the sample could be observed. The Vmh2-GFP based assay allowed quantification of thrombin in solution with a detection limit of 2.27aM. The specificity of the assay with respect to other proteases and proteins granted the measurement of thrombin added to healthy human plasma with same high sensitivity and a limit of detection of 2.3aM. Further advantages of the developed biosensor are the simplicity of its design and preparation, and the low requirements in terms of samples, reagents and time. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Rapid and non-destructive analysis of metallic dental restorations using X-ray fluorescence spectra and light-element sampling tools

    Science.gov (United States)

    Furuhashi, K.; Uo, M.; Kitagawa, Y.; Watari, F.

    2012-12-01

    IntroductionRecently, allergic diseases caused by dental metals have been increasing. Therefore, rapid and accurate analytical methods for the metal restorations in the oral cavities of patients are required. The purpose of this study was to develop a non-destructive extraction method for dental alloys, along with a subsequent, rapid and accurate elemental analysis. Materials and methodSamples were obtained by polishing the surfaces of metal restorations using a dental rotating tool with disposable buffs and polishing pastes. As materials for the analysis, three dental alloys were used. To compare the sampling and analysis efficiencies, two buffs and seven pastes were used. After polishing the surface of a metal restoration, the buff was analyzed using X-ray scanning analytical microscopy (XSAM). ResultsThe efficiency of the analysis was judged based on the sampling rate achieved and the absence of disturbing elements in the background in fluorescence X-ray spectra. The best results were obtained for the combination of TexMet as a buff with diamond as a paste. This combination produced a good collection efficiency and a plain background in the fluorescence X-ray spectra, resulting in a high precision of the analysis.

  3. Rapid quantification of viable Legionella in nuclear cooling tower waters using filter cultivation, fluorescent in situ hybridization and solid-phase cytometry.

    Science.gov (United States)

    Baudart, J; Guillaume, C; Mercier, A; Lebaron, P; Binet, M

    2015-05-01

    To develop a rapid and sensitive method to quantify viable Legionella spp. in cooling tower water samples. A rapid, culture-based method capable of quantifying as few as 600 Legionella microcolonies per litre within 2 days in industrial waters was developed. The method combines a short cultivation step of microcolonies on GVPC agar plate, specific detection of Legionella cells by a fluorescent in situ hybridization (FISH) approach, and a sensitive enumeration using a solid-phase cytometer. Following optimization of the cultivation conditions, the qualitative and quantitative performance of the method was assessed and the method was applied to 262 nuclear power plant cooling water samples. The performance of this method was in accordance with the culture method (NF-T 90-431) for Legionella enumeration. The rapid detection of viable Legionella in water is a major concern to the effective monitoring of this pathogenic bacterium in the main water sources involved in the transmission of legionellosis infection (Legionnaires' disease). The new method proposed here appears to be a robust, efficient and innovative means for rapidly quantifying cultivable Legionella in cooling tower water samples within 48 h. © 2015 The Society for Applied Microbiology.

  4. Rapid preparation of branched and degradable AIE-active fluorescent organic nanoparticles via formation of dynamic phenyl borate bond.

    Science.gov (United States)

    Long, Zi; Liu, Meiying; Mao, Liucheng; Zeng, Guangjian; Wan, Qing; Xu, Dazhuang; Deng, Fengjie; Huang, Hongye; Zhang, Xiaoyong; Wei, Yen

    2017-02-01

    The fluorescent organic nanoparticles (FNPs) with aggregation-induced emission (AIE) feature have received increasing attention for their advanced optical properties. Although many efforts have been devoted to the fabrication and biomedical applications of AIE-active FNPs, the preparation of branched AIE-active FNPs with degradability through formation of dynamic bonds have rarely been reported. In this work, branched AIE-active FNPs were fabricated via dynamic linkage of hydrophobic hyperbranched and degradable Boltorn H40 (H40) with phenylboronic acid terminated AIE dye (PhB(OH)2) and mPEG (mPEG-B(OH)2), which relied on a facile one-pot strategy between phenylboronic acid and diol group of H40. The branched H40-star-mPEG-PhB(OH)2 FNPs were characterized using nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy. Benefiting from their highly branched structure and amphiphilic properties, H40-star-mPEG-PhB(OH)2 could self-assemble into micelles and emit strong orange-red fluorescence. More importantly, cell viability results demonstrated that H40-star-mPEG-PhB(OH)2 FNPs showed good biocompatibility and promising candidates for bio-imaging. Taken together, we developed a one-pot strategy for preparation of branched AIE-active FNPs through the formation of dynamic phenyl borate. The resultant H40-star-mPEG-PhB(OH)2 FNPs should be promising biomaterials for different applications for biodegradability of H40 and responsiveness of phenyl borate. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Clinical validation and assessment of a modular fluorescent imaging system and algorithm for rapid detection and quantification of dental plaque.

    Science.gov (United States)

    Angelino, Keith; Shah, Pratik; Edlund, David A; Mohit, Mrinal; Yauney, Gregory

    2017-12-28

    Significant numbers of adults and children have untreated plaque due to poor oral hygiene and consequently suffer from associate dental and systemic diseases. A handheld device equipped with 405 nm light-emitting diodes was constructed to examine the prevalence of red fluorescence signatures associated with dental plaque. This device was used for in vivo imaging of all four incisors and all four canines of twenty-eight consenting human subjects. The same areas were further imaged under white light illumination with a commercial image-processing based plaque-imaging device, and evaluated by a hygienist and dentist. A custom computer vision algorithm using pixel information was developed to calculate plaque coverage ratios ranging from 0 (no plaque) to 1 (complete plaque coverage) for images captured by both devices. The algorithm calculated red fluorescence-based plaque coverage ratios ranging from 0.011 to 0.211 for the subjects imaged. Clinical assessment and statistical analyses of associated plaque ratios of the 405 nm device images indicated high sensitivity and specificity in detecting dental plaque by the experimental device compared to the commercial reference device. The low-cost and open source 405 nm device and the associated computer vision algorithm successfully captured red fluorescence signatures associated with dental plaque and demonstrated comparable performance to a commercially available device. Therefore, a proof of concept validation was provided for the construction and application of a sensitive cost-effective plaque-detecting device. A miniaturized mobile adaptable version of the device was also provided, together with and a step-by-step guide for device assembly and webhost the associated software, to facilitate open-source access to a cost-effective at-home, in-clinic oral care technology. ClinicalTrials.gov NCT03379337, December 19 2017. Retrospectively registered.

  6. A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

    Directory of Open Access Journals (Sweden)

    Lim Tit-Meng

    2010-10-01

    Full Text Available Abstract Background Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy. Results Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®. Conclusions The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic

  7. Microwave-assisted multicomponent reactions for rapid synthesis of AIE-active fluorescent polymeric nanoparticles by post-polymerization method.

    Science.gov (United States)

    Cao, Qian-Yong; Jiang, Ruming; Liu, Meiying; Wan, Qing; Xu, Dazhuang; Tian, Jianwen; Huang, Hongye; Wen, Yuanqing; Zhang, Xiaoyong; Wei, Yen

    2017-11-01

    The development of simple and effective methods for synthesis of fluorescent polymeric nanoparticles (FPNs) with aggregation-induced emission (AIE) plays an important role for the biomedical applications of AIE-active FPNs. In present work, we developed a facile strategy for the fabrication of AIE-active FPNs by a post-polymerization method based on the microwave-assisted Kabachnik-Fields (KF) reaction, which can conjugate with poly(PEGMA-NH2), AIE-active dye (TPE-CHO) and diethyl phosphate (DP) under microwave irradiation within 5min. The characterization results confirm that PEGMA-TPE FPNs are successfully prepared through the microwave-assisted KF reaction. The resultant AIE-active FPNs show high water dispersity, intensive fluorescence and low cytotoxicity. These features make these AIE-active FPNs great potential for biomedical applications. Moreover, the microwave-assisted KF reaction is simple, fast, atom economy that should be a general strategy for the fabrication of various multifunctional AIE-active FPNs. We believe this work will open up a new avenue for the preparation of AIE-active functional materials with great potential for different applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Rapid on-site detection of airborne asbestos fibers and potentially hazardous nanomaterials using fluorescence microscopy-based biosensing.

    Science.gov (United States)

    Kuroda, Akio; Alexandrov, Maxym; Nishimura, Tomoki; Ishida, Takenori

    2016-06-01

    A large number of peptides with binding affinity to various inorganic materials have been identified and used as linkers, catalysts, and building blocks in nanotechnology and nanobiotechnology. However, there have been few applications of material-binding peptides in the fluorescence microscopy-based biosensing (FM method) of environmental pollutants. A notable exception is the application of the FM method for the detection of asbestos, a dangerous industrial toxin that is still widely used in many developing countries. This review details the selection and isolation of asbestos-binding proteins and peptides with sufficient specificity to distinguish asbestos from a large variety of safer fibrous materials used as asbestos substitutes. High sensitivity to nanoscale asbestos fibers (30-35 nm in diameter) invisible under conventional phase contrast microscopy can be achieved. The FM method is the basis for developing an automated system for asbestos biosensing that can be used for on-site testing with a portable fluorescence microscope. In the future, the FM method could also become a useful tool for detecting other potentially hazardous nanomaterials in the environment. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Mapping the metal uptake in plants from Jasper Ridge Biological Preserve using synchrotron micro-focused X-ray fluorescence spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Allison [Univ. of California, Davis, CA (United States)

    2015-08-20

    Serpentine soil originates in the Earth’s mantle and contains high concentrations of potentially toxic transition metals. Although serpentine soil limits plant growth, endemic and adapted plants at Jasper Ridge Biological Preserve, located behind SLAC National Accelerator Laboratory, can tolerate these conditions. Serpentine soil and seeds belonging to native California and invasive plants were collected at Jasper Ridge. The seeds were grown hydroponically and on serpentine and potting soil to examine the uptake and distribution of ions in the roots and shoots using synchrotron micro-focused X-ray fluorescence spectroscopy. The results were used to determine differences between serpentine-tolerant plants. Rye grown on potting soil was enriched in Ni, Fe, Mn, and Cr compared to purple needlegrass grown on serpentine soil. Serpentine vegetation equally suppressed the uptake of Mn, Ni, and Fe in the roots and shoots. The uptake of Ca and Mg affected the uptake of other elements such as K, S, and P.

  10. Synthesis of Antibodies-Conjugated Fluorescent Dye-Doped Silica Nanoparticles for a Rapid Single Step Detection of Campylobacter jejuni in Live Poultry

    Directory of Open Access Journals (Sweden)

    Wachira Tansub

    2012-01-01

    Full Text Available The preparation of antibodies-conjugated fluorescent dye-doped silica nanoparticles (FDS-NPs was developed to detect Campylobacter jejuni cells under a fluorescence microscope. The particles prepared by sol-gel microemulsion techniques have a round shape with an average size of 43 ± 4 nm. They were highly photo stable and could emit strong orange fluorescent for 60 min. Both amine- and carboxyl-functionalized properties were evident from FTIR and FT Raman spectra. The FDS-NPs conjugated with antibodies against C. jejuni were well dispersed in PBS solution at 20 mM of NaCl. The conjugation with monoclonal antibodies against C. jejuni was successful. The direct observation of the antibodies-conjugated FDS-NPs- that bounds C. jejuni with Petroff Hausser counting chamber at 40x was clear. The different focus lengths clearly separated bound and unbound FDS-NPs under the microscope. We successfully synthesis the bio-conjugated dye doped silica nanoparticles for C. jejuni that are easy to use and giving clear detection in due time.

  11. The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002.

    Science.gov (United States)

    Schulze, Katja; Lang, Imke; Enke, Heike; Grohme, Diana; Frohme, Marcus

    2015-04-17

    Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein. Based on these findings we developed a method for fluorescence microscopy in order to distinguish producing and non-producing cells with respect to their pigmentation phenotype. By applying a specific filter set the emitted fluorescence of a producer cell with a reduced PC content appeared orange. The emitted fluorescence of a non-producing cell with a wt pigmentation phenotype was detected in red, and dead cells in green. In an automated process multiple images of each sample were taken and analyzed with a plugin for the image analysis software ImageJ to identify dead (green), non-producing (red) and producing (orange) cells. The results of the presented validation experiments revealed a good identification with 98 % red cells in the wt sample and 90 % orange cells in the producer sample. The detected wt pigmentation phenotype (red cells) in the producer sample were either not fully induced yet (in 48 h induced

  12. Rapid in-focus corrections on quantitative amplitude and phase imaging using transport of intensity equation method.

    Science.gov (United States)

    Meng, X; Tian, X; Kong, Y; Sun, A; Yu, W; Qian, W; Song, X; Cui, H; Xue, L; Liu, C; Wang, S

    2017-06-01

    Transport of intensity equation (TIE) method can acquire sample phase distributions with high speed and accuracy, offering another perspective for cellular observations and measurements. However, caused by incorrect focal plane determination, blurs and halos are induced, decreasing resolution and accuracy in both retrieved amplitude and phase information. In order to obtain high-accurate sample details, we propose TIE based in-focus correction technique for quantitative amplitude and phase imaging, which can locate focal plane and then retrieve both in-focus intensity and phase distributions combining with numerical wavefront extraction and propagation as well as physical image recorder translation. Certified by both numerical simulations and practical measurements, it is believed the proposed method not only captures high-accurate in-focus sample information, but also provides a potential way for fast autofocusing in microscopic system. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  13. Fluorescence In Situ Hybridization (FISH)-Based Karyotyping Reveals Rapid Evolution of Centromeric and Subtelomeric Repeats in Common Bean (Phaseolus vulgaris) and Relatives.

    Science.gov (United States)

    Iwata-Otsubo, Aiko; Radke, Brittany; Findley, Seth; Abernathy, Brian; Vallejos, C Eduardo; Jackson, Scott A

    2016-04-07

    Fluorescence in situ hybridization (FISH)-based karyotyping is a powerful cytogenetics tool to study chromosome organization, behavior, and chromosome evolution. Here, we developed a FISH-based karyotyping system using a probe mixture comprised of centromeric and subtelomeric satellite repeats, 5S rDNA, and chromosome-specific BAC clones in common bean, which enables one to unambiguously distinguish all 11 chromosome pairs. Furthermore, we applied the karyotyping system to several wild relatives and landraces of common bean from two distinct gene pools, as well as other related Phaseolus species, to investigate repeat evolution in the genus Phaseolus Comparison of karyotype maps within common bean indicates that chromosomal distribution of the centromeric and subtelomeric satellite repeats is stable, whereas the copy number of the repeats was variable, indicating rapid amplification/reduction of the repeats in specific genomic regions. In Phaseolus species that diverged approximately 2-4 million yr ago, copy numbers of centromeric repeats were largely reduced or diverged, and chromosomal distributions have changed, suggesting rapid evolution of centromeric repeats. We also detected variation in the distribution pattern of subtelomeric repeats in Phaseolus species. The FISH-based karyotyping system revealed that satellite repeats are actively and rapidly evolving, forming genomic features unique to individual common bean accessions and Phaseolus species. Copyright © 2016 Iwata-Otsubo et al.

  14. Fluorescence In Situ Hybridization (FISH-Based Karyotyping Reveals Rapid Evolution of Centromeric and Subtelomeric Repeats in Common Bean (Phaseolus vulgaris and Relatives

    Directory of Open Access Journals (Sweden)

    Aiko Iwata-Otsubo

    2016-04-01

    Full Text Available Fluorescence in situ hybridization (FISH-based karyotyping is a powerful cytogenetics tool to study chromosome organization, behavior, and chromosome evolution. Here, we developed a FISH-based karyotyping system using a probe mixture comprised of centromeric and subtelomeric satellite repeats, 5S rDNA, and chromosome-specific BAC clones in common bean, which enables one to unambiguously distinguish all 11 chromosome pairs. Furthermore, we applied the karyotyping system to several wild relatives and landraces of common bean from two distinct gene pools, as well as other related Phaseolus species, to investigate repeat evolution in the genus Phaseolus. Comparison of karyotype maps within common bean indicates that chromosomal distribution of the centromeric and subtelomeric satellite repeats is stable, whereas the copy number of the repeats was variable, indicating rapid amplification/reduction of the repeats in specific genomic regions. In Phaseolus species that diverged approximately 2–4 million yr ago, copy numbers of centromeric repeats were largely reduced or diverged, and chromosomal distributions have changed, suggesting rapid evolution of centromeric repeats. We also detected variation in the distribution pattern of subtelomeric repeats in Phaseolus species. The FISH-based karyotyping system revealed that satellite repeats are actively and rapidly evolving, forming genomic features unique to individual common bean accessions and Phaseolus species.

  15. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil.

    Science.gov (United States)

    Peng, Jun; Zhan, Yuanfeng; Zeng, Fanyun; Long, Haibo; Pei, Yuelin; Guo, Jianrong

    2013-12-01

    Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is one of the major limiting factors for watermelon production worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid and quantitative detection of Fon in soil. Positive products were amplified only from Fon isolates and not from any other species or formae speciales of F. oxysporum tested, showing a high specificity of the primer sets. The detection limit of the RealAmp assay was 1.2 pg μL(-1) genomic DNA or 10(3) spores g(-1) of artificially inoculated soil, whereas real-time PCR could detect as low as 12 fg μL(-1) or 10(2) spores g(-1). The RealAmp assay was further applied to detect eight artificially inoculated and 85 field soil samples. No significant differences were found between the results tested by the RealAmp and real-time PCR assays. The RealAmp assay is a simple, rapid and effective technique for the quantitative detection and monitoring of Fon in soil under natural conditions. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  16. Resolving colocalization of bacteria and metal(loid)s on plant root surfaces by combining fluorescence in situ hybridization (FISH) with multiple-energy micro-focused X-ray fluorescence (ME μXRF).

    Science.gov (United States)

    Honeker, Linnea K; Root, Robert A; Chorover, Jon; Maier, Raina M

    2016-12-01

    Metal(loid)-contamination of the environment due to anthropogenic activities is a global problem. Understanding the fate of contaminants requires elucidation of biotic and abiotic factors that influence metal(loid) speciation from molecular to field scales. Improved methods are needed to assess micro-scale processes, such as those occurring at biogeochemical interfaces between plant tissues, microbial cells, and metal(loid)s. Here we present an advanced method that combines fluorescence in situ hybridization (FISH) with synchrotron-based multiple-energy micro-focused X-ray fluorescence microprobe imaging (ME μXRF) to examine colocalization of bacteria and metal(loid)s on root surfaces of plants used to phytostabilize metalliferous mine tailings. Bacteria were visualized on a small root section using SytoBC nucleic acid stain and FISH probes targeting the domain Bacteria and a specific group (Alphaproteobacteria, Gammaproteobacteria, or Actinobacteria). The same root region was then analyzed for elemental distribution and metal(loid) speciation of As and Fe using ME μXRF. The FISH and ME μXRF images were aligned using ImageJ software to correlate microbiological and geochemical results. Results from quantitative analysis of colocalization show a significantly higher fraction of As colocalized with Fe-oxide plaques on the root surfaces (fraction of overlap 0.49±0.19) than to bacteria (0.072±0.052) (p<0.05). Of the bacteria that colocalized with metal(loid)s, Actinobacteria, known for their metal tolerance, had a higher correlation with both As and Fe than Alphaproteobacteria or Gammaproteobacteria. This method demonstrates how coupling these micro-techniques can expand our understanding of micro-scale interactions between roots, metal(loid)s and microbes, information that should lead to improved mechanistic models of metal(loid) speciation and fate. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Sensitive and rapid detection of endogenous hydrogen sulfide distributing in different mouse viscera via a two-photon fluorescent probe

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qian [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082 (China); Yang, Jinfeng [Tumor Hospital, Xiangya School of Medicine, Central South University, Changsha, 410013 (China); Li, Yinhui, E-mail: yinhuili16@163.com [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082 (China); Zheng, Jing [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082 (China); Yang, Ronghua, E-mail: yangrh@pku.edu.cn [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082 (China); School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, 410004 (China)

    2015-10-08

    Development of efficient methods for detection of endogenous H{sub 2}S in living cells and tissues is of considerable significance for better understanding the biological and pathological functions of H{sub 2}S. Two-photon (TP) fluorescent probes are favorable as powerful molecular tools for studying physiological process due to its non-invasiveness, high spatiotemporal resolution and deep-tissues imaging. Up to date, several TP probes for intracellular H{sub 2}S imaging have been designed, but real-time imaging of endogenous H{sub 2}S-related biological processes in tissues is hampered due to low sensitivity, long response time and interference from other biothiols. To address this issue, we herein report a novel two-photon fluorescent probe (TPP-H{sub 2}S) for highly sensitive and fast monitoring and imaging H{sub 2}S levels in living cells and tissues. In the presence of H{sub 2}S, it exhibits obviously improved sensitivity (LOD: 0.12 μM) and fast response time (about 2 min) compared with the reported two-photon H{sub 2}S probes. With two-photon excitation, TPP-H{sub 2}S displays high signal-to-noise ratio and sensitivity even no interference in cell growth media. As further application, TPP-H{sub 2}S is applied for fast imaging of H{sub 2}S in living cells and different fresh tissues by two-photon confocal microscope. Most importantly we first measured the endogenous H{sub 2}S level in different viscera by vivisection and found that the distribution of endogenous H{sub 2}S mostly in brain, liver and lung. The excellent sensing properties of TPP-H{sub 2}S make it a practically useful tool for further studying biological roles of H{sub 2}S. - Highlights: • This two-photon probe exhibits an improved sensitivity and response time to H{sub 2}S. • This probe shows excellent membrane permeability and fast visualization of H{sub 2}S in living cells and tissues. • This probe is successfully applied to measure the endogenously produced H{sub 2}S levels in

  18. Chemical-Induced Read-Through at Premature Termination Codons Determined by a Rapid Dual-Fluorescence System Based on S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Emiliano Altamura

    Full Text Available Nonsense mutations generate in-frame stop codons in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs include the synthesis of truncated proteins with loss of protein function causing severe inherited or acquired diseases. A therapeutic approach has been recently developed that is based on the use of chemical agents with the ability to suppress PTCs (read-through restoring the synthesis of a functional full-length protein. Research interest for compounds able to induce read-through requires an efficient high throughput large scale screening system. We present a rapid, sensitive and quantitative method based on a dual-fluorescence reporter expressed in the yeast Saccharomyces cerevisiae to monitor and quantitate read-through at PTCs. We have shown that our novel system works equally well in detecting read-through at all three PTCs UGA, UAG and UAA.

  19. Fluorescence Imaging of Streptococcus pneumoniae with the Helix pomatia agglutinin (HPA) As a Potential, Rapid Diagnostic Tool

    Science.gov (United States)

    Domenech, Mirian; García, Ernesto

    2017-01-01

    Streptococcus pneumoniae is a common human pathogen and a major causal agent of life-threatening infections that can either be respiratory or non-respiratory. It is well known that the Helix pomatia (edible snail) agglutinin (HPA) lectin shows specificity for terminal αGalNAc residues present, among other locations, in the Forssman pentasaccharide (αGalNAc1→3βGalNAc1→3αGal1→4βGal1→4βGlc). Based on experiments involving choline-independent mutants and different growth conditions, we propose here that HPA recognizes the αGalNAc terminal residues of the cell wall teichoic and lipoteichoic acids of S. pneumoniae. In addition, experimental evidence showing that pneumococci can be specifically labeled with HPA when growing as planktonic cultures as well as in mixed biofilms of S. pneumoniae and Haemophilus influenzae has been obtained. It should be underlined that pneumococci were HPA-labeled despite of the presence of a capsule. Although some non-pneumococcal species also bind the agglutinin, HPA-binding combined with fluorescence microscopy constitutes a suitable tool for identifying S. pneumoniae and, if used in conjunction with Gram staining and/or other suitable technique like antigen detection, it may potentially facilitate a fast and accurate diagnosis of pneumococcal infections. PMID:28769901

  20. Simultaneous measurements of velocity, temperature, and pressure using rapid CW wavelength-modulation laser-induced fluorescence of OH

    Science.gov (United States)

    Chang, A. Y.; Battles, B. E.; Hanson, R. K.

    1990-01-01

    In high speed flows, laser induced fluorescence (LIF) on Doppler shifted transitions is an attractive technique for velocity measurement. LIF velocimetry was applied to combined single-point measurements of velocity, temperature, and pressure and 2-D imaging of velocity and pressure. Prior to recent research using NO, LIF velocimetry in combustion related flows relied largely on the use of seed molecules. Simultaneous, single-point LIF measurements is reported of velocity, temperature, and pressure using the naturally occurring combustion species OH. This experiment is an extension of earlier research in which a modified ring dye laser was used to make time resolved temperature measurements behind reflected shock waves by using OH absorption an in postflame gases by using OH LIF. A pair of fused-silica rhombs mounted on a single galvanonmeter in an intracavity-doubled Spectra-Physics 380 ring laser permit the UV output to be swept continuously over a few wave numbers at an effective frequency of 3kHz.

  1. Rapid identification of polystyrene foam wastes containing hexabromocyclododecane or its alternative polymeric brominated flame retardant by X-ray fluorescence spectroscopy.

    Science.gov (United States)

    Schlummer, Martin; Vogelsang, Jörg; Fiedler, Dominik; Gruber, Ludwig; Wolz, Gerd

    2015-07-01

    The brominated flame retardant hexabromocyclododecane (HBCDD) was added to Annex A of the list of persistent organic pollutants (POPs) of the Stockholm Convention. Thus, production and use of HBCDD will be banned, and the recycling of HBCDD-containing foam waste will be restricted. In reaction a special polymeric brominated flame retardant (PolyFR) was developed to replace HBCDD in expanded and extruded polystyrene foams for building and construction applications. A decision has to be made at some future time whether expanded and extruded polystyrene foam waste is to be subjected to incineration (with HBCDD) or to recycling (without HBCDD). Therefore, an appropriate and rapid field method is required to distinguish between foams containing HBCDD and foams free from HBCDD. Here we present a screening method for identifying HBCDD containing expanded and extruded polystyrene foams. The test principle is based on the fact that PolyFR (a brominated polymeric macromolecule) is not extractable whereas HBCDD (a low molecular weight substance) is extractable. Following rapid extraction of HBCDD the brominated flame retardant is identified and quantified via bromine analysis using a handheld X-ray fluorescence instrument. The method was applied successfully to 27 expanded and extruded polystyrene foam samples (foams and extruded polystyrene foam raw materials), which were provided without any information about the applied flame retardant. The presence of HBCDD was confirmed for all HBCDD-positive samples in the test. A robustness test revealed a high degree of correctness and a high repeatability for the test system: samples containing HBCDD and HBCDD-free samples were identified correctly with relative standard deviations of quantitative results below 14%. Moreover, X-ray fluorescence spectroscopy test results agree well with HBCDD determinations performed in a laboratory with a gas chromatograph coupled to a flame ionisation detector. © The Author(s) 2015.

  2. Rapid real-time diagnostic PCR for Trichophyton rubrum and Trichophyton mentagrophytes in patients with tinea unguium and tinea pedis using specific fluorescent probes.

    Science.gov (United States)

    Miyajima, Yoshiharu; Satoh, Kazuo; Uchida, Takao; Yamada, Tsuyoshi; Abe, Michiko; Watanabe, Shin-ichi; Makimura, Miho; Makimura, Koichi

    2013-03-01

    Trichophyton rubrum and Trichophyton mentagrophytes human-type (synonym, Trichophyton interdigitale (anthropophilic)) are major causative pathogens of tinea unguium. For suitable diagnosis and treatment, rapid and accurate identification of etiologic agents in clinical samples using reliable molecular based method is required. For identification of organisms causing tinea unguium, we developed a new real-time polymerase chain reaction (PCR) with a pan-fungal primer set and probe, as well as specific primer sets and probes for T. rubrum and T. mentagrophytes human-type. We designed two sets of primers from the internal transcribed spacer 1 (ITS1) region of fungal ribosomal DNA (rDNA) and three quadruple fluorescent probes, one for detection wide range pathogenic fungi and two for classification of T. rubrum and T. mentagrophytes by specific binding to different sites in the ITS1 region. We investigated the specificity of these primer sets and probes using fungal genomic DNA, and also examined 42 clinical specimens with our real-time PCR. The primers and probes specifically detected T. rubrum, T. mentagrophytes, and a wide range of pathogenic fungi. The causative pathogens were identified in 42 nail and skin samples from 32 patients. The total time required for identification of fungal species in each clinical specimen was about 3h. The copy number of each fungal DNA in the clinical specimens was estimated from the intensity of fluorescence simultaneously. This PCR system is one of the most rapid and sensitive methods available for diagnosing dermatophytosis, including tinea unguium and tinea pedis. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  3. Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

    Science.gov (United States)

    2013-01-01

    Background Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina. PMID:23586331

  4. Rapid and accurate detection of Escherichia coli growth by fluorescent pH-sensitive organic nanoparticles for high-throughput screening applications.

    Science.gov (United States)

    Si, Yang; Grazon, Chloé; Clavier, Gilles; Rieger, Jutta; Audibert, Jean-Frédéric; Sclavi, Bianca; Méallet-Renault, Rachel

    2016-01-15

    Rapid detection of bacterial growth is an important issue in the food industry and for medical research. Here we present a novel kind of pH-sensitive fluorescent nanoparticles (FANPs) that can be used for the rapid and accurate real-time detection of Escherichia coli growth. These organic particles are designed to be non-toxic and highly water-soluble. Here we show that the coupling of pH sensitive fluoresceinamine to the nanoparticles results in an increased sensitivity to changes in pH within a physiologically relevant range that can be used to monitor the presence of live bacteria. In addition, these FANPs do not influence bacterial growth and are stable over several hours in a complex medium and in the presence of bacteria. The use of these FANPs allows for continuous monitoring of bacterial growth via real-time detection over long time scales in small volumes and can thus be used for the screening of a large number of samples for high-throughput applications such as screening for the presence of antibiotic resistant strains. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs.

    Science.gov (United States)

    Tian, C J; Lin, Z X; He, X M; Luo, Q; Luo, C B; Yu, H Q; Chen, R; Wu, X W; Zhu, D Z; Ren, Z J; Bi, Y Z; Ji, J

    2012-08-01

    The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter.

  6. Independent Predictors for Lifetime and Recent Substance Use Disorders in Patients with Rapid Cycling Bipolar Disorder: Focus on Anxiety Disorders

    Science.gov (United States)

    Gao, Keming; Chan, Philip K.; Verduin, Marcia L.; Kemp, David E.; Tolliver, Bryan K.; Ganocy, Stephen J.; Bilali, Sarah; Brady, Kathleen T.; Findling, Robert L.; Calabrese, Joseph R.

    2010-01-01

    We set out to study independent predictor(s) for lifetime and recent substance use disorder (SUDs) in patients with rapid cycling bipolar disorder (RCBD). Extensive Clinical Interview and Mini International Neuropsychiatric Interview were used to ascertain DSM-IV Axis I diagnoses of RCBD, anxiety disorders, and SUDs. Data from patients enrolling into four similar clinical trials were used. Where appropriate, univariate analyses with t-test or Chi-Square were applied. Stepwise logistic regression was used to examine the relationship among predictor variables and lifetime and recent SUDs. Univariate analysis showed that patients with co-occurring anxiety disorders (n=261) had significantly increased rates of lifetime (OR=2.1) and recent (OR=1.9) alcohol dependence as well as lifetime (OR=3.4) and recent (OR=2.5) marijuana dependence compared to those without co-occurring anxiety disorder (n=303). In logistic regression analyses, generalized anxiety disorder (GAD) was associated with increased risk for lifetime SUDs (OR=2.34), alcohol dependence (OR=1.73), and marijuana dependence (OR=3.36), and recent marijuana dependence (OR=3.28). A history of physical abuse was associated with increased risk for lifetime SUDs (OR=1.71) and recent marijuana dependence (OR=3.47). Earlier onset of first mania/hypomania was associated with increased risk for lifetime SUDs (5% per year) and recent marijuana dependence (12% per year) and later treatment with a mood stabilizer were also associated with increased risk for recent SUDs (8% per year). Positive associations between generalized anxiety disorder, later treatment with a mood stabilizer, and early childhood trauma and history of SUDs suggests that adequate treatment of comorbid anxiety, early treatment with a mood stabilizer, and prevention of childhood trauma may reduce the risk for the development of SUDs in patients with bipolar disorder. PMID:20716307

  7. Rapid, one-step fabrication and loading of nanoscale 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes in a simple, double flow-focusing microfluidic device.

    Science.gov (United States)

    Tien Sing Young, Ryan V; Tabrizian, Maryam

    2015-07-01

    Liposomes are currently well-established as biocompatible delivery vehicles for numerous compounds. However, conventional manufacturing tends to rely on time-consuming processes, costly equipment, unstable reaction parameters, and numerous pre- and post-processing steps. Herein, we demonstrate a microscope-slide-sized alternative: a double flow-focusing microfluidic geometry capable of sub-hour synthesis and controlled loading of tunable liposomes. Using phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine as the bilayer constituent, the effect of varying the dissolved lipid concentration and flow rate ratio on synthesized liposome diameters was investigated and the encapsulation of fluorescent hydrophobic drug model ergost-5,7,9(11),22-tetraen-3β-ol was performed to ascertain the potential of this device as a loading platform.

  8. Rapid, low-cost fluorescent assay of β-lactamase-derived antibiotic resistance and related antibiotic susceptibility

    Science.gov (United States)

    Erdem, S. Sibel; Khan, Shazia; Palanisami, Akilan; Hasan, Tayyaba

    2014-10-01

    Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of β-lactamase resistance (the most common form of AR) that uses a modified β-lactam structure decorated with two fluorophores quenched due to their close proximity. When the β-lactam core is cleaved by β-lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the β-lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of β-lactamase resistance, both for epidemiological purposes as well as individualized patient care.

  9. Use of "biokit HSV-2 Rapid Assay" to improve the positive predictive value of Focus HerpeSelect HSV-2 ELISA.

    Science.gov (United States)

    Morrow, Rhoda Ashley; Friedrich, David; Meier, Amalia; Corey, Lawrence

    2005-10-14

    Commercially available assays to detect antibodies to the herpes simplex virus type 2 (HSV-2)-specific glycoprotein gG-2 have markedly improved serologic diagnosis of HSV-2 infection. However, even tests with high specificity can have low positive predictive values in low prevalence populations. HSV-2 is a chronic, life-long viral infection that requires both medical attention and potential alterations in health care strategy. As such, the concern for false positive diagnoses is high confirmatory testing is routine for other viral serologies such as HIV and hepatitis C. We evaluated such a strategy for HSV-2 serology by using an easily performed commercial test, biokitHSV-2 rapid test ("Biokit"; Biokit USA, Lexington MA) as a confirmatory test for the widely used gG-2 specific serology ("Focus;" HerpeSelect HSV-2 ELISA; Focus Diagnostics, Cypress CA). We tested 782 sera by Focus HSV-2 ELISA, Biokit, and the current gold standard test, Western blot (WB). The positive predictive value of the Focus HSV-2 ELISA increased from 80.5% to 95.6% when Biokit testing was performed on sera that were initially positive by Focus HSV-2 ELISA. Confirmatory testing increased the specificity markedly among sera with Focus EIA values between 1.1 and 3.5: only 35% of low positive (index values 1.1-3.5) Focus HSV-2 ELISA results confirmed as positive by Biokit and WB compared with 92% of those with index values >3.5. Mathematical modeling of the data resulted in expected positive predictive values over 98% for populations with antibody prevalences typical of clinical practices in the US and Europe. Confirmatory Biokit testing of positive Focus HSV-2 ELISA results is fast, easy, and effective in reducing falsely positive HSV-2 antibody results. Patients, clinicians, and laboratories could benefit from the enhanced specificity of this simple HSV-2 serologic test combination.

  10. Use of "biokit HSV-2 Rapid Assay" to improve the positive predictive value of Focus HerpeSelect HSV-2 ELISA

    Directory of Open Access Journals (Sweden)

    Friedrich David

    2005-10-01

    Full Text Available Abstract Background Commercially available assays to detect antibodies to the herpes simplex virus type 2 (HSV-2-specific glycoprotein gG-2 have markedly improved serologic diagnosis of HSV-2 infection. However, even tests with high specificity can have low positive predictive values in low prevalence populations. HSV-2 is a chronic, life-long viral infection that requires both medical attention and potential alterations in health care strategy. As such, the concern for false positive diagnoses is high confirmatory testing is routine for other viral serologies such as HIV and hepatitis C. We evaluated such a strategy for HSV-2 serology by using an easily performed commercial test, biokitHSV-2 rapid test ("Biokit"; Biokit USA, Lexington MA as a confirmatory test for the widely used gG-2 specific serology ("Focus;" HerpeSelect HSV-2 ELISA; Focus Diagnostics, Cypress CA. Methods We tested 782 sera by Focus HSV-2 ELISA, Biokit, and the current gold standard test, Western blot (WB. Results The positive predictive value of the Focus HSV-2 ELISA increased from 80.5% to 95.6% when Biokit testing was performed on sera that were initially positive by Focus HSV-2 ELISA. Confirmatory testing increased the specificity markedly among sera with Focus EIA values between 1.1 and 3.5: only 35% of low positive (index values 1.1–3.5 Focus HSV-2 ELISA results confirmed as positive by Biokit and WB compared with 92% of those with index values >3.5. Mathematical modeling of the data resulted in expected positive predictive values over 98% for populations with antibody prevalences typical of clinical practices in the US and Europe. Conclusion Confirmatory Biokit testing of positive Focus HSV-2 ELISA results is fast, easy, and effective in reducing falsely positive HSV-2 antibody results. Patients, clinicians, and laboratories could benefit from the enhanced specificity of this simple HSV-2 serologic test combination.

  11. Verification of Rapid Focused-Recharge in Depressions of Kuwait and the Arabian Peninsula Using Thermal and VNIR Remote Sensing

    Science.gov (United States)

    Rotz, R. R.; Milewski, A.

    2013-12-01

    In the Arabian Peninsula, freshwater recharge from rainfall is infrequent. Recharge is typically focused in small depressions that fill with seasonal runoff and potentially form freshwater lenses. This phenomenon has been verified in the Raudhatain watershed in Kuwait. This study aims to substantiate previously hypothesized lens locations and detect water in the subsurface by using thermal remote sensing and rainfall data. Potential freshwater lenses (~142) have been previously postulated throughout Kuwait and Saudi Arabia, but lack verification due to inadequate monitoring networks. We hypothesize that due to water's unique heat capacity, recharge zones can be detected by identifying areas with lower changes in surface radiance values than neighboring dry areas between day and night after peak or sustained rainfall. If successful, recharge zones and freshwater lenses can be identified and verified in remote hyper-arid regions. We collected 320 high-resolution (15m - 90m), low cloud cover (images in the visible near-infrared (VNIR) and thermal infrared (TIR) wavelengths obtained from the Advanced Spaceborne Thermal Emission and Reflection Radiometer sensor (ASTER) between 2004 and 2012. Overlapping day and night images were subtracted from each other to show surface radiance fluctuations and difference images were compared with rainfall data from Daily TRMM_3B42v7a between 2004 and 2012. Several lens locations, runoff channels, agricultural regions, and wetlands were detected in areas where radiance values change between 0.067 - 2.25 Wsr-1m-2 from day to night scenes and verified by Google Earth (15m), Landsat (30m), and ASTER VNIR (15m) images. Additionally, two seasonal peak rainfall (~35mm/day) events positively correlate with the surface radiance difference values. Surface radiance values for dry areas adjacent to the postulated lens locations range between 2.25 - 12.2 Wsr-1m-2. Results demonstrate the potential for shallow groundwater detection through the

  12. A new turn-on fluorimetric method for the rapid speciation of Cr(III)/Cr(VI) species in tea samples with rhodamine-based fluorescent reagent

    Science.gov (United States)

    Özyol, Esra; Saçmacı, Şerife; Saçmacı, Mustafa; Ülgen, Ahmet

    2018-02-01

    A new fluorimetric method with rhodamine-based fluorescent agent was developed for the rapid speciation of Cr(III)/Cr(VI) in tea, soil and water samples. The system, which utilizes a fluorescent reagent, was used for the first time after synthesis/characterization of 3‧,6‧-bis(diethylamino)-2-{[(1E)-(2,4-dimethoxyphenyl)methylene] amino}spiro[isoindole-1,9‧-xanthen]-3(2H)-one (BDAS). The reagent responds instantaneously at room temperature in a 1:1 stoichiometric manner to the amount of Cr(III). The selectivity of this system for Cr(III) over other metal ions is remarkably high, and its sensitivity is below 0.01 mg L- 1 in aqueous solutions which enables a simplification without any pretreatment of the real sample. The method has a wide linear range of 0.1-10 mg L- 1 and a detection limit of 0.15 μg L- 1 for Cr(III) while the relative standard deviation was 0.1% for 0.1 mg L- 1 Cr(III) concentration. The results of detection and recovery experiments for Cr(III) in tea, soil and water were satisfactory, indicating that the method has better feasibility and application potential in the routine determination and speciation of Cr(III)/Cr(VI). The results of analysis of the certified reference material (INCT-TL-1 tea sample and CWW-TM-D waste water) are in good agreement with the certified value.

  13. Rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 in soil by real-time fluorescence loop-mediated isothermal amplification.

    Science.gov (United States)

    Peng, Jun; Zhang, He; Chen, Fengping; Zhang, Xin; Xie, Yixian; Hou, Xianwen; Li, Guangyi; Pu, Jinji

    2014-12-01

    In this study, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) was developed and evaluated for the rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 (R4) in soil. The LAMP primer set was designed based on previously verified RAPD marker sequences, and the RealAmp assay could specifically detect and distinguish R4 isolates from other related species. The detection sensitivity of the RealAmp assay was approx. 3·82 × 10(3) copies of plasmid DNA or 10(3) of spores per gram in artificially infested soil, indicating that the method is highly tolerant to inhibitor substances in soil compared to real-time PCR. Combining previously published TR4-specific detection methods with the newly established R4-specific RealAmp assay, an indirect approach to detect and differentiate ST4 isolates was achieved by comparing the detection results of R4 and TR4 simultaneously. The existence of ST4 isolates in China was subsequently confirmed through the developed approach. The developed RealAmp assay has been confirmed to be a simple, rapid and effective method to detect R4 in soil, which facilitates to further identify and distinguish ST4 isolates through the comparative analysis of detection results between TR4 and R4 simultaneously. The technique is an alternative quantitative detection method, which will be used for a routine detection service for the soil-borne pathogen in China. © 2014 The Society for Applied Microbiology.

  14. Rapid and sensitive detection of Cronobacter spp. (previously Enterobacter sakazakii) in food by duplex PCR combined with capillary electrophoresis-laser-induced fluorescence detector.

    Science.gov (United States)

    Ruan, Jia; Li, Ming; Liu, Ya-Pan; Li, Yuan-Qian; Li, Yong-Xin

    2013-03-15

    Cronobacter spp. (Enterobacter sakazakii) is an emerging opportunistic pathogen with a 40-80% mortality rate in infants and immunocompromised crowd resulting from the consumption of contaminated food. A novel method for detecting Cronobacter spp. in food samples by duplex polymerase chain reaction (PCR) in combination with capillary electrophoresis-laser induced fluorescence (CE-LIF) detector has been developed. The specific gene sequences of 16S-23S rDNA internal transcribed spacer (ITS) and the outer membrane protein A (OmpA) of Cronobacter spp. were amplified by duplex PCR. The PCR products were separated and determined sensitively by CE-LIF within 12min. The relative standard deviations of migration time for the detected DNA fragments were 2.01-2.91%. The detection limit was as low as 1.6×10(1)cfu/mL of Cronobacter spp. Besides, the specificity of the method was verified by 24 non-Cronobacter bacterial strains. A total of 120 commercial infant food formula were tested for the presence of Cronobacter spp. by using the proposed method. This current study demonstrates that the combination of CE-LIF method with duplex PCR is rapid, sensitive and environmental friendly, and has the potential to be adapted for the routine detection of Cronobacter spp. in food samples. To the best of our knowledge, this is the first use of CE-LIF for the detection of Cronobacter spp. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  15. Sensitive, accurate and rapid detection of trace aliphatic amines in environmental samples with ultrasonic-assisted derivatization microextraction using a new fluorescent reagent for high performance liquid chromatography.

    Science.gov (United States)

    Chen, Guang; Liu, Jianjun; Liu, Mengge; Li, Guoliang; Sun, Zhiwei; Zhang, Shijuan; Song, Cuihua; Wang, Hua; Suo, Yourui; You, Jinmao

    2014-07-25

    A new fluorescent reagent, 1-(1H-imidazol-1-yl)-2-(2-phenyl-1H-phenanthro[9,10-d]imidazol-1-yl)ethanone (IPPIE), is synthesized, and a simple pretreatment based on ultrasonic-assisted derivatization microextraction (UDME) with IPPIE is proposed for the selective derivatization of 12 aliphatic amines (C1: methylamine-C12: dodecylamine) in complex matrix samples (irrigation water, river water, waste water, cultivated soil, riverbank soil and riverbed soil). Under the optimal experimental conditions (solvent: ACN-HCl, catalyst: none, molar ratio: 4.3, time: 8 min and temperature: 80°C), micro amount of sample (40 μL; 5mg) can be pretreated in only 10 min, with no preconcentration, evaporation or other additional manual operations required. The interfering substances (aromatic amines, aliphatic alcohols and phenols) get the derivatization yields of impurities. With this UDME-HPLC-FD-MS method, the accuracy (-0.73-2.12%), precision (intra-day: 0.87-3.39%; inter-day: 0.16-4.12%), recovery (97.01-104.10%) and sensitivity were significantly improved. Successful applications in environmental samples demonstrate the superiority of this method in the sensitive, accurate and rapid determination of trace aliphatic amines in micro amount of complex samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients.

    Science.gov (United States)

    Steensels, Deborah; Reynders, Marijke; Descheemaeker, Patrick; Curran, Martin D; Jacobs, Frédérique; Denis, Olivier; Delforge, Marie-Luce; Montesinos, Isabel

    2017-07-01

    Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (ptesting were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly. Copyright © 2017 Elsevier B.V. All

  17. Short communication: A novel method using immunomagnetic separation with a fluorescent nanobeads lateral flow assay for the rapid detection of low-concentration Escherichia coli O157:H7 in raw milk.

    Science.gov (United States)

    Huang, Zhen; Cui, Xi; Xie, Quan-Yuan; Liu, Dao-Feng; Lai, Wei-Hua

    2016-12-01

    Escherichia coli O157:H7 is an important serotype of enterohemorrhagic E. coli that was first identified as a human pathogen in 1982. This pathogen causes several serious diseases. In this study, immunomagnetic separation was coupled with a fluorescent nanobeads lateral flow assay to establish a sensitive and rapid detection method for Escherichia coli O157:H7 in raw milk. The pathogen was captured from raw milk by immunomagnetic separation with immunomagnetic nanobeads and then detected using a fluorescent nanobeads lateral flow assay. A fluorescent line was formed in the test line of the test strip and quantitatively detected using a fluorescent reader. Screening times, which included immunomagnetic separation and the fluorescent nanobeads lateral flow assay, were 8, 7, 6, and 5h when 1, 5, 25, and 125 cfu of E. coli O157:H7, respectively, were inoculated into 25mL of raw milk. The established method could be widely applied to the rapid onsite detection of other pathogens to ensure food safety. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  18. Establishment of a green fluorescent protein tracing murine model focused on the functions of host components in necrosis repair and the niche of subcutaneously implanted glioma.

    Science.gov (United States)

    Lu, Zhao-Hui; Lv, Ke; Zhang, Jin-Shi; Dai, Chun-Gang; Liu, Bin; Ma, Xiao-Yu; He, Lin-Ming; Jia, Jing-Yun; Chen, Yan-Ming; Dai, Xing-Liang; Wang, Ai-Dong; Dong, Jun; Zhang, Quan-Bin; Lan, Qing; Huang, Qiang

    2014-02-01

    Due to progress in the research of glioma stem cells and the glioma niche, development of an animal model that facilitates the elucidation of the roles of the host tissue and cells is necessary. The aim of the present study was to develop a subcutaneous xenograft green fluorescent protein nude mouse model and use this model to analyze the roles of host cells in tumor necrosis repair. Tumors derived from the human glioma stem/progenitor cell line SU3 were subcutaneously implanted in green fluorescent protein nude mice. The implanted tumors were then passed from animal to animal for 10 generations. Finally, subcutaneous xenografts were assayed with traditional pathology, immunopathological techniques and fluorescence photography. For each generation, the tumorigenicity rate was 100%. Subcutaneous xenografts were rich in blood vessels, and necrotic and hemorrhagic foci, which highly expressed hypoxia-inducible factor-1α, tumor necrosis factor, Ki-67, CD68 and CD11b. In the interstitial tissue, particularly in old hemorrhagic foci, there were numerous cells expressing green fluorescent protein, CD68 and CD11b. Green fluorescent protein nude mouse subcutaneous xenografts not only consistently maintained the high invasiveness and tumorigenicity of glioma stem/progenitor cells, but also consisted of a high concentration of tumor blood vessels and necrotic and hemorrhagic foci. Subcutaneous xenografts also expressed high levels of tumor microenvironment-related proteins and host-derived tumor interstitial molecules. The model has significant potential for further research on tumor tissue remodeling and the tumor microenvironment.

  19. Establishment of a Bioenergy-Focused Microalgae Strain Collection Using Rapid, High-Throughput Methodologies: Cooperative Research and Development Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Pienkos, Philip T. [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2013-11-01

    This project is part of the overall effort by and among NREL, Colorado State University, University of Colorado, and Colorado School of Mines known as the Colorado Center for Biorefining and Biofuels. This is part of a larger statewide effort provided for in House Bill 06-1322, establishing a Colorado Collaboratory that envisions these four institutions working together as part of the state'senergy plan. This individual project with Colorado School of Mines is the first of many envisioned in this overall effort. The project focuses on development of high throughput procedures aimed at rapidly isolating and purifying novel microalgal strains (specifically green alga and diatoms) from water samples obtained from unique aquatic environments.

  20. Rapid Characterization of Microcystin-Producing Cyanobacteria in Freshwater Lakes by TSA-FISH (Tyramid Signal Amplification-Fluorescent In Situ Hybridization

    Directory of Open Access Journals (Sweden)

    Luc Brient

    2017-07-01

    Full Text Available Microcystin (MC is a common and widespread toxin which represents a health hazard to humans and animals. MC toxin concentrations are monitored by various direct or proxy techniques (HPLC, LC-MS/MS, ELISA, PPIA, however, these techniques do not discriminate producing species from non-producing ones. In order to simultaneously provide the identity and activity of cyanotoxin producing species in freshwater lakes, we applied simple, and fully detailed, whole cell fluorescent in situ hybridization enhanced by tyramid signal amplification (TSA-FISH. DNA oligonucleotide probes MICR3 and MCYA were targeting 16S rRNA and mcyA-mRNA, respectively. The mcyA gene is coding for the MC synthetase enzyme involved in MC synthesis. Controls were acquired with the general eubacterial 16S rRNA probe EUB338, for TSA-FISH assay, and standard HPLC and LC-MS/MS as standard methods for the measurements of MC concentration. Results obtained from monoclonal strains and natural samples demonstrated a specific identification of Microcystis species and were able to discriminate MC producing from non-producing ones. In addition, the MCYA probe allowed the specific detection of MC-synthetase mRNA within Planktothrix isothrix (Oscillatoriale filaments. Two kinds of mcyA-mRNA labeling were observed in these cells, spots like and plasmid like, which illustrates the well-known plasticity of microbial genome to adapt to environmental stresses. We demonstrated that a simple TSA-FISH assay allows acquiring rapidly dual information of the presence and abundance of potentially toxic species, while identifying species actively producing MC-synthetase mRNA, a proxy of MC toxin. This technique has the potential to be developed into an effective environmental monitoring tool. In addition, detail visualization of cellular mRNAs is powerful for the acquisition of ecological and biomolecular studies of toxic cyanobacteria.

  1. Establishment of a novel immunoassay system for rapid detection of 2,4-dichlorophenoxyacetic acid residues based on magnetic-fluorescent probes

    Directory of Open Access Journals (Sweden)

    WANG Yuanfeng

    2014-12-01

    Full Text Available A novel immunoassay system based on magnetic-fluorescent probes was established to detect 2.4-dichlorophenoxyacetic acid (2,4-D residue in liquid system in food and agricultural products.The composites of anti-2,4-D antibody bound to Fe3O4@SiO2-NH2 was employed as the solid phase as well as magnetic probe.The composites composed of 2,4-D-OVA labeled with CdTe@SiO2-NH2 as the fluorescent probe was used to produce fluorescent signal.2,4-D and its fluorescent probe competed binding the antibody on the surface of the magnetic probe.The optimization of 2,4-D-OVA dosage,coupling PH and reaction time in preparing the fluorescent probe were investigated.It showed that in the synthesis of fluorescent probe 8.2 was the optimal pH,70 min was the optimal coupling time,500 μL amount of 2,4-D-OVA.The standard curve was obtained with the concentration of 2,4-D and the maximum fluorescence intensity.The detection limit of the assay was gotten and it was 3.55×10-8.One reaction step and one washing step were needed.The assay significantly shortened the testing time and amplified the detection signal compared with classic ELISA.

  2. An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea.

    Science.gov (United States)

    Rao, C Durga; Reddy, Harikrishna; Naidu, Jagadish R; Raghavendra, A; Radhika, N S; Karande, Anjali

    2015-11-01

    We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea. The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. A selective and label-free strategy for rapid screening of telomere-binding Ligands via fluorescence regulation of DNA/silver nanocluster

    National Research Council Canada - National Science Library

    Rui Cheng; Jing Xu; Xiafei Zhang; Zhilu Shi; Qi Zhang; Yan Jin

    2017-01-01

    Herein, the conformational switch of G-rich oligonucleotide (GDNA) demonstrated the obvious functional switch of GDNA which was found to significantly affect the fluorescence of the in-situ synthesized DNA/silver nanocluster (DNA-AgNC...

  4. Fundamentals of fluorescence and fluorescence microscopy.

    Science.gov (United States)

    Wolf, David E

    2013-01-01

    This chapter discusses the fundamental physics of fluorescence. The application of fluorescence to microscopy represents an important transition in the development of microscopy, particularly as it applies to biology. It enables quantitating the amounts of specific molecules within a cell, determining whether molecules are complexing on a molecular level, measuring changes in ionic concentrations within cells and organelles, and measuring molecular dynamics. This chapter also discusses the issues important to quantitative measurement of fluorescence and focuses on four of quantitative measurements of fluorescence--boxcar-gated detection, streak cameras, photon correlation, and phase modulation. Although quantitative measurement presents many pitfalls to the beginner, it also presents significant opportunities to one skilled in the art. This chapter also examines how fluorescence is measured in the steady state and time domain and how fluorescence is applied in the modern epifluorescence microscope. Copyright © 2007 Elsevier Inc. All rights reserved.

  5. Photophysical Diversity of Water-Soluble Fluorescent Conjugated Polymers Induced by Surfactant Stabilizers for Rapid and Highly Selective Determination of 2,4,6-Trinitrotoluene Traces.

    Science.gov (United States)

    Alizadeh, Naader; Akbarinejad, Alireza; Ghoorchian, Arash

    2016-09-21

    The increasing application of fluorescence spectroscopy in development of reliable sensing platforms has triggered a lot of research interest for the synthesis of advanced fluorescent materials. Herein, we report a simple, low-cost strategy for the synthesis of a series of water-soluble conjugated polymer nanoparticles with diverse emission range using cationic (hexadecyltrimethylammonium bromide, CTAB), anionic (sodium dodecylbenzenesulfonate, SDBS), and nonionic (TX114) surfactants as the stabilizing agents. The role of surfactant type on the photophisical and sensing properties of resultant polymers has been investigated using dynamic light scattering (DLS), FT-IR, UV-vis, fluorescence, and energy dispersive X-ray (EDS) spectroscopies. The results show that the surface polarity, size, and spectroscopic and sensing properties of conjugated polymers could be well controlled by the proper selection of the stabilizer type. The fluorescent conjugated polymers exhibited fluorescence quenching toward nitroaromatic compounds. Further studies on the fluorescence properties of conjugated polymers revealed that the emission of the SDBS stabilized polymer, N-methylpolypyrrole-SDBS (NMPPY-SDBS), is strongly quenched by 2,4,6-trinitrotoluene molecule with a large Stern -Volmer constant of 59 526 M(-1) and an excellent detection limit of 100 nM. UV-vis and cyclic voltammetry measurements unveiled that fluorescence quenching occurs through a charge transfer mechanism between electron rich NMPPY-SDBS and electron deficient 2,4,6-trinitrotoluene molecules. Finally, the as-prepared conjugated polymer and approach were successfully applied to the determination of 2,4,6-trinitrotoluene in real water samples.

  6. Micellar Enhanced Three-Dimensional Excitation-Emission Matrix Fluorescence for Rapid Determination of Antihypertensives in Human Plasma with Aid of Second-Order Calibration Methods

    Directory of Open Access Journals (Sweden)

    Hai-Yan Fu

    2015-01-01

    Full Text Available A highly sensitive three-dimensional excitation-emission fluorescence method was proposed to determine antihypertensives including valsartan and amlodipine besylate in human plasma with the aid of second-order calibration methods based on parallel factor analysis (PARAFAC and alternating trilinear decomposition (ATLD algorithms. Antihypertensives with weak fluorescent can be transformed into a strong fluorescent property by changing microenvironment in samples using micellar enhanced surfactant. Both the adopted algorithms with second-order advantage can improve the resolution and directly attain antihypertensives concentration even in the presence of potential strong intrinsic fluorescence from human plasma. The satisfactory results can be achieved for valsartan and amlodipine besylate in complicated human plasma. Furthermore, some statistical parameters and figures of merit were evaluated to investigate the performance of the proposed method, and the accuracy and precision of the proposed method were also validated by the elliptical joint confidence region (EJCR test and repeatability analysis of intraday and interday assay. The proposed method could not only light a new avenue to directly determine valsartan or amlodipine besylate in human plasma, but also hold great potential to be extended as a promising alternative for more practical applications in the determination of weak fluorescent drugs.

  7. Development of Methods for the Real-Time and Rapid Identification and Detection of TSE in Living Animals Using Fluorescence Spectroscopy of the Eye

    Science.gov (United States)

    2006-07-01

    the eye, especially in the diseased eye. An increase in lipofuscin accumulation is known to occur in human Creutzfeldt-Jakob disease victims and in...retina is homogeneously weakly emmissive or whether it is heterogeneous, containing very bright regions of highly fluorescent material resulting from

  8. Development of an Immunomagnetic Bead-Immunoliposome Fluorescence Assay for Rapid Detection of Escherichia coli O157:H7 in Aqueous Samples and Comparison of the Assay with a Standard Microbiological Method

    OpenAIRE

    DeCory, Thomas R.; Durst, Richard A.; Zimmerman, Scott J.; Garringer, Linda A.; Paluca, Gary; DeCory,Heleen H.; Montagna, Richard A.

    2005-01-01

    The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic b...

  9. Rapid, sensitive, and selective fluorescent DNA detection using iron-based metal-organic framework nanorods: Synergies of the metal center and organic linker.

    Science.gov (United States)

    Tian, Jingqi; Liu, Qian; Shi, Jinle; Hu, Jianming; Asiri, Abdullah M; Sun, Xuping; He, Yuquan

    2015-09-15

    Considerable recent attention has been paid to homogeneous fluorescent DNA detection with the use of nanostructures as a universal "quencher", but it still remains a great challenge to develop such nanosensor with the benefits of low cost, high speed, sensitivity, and selectivity. In this work, we report the use of iron-based metal-organic framework nanorods as a high-efficient sensing platform for fluorescent DNA detection. It only takes about 4 min to complete the whole "mix-and-detect" process with a low detection limit of 10 pM and a strong discrimination of single point mutation. Control experiments reveal the remarkable sensing behavior is a consequence of the synergies of the metal center and organic linker. This work elucidates how composition control of nanostructures can significantly impact their sensing properties, enabling new opportunities for the rational design of functional materials for analytical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Investigating portable fluorescent microscopy (CyScope® as an alternative rapid diagnostic test for malaria in children and women of child-bearing age

    Directory of Open Access Journals (Sweden)

    Sousa-Figueiredo José

    2010-08-01

    Full Text Available Abstract Background Prompt and correct diagnosis of malaria is crucial for accurate epidemiological assessment and better case management, and while the gold standard of light microscopy is often available, it requires both expertise and time. Portable fluorescent microscopy using the CyScope® offers a potentially quicker, easier and more field-applicable alternative. This article reports on the strengths, limitations of this methodology and its diagnostic performance in cross-sectional surveys on young children and women of child-bearing age. Methods 552 adults (99% women of child-bearing age and 980 children (99% ≤ 5 years of age from rural and peri-urban regions of Ugandan were examined for malaria using light microscopy (Giemsa-stain, a lateral-flow test (Paracheck-Pf® and the CyScope®. Results from the surveys were used to calculate diagnostic performance (sensitivity and specificity as well as to perform a receiver operating characteristics (ROC analyses, using light microscopy as the gold-standard. Results Fluorescent microscopy (qualitative reads showed reduced specificity (400 parasites/μL blood: sensitivity of 64.2% and specificity of 86.0%. Overall, the diagnostic performance of the CyScope was found inferior to that of Paracheck-Pf®. Discussion Fluorescent microscopy using the CyScope® is certainly a field-applicable and relatively affordable solution for malaria diagnoses especially in areas where electrical supplies may be lacking. While it is unlikely to miss higher parasitaemia, its application in cross-sectional community-based studies leads to many false positives (i.e. small fluorescent bodies of presently unknown origin mistaken as malaria parasites. Without recourse to other technologies, arbitration of these false positives is presently equivocal, which could ultimately lead to over-treatment; something that should be further explored in future investigations if the CyScope® is to be more widely implemented.

  11. Europium-decorated graphene quantum dots as a fluorescent probe for label-free, rapid and sensitive detection of Cu(2+) and L-cysteine.

    Science.gov (United States)

    Lin, Liping; Song, Xinhong; Chen, Yiying; Rong, Mingcong; Wang, Yiru; Zhao, Li; Zhao, Tingting; Chen, Xi

    2015-09-03

    In this work, europium-decorated graphene quantum dots (Eu-GQDs) were prepared by treating three-dimensional Eu-decorated graphene (3D Eu-graphene) via a strong acid treatment. Various characterizations revealed that Eu atoms were successfully complexed with the oxygen functional groups on the surface of graphene quantum dots (GQDs) with the atomic ratio of 2.54%. Compared with Eu free GQDs, the introduction of Eu atoms enhanced the electron density and improved the surface chemical activities of Eu-GQDs. Therefore, the obtained Eu-GQDs were used as a novel "off-on" fluorescent probe for the label-free determination of Cu(2+) and l-cysteine (L-Cys) with high sensitivity and selectivity. The fluorescence intensity of Eu-GQDs was quenched in the presence of Cu(2+) owing to the coordination reaction between Cu(2+) and carboxyl groups on the surface of the Eu-GQDs. The fluorescence intensity of Eu-GQDs recovered with the subsequent addition of L-Cys because of the strong affinity of Cu(2+) to L-Cys via the Cu-S bond. The experimental results showed that the fluorescence variation of the proposed approach had a good linear relationship in the range of 0.1-10 μM for Cu(2+) and 0.5-50 μM for L-Cys with corresponding detection limits of 0.056 μM for Cu(2+) and 0.31 μM for L-Cys. The current approach also displayed a special response to Cu(2+) and L-Cys over the other co-existing metal ions and amino acids, and the results obtained from buffer-diluted serum samples suggested its applicability in biological samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. A selective and label-free strategy for rapid screening of telomere-binding Ligands via fluorescence regulation of DNA/silver nanocluster

    Science.gov (United States)

    Cheng, Rui; Xu, Jing; Zhang, Xiafei; Shi, Zhilu; Zhang, Qi; Jin, Yan

    2017-03-01

    Herein, the conformational switch of G-rich oligonucleotide (GDNA) demonstrated the obvious functional switch of GDNA which was found to significantly affect the fluorescence of the in-situ synthesized DNA/silver nanocluster (DNA-AgNC) in homogeneous solution. We envisioned that the allosteric interaction between GDNA and DNA-AgNC would be possible to be used for screening telomere-binding ligands. A unimolecular probe (12C5TG) is ingeniously designed consisting of three contiguous DNA elements: G-rich telomeric DNA (GDNA) as molecular recognition sequence, T-rich DNA as linker and C-rich DNA as template of DNA-AgNC. The quantum yield and stability of 12C5TG-AgNC is greatly improved because the nearby deoxyguanosines tended to protect DNA/AgNC against oxidation. However, in the presence of ligands, the formation of G-quadruplex obviously quenched the fluorescence of DNA-AgNC. By taking full advantage of intramolecular allosteric effect, telomere-binding ligands were selectively and label-free screened by using deoxyguanines and G-quadruplex as natural fluorescence enhancer and quencher of DNA-AgNC respectively. Therefore, the functional switching of G-rich structure offers a cost-effective, facile and reliable way to screen drugs, which holds a great potential in bioanalysis as well.

  13. Europium-decorated graphene quantum dots as a fluorescent probe for label-free, rapid and sensitive detection of Cu{sup 2+} and L-cysteine

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Liping [College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002 (China); Song, Xinhong; Chen, Yiying; Rong, Mingcong; Wang, Yiru [Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005 (China); Zhao, Li; Zhao, Tingting [Xiamen Huaxia College, Xiamen, 361024 (China); Chen, Xi, E-mail: xichen@xmu.edu.cn [Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005 (China); State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, 361005 (China)

    2015-09-03

    In this work, europium-decorated graphene quantum dots (Eu-GQDs) were prepared by treating three-dimensional Eu-decorated graphene (3D Eu-graphene) via a strong acid treatment. Various characterizations revealed that Eu atoms were successfully complexed with the oxygen functional groups on the surface of graphene quantum dots (GQDs) with the atomic ratio of 2.54%. Compared with Eu free GQDs, the introduction of Eu atoms enhanced the electron density and improved the surface chemical activities of Eu-GQDs. Therefore, the obtained Eu-GQDs were used as a novel “off-on” fluorescent probe for the label-free determination of Cu{sup 2+} and L-cysteine (L-Cys) with high sensitivity and selectivity. The fluorescence intensity of Eu-GQDs was quenched in the presence of Cu{sup 2+} owing to the coordination reaction between Cu{sup 2+} and carboxyl groups on the surface of the Eu-GQDs. The fluorescence intensity of Eu-GQDs recovered with the subsequent addition of L-Cys because of the strong affinity of Cu{sup 2+} to L-Cys via the Cu–S bond. The experimental results showed that the fluorescence variation of the proposed approach had a good linear relationship in the range of 0.1–10 μM for Cu{sup 2+} and 0.5–50 μM for L-Cys with corresponding detection limits of 0.056 μM for Cu{sup 2+} and 0.31 μM for L-Cys. The current approach also displayed a special response to Cu{sup 2+} and L-Cys over the other co-existing metal ions and amino acids, and the results obtained from buffer-diluted serum samples suggested its applicability in biological samples. - Highlights: • The europium-decorated graphene quantum dots (Eu-GQDs) have been successfully prepared. • Various characterizations results proved that Eu atoms were successfully introduced into graphene quantum dots. • The introduced Eu atoms changed the electron density and surface chemical activities of Eu-GQDs. • Eu-GQDs were used as an “off-on” fluorescent probe for Cu{sup 2+} and L-cysteine detection

  14. Rapid Quantification of Bacteria in Infected Root Canals Using Fluorescence Reagents and a Membrane Filter: A Pilot Study on Its Clinical Application to the Evaluation of the Outcomes of Endodontic Treatment

    Directory of Open Access Journals (Sweden)

    Takuichi Sato

    2012-01-01

    Full Text Available Objective. The bacterial examination has been performed during the course of the root canal treatment. In the present pilot study, the new developed method, using fluorescence reagents and a membrane filter, was applied to the detection and quantification of bacteria in infected root canals, in order to evaluate the outcomes of the treatment. Methods. Six infected root canals with periapical lesions from 5 subjects were included. Informed consent was obtained from all subjects (age ranges, 23–79 years. Samples from infected root canals were collected at the beginning of the treatment (termed #25 First, the end of the first day of treatment (termed #55 First, and the next appointment day (termed #55 Second. Then, the bacterial count (CFU was measured using fluorescence reagents (4′,6′-diamidino-2-phenylindole and propidium iodide and the polycarbonate membrane filter by Bioplorer. Results. The mean ± SD of CFU in the sample of “#25 First” was (1.0±1.4×105. As the root canal treatment progressed, the CFU decreased as 7.9×103 (#55 First and 4.3×102 (#55 Second. Conclusion. In the present pilot study, rapid detection and quantification of bacteria in infected root canals were found to be successfully performed using fluorescence reagents and a membrane filter (Bioplorer analysis.

  15. Fluorescent discharge lamp

    Science.gov (United States)

    Mukai, E.; Otsuka, H.; Nomi, K.; Honmo, I.

    1982-01-01

    A rapidly illuminating fluorescent lamp 1,200 mm long and 32.5 mm in diameter with an interior conducting strip which is compatible with conventional fixtures and ballasts is described. The fluorescent lamp is composed of a linear glass tube, electrodes sealed at both ends, mercury and raregas sealed in the glass tube, a fluorescent substance clad on the inner walls of the glass tube, and a clad conducting strip extending the entire length of the glass tube in the axial direction on the inner surface of the tube.

  16. Fluorescent discharge lamp

    Science.gov (United States)

    Mukai, E.; Otsuka, H.; Nomi, K.; Honmo, I.

    1982-07-01

    A rapidly illuminating fluorescent lamp 1,200 mm long and 32.5 mm in diameter with an interior conducting strip which is compatible with conventional fixtures and ballasts is described. The fluorescent lamp is composed of a linear glass tube, electrodes sealed at both ends, mercury and raregas sealed in the glass tube, a fluorescent substance clad on the inner walls of the glass tube, and a clad conducting strip extending the entire length of the glass tube in the axial direction on the inner surface of the tube.

  17. Rapid, high-throughput detection of azalea lace bug (Hemiptera: Tingidae) predation by Chrysoperla rufilabris (Neuroptera: Chrysopidae), using fluorescent-polymerase chain reaction primers.

    Science.gov (United States)

    Rinehart, Timothy A; Boyd, David W

    2006-12-01

    Azalea lace bugs, Stephanitis pyrioides (Scott) (Hemiptera: Tingidae), are the most common pest of azaleas (Rhododendron spp.) in nursery production and the landscape. Although pesticides are commonly used to control lace bugs, natural enemies can be a significant source of lace bug mortality. Lacewings (Neuroptera: Chrysopidae) are natural enemies of lace bugs and easily consume them in laboratory studies. Field studies on lacewing biocontrol of azalea lace bugs are underway; however, monitoring lacewing predation in a nursery environment by direct observation is impractical. Here, we describe a fluorescent-polymerase chain reaction method to estimate S. pyrioides consumption based on the gut contents of lacewing predators. Lace bug DNA was detected in fed lacewings up to 32 h after ingestion. More than 80% of the ingested lace bugs were detected using our method with only one false positive result. The assay is both high-throughput and relatively inexpensive, making it a practical approach to documenting lace bug predation in the field.

  18. Sensitive and rapid detection of endogenous hydrogen sulfide distributing in different mouse viscera via a two-photon fluorescent probe.

    Science.gov (United States)

    Chen, Qian; Yang, Jinfeng; Li, Yinhui; Zheng, Jing; Yang, Ronghua

    2015-10-08

    Development of efficient methods for detection of endogenous H2S in living cells and tissues is of considerable significance for better understanding the biological and pathological functions of H2S. Two-photon (TP) fluorescent probes are favorable as powerful molecular tools for studying physiological process due to its non-invasiveness, high spatiotemporal resolution and deep-tissues imaging. Up to date, several TP probes for intracellular H2S imaging have been designed, but real-time imaging of endogenous H2S-related biological processes in tissues is hampered due to low sensitivity, long response time and interference from other biothiols. To address this issue, we herein report a novel two-photon fluorescent probe (TPP-H2S) for highly sensitive and fast monitoring and imaging H2S levels in living cells and tissues. In the presence of H2S, it exhibits obviously improved sensitivity (LOD: 0.12 μM) and fast response time (about 2 min) compared with the reported two-photon H2S probes. With two-photon excitation, TPP-H2S displays high signal-to-noise ratio and sensitivity even no interference in cell growth media. As further application, TPP-H2S is applied for fast imaging of H2S in living cells and different fresh tissues by two-photon confocal microscope. Most importantly we first measured the endogenous H2S level in different viscera by vivisection and found that the distribution of endogenous H2S mostly in brain, liver and lung. The excellent sensing properties of TPP-H2S make it a practically useful tool for further studying biological roles of H2S. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. The fluorescence spectroscopy of Dissolved Organic Matter (DOM): a tool to characterize rapid infiltration flows and vulnerability in karst systems. Application to the Lez hydrosystem (Hérault, France)

    Science.gov (United States)

    Quiers, M.; Batiot-Guilhe, C.; Seidel, J.; Bicalho, C. C.; Perrette, Y.; Jourde, H.

    2011-12-01

    Due to the fundamental role the DOM plays in the pollutant transport, this tracer is already used in different aquatic systems but very few studies concern karst systems. Water transit times and recharge period can be estimated using the fluorescence property of DOM. The aim of this study is to apply this approach in order to improve the karst aquifers functioning and to obtain essential information to preserve water resource. This method was applied to the Lez karst hydrosystem which belong to the MEDYCYSS observatory (Multi scalE observatory of flooD dYnamiCs and hYdrodynamicS in karSt). Its main outlet, the Lez spring, supplies drinking water to the city of Montpellier (France). Since March 2006, the hydrodynamic and hydrochemical parameters of the karstic spring have been monitored (turbidity, temperature, electrical conductivity, pH, major and trace elements, Total Organic Carbon :TOC, DOM and bacteria). The DOM fluorescence was measured with a spectrofluorimeter using the Excitation-Emission Matrix (EEM). Each EEM was generated with an excitation from 220 to 450 nm, detecting the emission spectra between 250 and 550 nm. The fluorescence spectra are characterized by 3 excitation-emission domains. Two correspond to humic compounds fluorophores (H1, H2) with a pedogenic origin (mature OM). The third region of fluorescence is related to protein-like compounds (P1) with a pedogenic (fresh OM) or an anthropogenic origin. The Fluorescence Intensity (FI) of these three domains increases with the spring discharge and is correlated with rapid infiltration flow markers (especially TOC and turbidity). So FI can be used as a tracer of infiltration flows in karst systems. Moreover, FI peaks emitted by P1 are also correlated to peaks of faecal coliforms. The survival time of these bacteria is lower than one week in a karst aquifer. Consequently, fluorophore P1 is a tracer of short transit time and anthropic contamination. FI emitted by H1 and H2 are related to the

  20. Fluorescent lamp and ballast options

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2002-07-01

    This paper reviews some of the current technologies for fluorescent lamps and ballasts with particular focus on the most common configuration in Canada - the F32T8, 4 ft length, two-lamp ballast combination. Fluorescent lamps require a high voltage surge for start-up. Technical specifications for the F32T8 lamp were provided along with reasons why they are the preferred choice. The three types of ballasts include electromagnetic, electronic and hybrid. While electromagnetic ballasts perform the same start-up duty, they are not as efficient as electronic or hybrid ballasts. Hybrid ballasts are energy efficient, but they have problems with lamp flicker, tar leakage and shorter life expectancy. Electronic ballasts eliminate flicker, do not leak and have a life expectancy of 25 years. Electronic ballasts can be instant, rapid start, or dimmable. Energy information on different types of ballast systems was presented along with a comparison of the type of light produced according to lamp and ballast combinations. This paper also presents a case study in which the lighting system of a 25-storey building was retrofitted with energy efficient fluorescent lamps and ballasts for an energy savings of about $100,000 per year and a 5 year payback period. 2 tabs., 4 figs.

  1. Auramine orange stain with fluorescence microscopy is a rapid and sensitive technique for the detection of cervical lymphadenitis due to mycobacterial infection using fine needle aspiration cytology: a case series.

    Science.gov (United States)

    Cheng, Alan G; Chang, Anthony; Farwell, D Greg; Agoff, S Nicholas

    2005-09-01

    We sought to evaluate the effectiveness of the auramine orange (AO) stain in diagnosing mycobacterial cervical adenitis (MCA) from fine needle aspiration (FNA) cytology. A retrospective review of 19 patients evaluated at 2 urban hospitals from 2000 to 2003 for suspected MCA. FNA specimens were inoculated to culture media and had direct smears stained by the auramine acid fast method. Mycobacteria were identified in 16 (84.2%) of 19 AO-stained FNA specimens, with results available within 4 hours. Corresponding cultures were positive for mycobacteria in 12 specimens, 9 tuberculous and 3 nontuberculous, and grew Mycobacterium tuberculosis from the 3 AO-negative specimens. Three of the 4 patients with negative cultures had previously taken anti-mycobacterial medications. The AO stain with fluorescence microscopy is a sensitive and rapid method for detecting tuberculous and nontuberculous mycobacteria. It is a valuable tool for the otolaryngologists and pathologists in the diagnosis of MCA.

  2. Ultrasound-assisted single extraction tests for rapid assessment of metal extractability from soils by total reflection X-ray fluorescence.

    Science.gov (United States)

    De La Calle, I; Cabaleiro, N; Lavilla, I; Bendicho, C

    2013-09-15

    In this work, ultrasound-assisted extraction (UAE) was employed for acceleration of metal extraction from soil samples. After extraction, multielemental analysis (Cr, Mn, Fe, Ni, Cu, Zn and Pb) of EDTA and acetic acid extracts was performed by total reflection X-ray fluorescence spectrometry (TXRF). High-intensity ultrasonic processors, i.e. the ultrasonic probe (50W) and the cup-horn sonoreactor (200W) were applied. Both ultrasonic procedures were compared with a miniaturized version of the single extraction scheme proposed by the Standards, Measurements and Testing program (SM&T). The extraction time with EDTA was reduced from 1h (conventional procedure) to 2 min (ultrasonic probe) or to 10 min (cup-horn sonoreactor). The time required for acetic acid extraction was also reduced from 16 h (conventional procedure) to 6 min (ultrasonic probe) or to 30 min (cup-horn sonoreactor). In addition, the amount of sample and extractants was drastically reduced as a result of the miniaturization implemented in the developed approaches. The combination of UAE and TXRF allows assessing the potential metal mobility and bioavailability in a simple way. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Rapid trace level determination of sulfonamide residues in honey with online extraction using short C-18 column by high-performance liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Sajid, Muhammad; Na, Na; Safdar, Muhammad; Lu, Xin; Ma, Lin; He, Lan; Ouyang, Jin

    2013-11-01

    A sensitive and inexpensive quantification method with online extraction using a short C-18 column for sulfonamide residues in honey by high performance liquid chromatography with fluorescence detector was developed and validated. In sample preparation, acid hydrolysis was used to break the N-glycoside bond between the honey sugar and sulfonamide drugs and derivatization of sulfonamide residues with fluorescamine was conducted at pH 3.5 using a citrate buffer (0.5M) in the honey matrix. The chromatography was carried out on Zorbax Extended C-18 (250mm×4.6mm; 5μm) column, using a mixture of acetonitrile and an acetate buffer (pH 4.50, 20mM) as a mobile phase. A Zorbax Extended C-18 (12mm×4.6mm; 5μm) column was used for online extraction of fifteen sulfonamide residues from honey sample with the help of a two position valve. The limit of quantification of sulfonamide residues in honey was less than 3ngg(-1), and the percentage recovery of study compounds in spiked honey sample was from 80% for sulfacetamide to 100% of sulfachloropyridazine. The developed method has excellent linearity for all studied sulfonamides with a correlation coefficient 0.993. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Chemometrics-assisted excitation-emission fluorescence analytical data for rapid and selective determination of optical brighteners in the presence of uncalibrated interferences

    Science.gov (United States)

    Gholami, Ali; Masoum, Saeed; Mohsenikia, Atefeh; Abbasi, Saleheh

    2016-01-01

    This study describes a novel approach for the simultaneous determination of CBS-X and CXT as widely used optical brighteners in household detergent, by combining the advantage of the high sensitivity of molecular fluorescence, and the selectivity of second-order chemometric methods. The proposed method is assisted by second-order chemometric analyses employing the PARAFAC, SWATLD and APTLD that help us to determine CBS-X and CXT in laundry powders and environmental samples, through the unique decomposition of the three-way data array. Proposed method can provide the extraction of relative concentrations of the analytes, as well as the spectral profiles. This approach achieves the second-order advantage and in principle could be able to overcome the spectral uncalibrated interference problems in the determination of CBS-X and CXT at the ng g- 1 level. By spiking the known concentrations of these compounds to the real samples, the accuracy of the proposed methods was validated and recoveries of the spiked values were calculated. High recoveries (90.00%-113.33%) for the spiked laundry powders and real environmental samples indicate the present method successfully faces this complex challenge without the necessity of applying separation and preconcentration steps in environmental contaminations.

  5. Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil

    Science.gov (United States)

    Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

    Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 103 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China. PMID:24376590

  6. Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method.

    Science.gov (United States)

    DeCory, Thomas R; Durst, Richard A; Zimmerman, Scott J; Garringer, Linda A; Paluca, Gary; DeCory, Heleen H; Montagna, Richard A

    2005-04-01

    The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.

  7. Methodology using a portable X-ray fluorescence device for on-site and rapid evaluation of heavy-atom contamination in wounds: a model study for application to plutonium contamination.

    Directory of Open Access Journals (Sweden)

    Hiroshi Yoshii

    Full Text Available Workers decommissioning the Fukushima-Daiichi nuclear power plant damaged from the Great East Japan Earthquake and resulting tsunami are at risk of injury with possible contamination from radioactive heavy atoms including actinides, such as plutonium. We propose a new methodology for on-site and rapid evaluation of heavy-atom contamination in wounds using a portable X-ray fluorescence (XRF device. In the present study, stable lead was used as the model contaminant substitute for radioactive heavy atoms. First, the wound model was developed by placing a liquid blood phantom on an epoxy resin wound phantom contaminated with lead. Next, the correlation between the concentration of contaminant and the XRF peak intensity was formulated considering the thickness of blood exiting the wound. Methods to determine the minimum detection limit (MDL of contaminants at any maximal equivalent dose to the wound by XRF measurement were also established. For example, in this system, at a maximal equivalent dose of 16.5 mSv to the wound and blood thickness of 0.5 mm, the MDL value for lead was 1.2 ppm (3.1 nmol. The radioactivity of 239Pu corresponding to 3.1 nmol is 1.7 kBq, which is lower than the radioactivity of 239Pu contaminating puncture wounds in previous severe accidents. In conclusion, the established methodology could be beneficial for future development of a method to evaluate plutonium contamination in wounds. Highlights: Methodology for evaluation of heavy-atom contamination in a wound was established. A portable X-ray fluorescence device enables on-site, rapid and direct evaluation. This method is expected to be used for evaluation of plutonium contamination in wounds.

  8. Methodology using a portable X-ray fluorescence device for on-site and rapid evaluation of heavy-atom contamination in wounds: a model study for application to plutonium contamination.

    Science.gov (United States)

    Yoshii, Hiroshi; Yanagihara, Kouta; Imaseki, Hitoshi; Hamano, Tsuyoshi; Yamanishi, Hirokuni; Inagaki, Masayo; Sakai, Yasuhiro; Sugiura, Nobuyuki; Kurihara, Osamu; Sakai, Kazuo

    2014-01-01

    Workers decommissioning the Fukushima-Daiichi nuclear power plant damaged from the Great East Japan Earthquake and resulting tsunami are at risk of injury with possible contamination from radioactive heavy atoms including actinides, such as plutonium. We propose a new methodology for on-site and rapid evaluation of heavy-atom contamination in wounds using a portable X-ray fluorescence (XRF) device. In the present study, stable lead was used as the model contaminant substitute for radioactive heavy atoms. First, the wound model was developed by placing a liquid blood phantom on an epoxy resin wound phantom contaminated with lead. Next, the correlation between the concentration of contaminant and the XRF peak intensity was formulated considering the thickness of blood exiting the wound. Methods to determine the minimum detection limit (MDL) of contaminants at any maximal equivalent dose to the wound by XRF measurement were also established. For example, in this system, at a maximal equivalent dose of 16.5 mSv to the wound and blood thickness of 0.5 mm, the MDL value for lead was 1.2 ppm (3.1 nmol). The radioactivity of 239Pu corresponding to 3.1 nmol is 1.7 kBq, which is lower than the radioactivity of 239Pu contaminating puncture wounds in previous severe accidents. In conclusion, the established methodology could be beneficial for future development of a method to evaluate plutonium contamination in wounds. Highlights: Methodology for evaluation of heavy-atom contamination in a wound was established. A portable X-ray fluorescence device enables on-site, rapid and direct evaluation. This method is expected to be used for evaluation of plutonium contamination in wounds.

  9. Addressing the need for rapid treatment of agitation in schizophrenia and bipolar disorder: focus on inhaled loxapine as an alternative to injectable agents

    Directory of Open Access Journals (Sweden)

    Citrome L

    2013-05-01

    Full Text Available Leslie CitromeDepartment of Psychiatry and Behavioral Sciences, New York Medical College, Valhalla, NY, USAAbstract: Agitation (excessive motor or verbal activity can be associated with schizophrenia or bipolar mania, and can further escalate into aggressive behavior and potentially lead to injuries in patients and staff. Medications used to treat agitation include antipsychotics and benzodiazepines, usually administered intramuscularly when rapid action is desired. Loxapine, a first-generation antipsychotic, has recently been reformulated into an inhaled powder that allows for direct administration to the lungs, resulting in rapid absorption into the systemic circulation. Administered via a single-use device, inhaled loxapine was tested in randomized controlled trials in agitation associated with schizophrenia or bipolar mania; doses of 5 mg and 10 mg were found to be efficacious, with an apparent dose response. In the Phase III studies, number needed to treat versus placebo for a ≥40% reduction from baseline on the Positive and Negative Syndrome Scale – Excited Component (PANSS-EC at 2 hours was three for patients with bipolar disorder, and five for 5 mg and four for 10 mg for patients with schizophrenia, with effect sizes comparable to what has been observed in analogous studies of intramuscular injection of antipsychotics or lorazepam. Separation from placebo on the PANSS-EC was as early as 10 minutes postinhalation, the first time point where this was measured. Dysgeusia was the most commonly encountered spontaneously reported adverse event. Adverse events related to extrapyramidal symptoms and akathisia were relatively rare. Spirometry studies identified the potential for bronchospasm particularly in persons with asthma. Because of concerns over pulmonary safety, inhaled loxapine is restricted to use in hospitals and patients need to be prescreened for the presence of pulmonary disease, as well as monitored for signs and symptoms of

  10. Rapid mitochondrial DNA typing using restriction enzyme digestion of polymerase chain reaction amplicons followed by capillary electrophoresis separation with laser-induced fluorescence detection.

    Science.gov (United States)

    Butler, J M; Wilson, M R; Reeder, D J

    1998-01-01

    The polymorphic control region of mitochondrial DNA (mtDNA) is becoming more commonly used in forensic applications to differentiate among individuals in a population. Two hypervariable regions (HV1 and HV2) are often sequenced following amplification of the mtDNA via the polymerase chain reaction (PCR). More rapid screening assays would reduce both the effort and the expense of comparing two samples. A methodology has been developed that first uses restriction endonuclease digestion of the PCR-amplified mtDNA using RsaI and MnlI and then capillary electrophoresis (CE) to separate and size the PCR-RFLP fragments. This rapid procedure offers an alternative method for screening of polymorphisms in amplified mtDNA samples. In addition, the presence of a T-->C transition at position 16189, which gives rise to the so-called "C-stretch" in HV1, may be predicted from the presence of nonspecific PCR products in the CE results.

  11. The rapid prototyping of textured amorphous surfaces for the graphoepitaxial deposition of CdTe films using focused ion beam lithography

    Energy Technology Data Exchange (ETDEWEB)

    Neretina, S. [McMaster University, Department of Engineering Physics and Centre for Emerging Device Technologies, Hamilton, Ontario (Canada); Temple University, Department of Mechanical Engineering, Philadelphia, PA (United States); Hughes, R.A. [McMaster University, Brockhouse Institute for Materials Research, Hamilton, Ontario (Canada); Temple University, Department of Mechanical Engineering, Philadelphia, PA (United States); Stortz, G.; Mascher, P. [McMaster University, Department of Engineering Physics and Centre for Emerging Device Technologies, Hamilton, Ontario (Canada); Preston, J.S. [McMaster University, Department of Engineering Physics and Centre for Emerging Device Technologies, Hamilton, Ontario (Canada); McMaster University, Brockhouse Institute for Materials Research, Hamilton, Ontario (Canada)

    2011-02-15

    Cadmium telluride films deposited on amorphous substrates exhibit a grain structure characterized by [111]-oriented grains, but where the in-plane grain orientation is randomized due to the absence of epitaxy. Here, we explore the viability of promoting an in-plane grain alignment through graphoepitaxy. Fifteen different substrate surface textures were fabricated using focused ion beam lithography. This approach allows for the side-by-side deposition of surface textures where both the areal extent and depth of the surface features are varied in a systematic manner. CdTe films deposited overtop these textures show grain structures with dramatic variations, revealing that particular length scales have the most pronounced effect on the grain structure. (orig.)

  12. Research of the fluorescence detection apparatus for nutrients

    Science.gov (United States)

    Wang, Yu; Yan, Huimin; Ni, Xuxiang; Xu, Xiaoyi; Chen, Shibing

    2015-10-01

    The research of the multifunctional analyzer of Clinical Nutrition, which integrates the absorbance, luminescence, fluorescence and other optical detection methods, can overcome the functional limitations of a single technology on human nutrition analysis, and realize a rapid and accurate analysis of the nutrients. This article focuses on the design of fluorescence detection module that uses a photomultiplier tube(PMT) to detect weak fluorescence, and utilizes the single photon counting method to measure the fluorescence intensity, and then according to the relationship between the fluorescent marker and fluorescence intensity, the concentration of the analyte can be derived. Using fluorescein isothiocyanate(FITC, the most widely used fluorescein currently)to mark antibodies in the experiment, therefore, according to the maximum absorption wavelength and the maximum emission wavelength of the fluorescein isothiocyanate, to select the appropriate filters to set up the optical path. In addition, the fluorescence detection apparatus proposed in this paper uses an aspherical lens with large numerical aperture, in order to improve the capacity of signal acquisition more effectively, and the selective adoption of flexible optical fiber can realize a compact opto-mechanical structure, which is also conducive to the miniaturization of the device. The experimental results show that this apparatus has a high sensitivity, can be used for the detection and analysis of human nutrition.

  13. Non-invasive rapid harvest time determination of oil-producing microalgae cultivations for bio-diesel production by using Chlorophyll fluorescence

    Directory of Open Access Journals (Sweden)

    Yaqin eQiao

    2015-10-01

    Full Text Available For the large-scale cultivation of microalgae for biodiesel production, one of the key problems is the determination of the optimum time for algal harvest when algae cells are saturated with neutral lipids. In this study, a method to determine the optimum harvest time in oil-producing microalgal cultivations by measuring the maximum photochemical efficiency of photosystem II (PSII, also called Fv/Fm, was established. When oil-producing Chlorella strains were cultivated and then treated with nitrogen starvation, it not only stimulated neutral lipid accumulation, but also affected the photosynthesis system, with the neutral lipid contents in all four algae strains – Chlorella sorokiniana C1, Chlorella sp. C2, C. sorokiniana C3, C. sorokiniana C7 – correlating negatively with the Fv/Fm values. Thus, for the given oil-producing algae, in which a significant relationship between the neutral lipid content and Fv/Fm value under nutrient stress can be established, the optimum harvest time can be determined by measuring the value of Fv/Fm. It is hoped that this method can provide an efficient way to determine the harvest time rapidly and expediently in large-scale oil-producing microalgae cultivations for biodiesel production.

  14. SynPAnal: software for rapid quantification of the density and intensity of protein puncta from fluorescence microscopy images of neurons.

    Directory of Open Access Journals (Sweden)

    Eric Danielson

    Full Text Available Continuous modification of the protein composition at synapses is a driving force for the plastic changes of synaptic strength, and provides the fundamental molecular mechanism of synaptic plasticity and information storage in the brain. Studying synaptic protein turnover is not only important for understanding learning and memory, but also has direct implication for understanding pathological conditions like aging, neurodegenerative diseases, and psychiatric disorders. Proteins involved in synaptic transmission and synaptic plasticity are typically concentrated at synapses of neurons and thus appear as puncta (clusters in immunofluorescence microscopy images. Quantitative measurement of the changes in puncta density, intensity, and sizes of specific proteins provide valuable information on their function in synaptic transmission, circuit development, synaptic plasticity, and synaptopathy. Unfortunately, puncta quantification is very labor intensive and time consuming. In this article, we describe a software tool designed for the rapid semi-automatic detection and quantification of synaptic protein puncta from 2D immunofluorescence images generated by confocal laser scanning microscopy. The software, dubbed as SynPAnal (for Synaptic Puncta Analysis, streamlines data quantification for puncta density and average intensity, thereby increases data analysis throughput compared to a manual method. SynPAnal is stand-alone software written using the JAVA programming language, and thus is portable and platform-free.

  15. Diffraction phase and fluorescence microscopy.

    Science.gov (United States)

    Park, Yongkeun; Popescu, Gabriel; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

    2006-09-04

    We have developed diffraction phase and fluorescence (DPF) microscopy as a new technique for simultaneous quantitative phase imaging and epi-fluorescence investigation of live cells. The DPF instrument consists of an interference microscope, which is incorporated into a conventional inverted fluorescence microscope. The quantitative phase images are characterized by sub-nanometer optical path-length stability over periods from milliseconds to a cell lifetime. The potential of the technique for quantifying rapid nanoscale motions in live cells is demonstrated by experiments on red blood cells, while the composite phase-fluorescence imaging mode is exemplified with mitotic kidney cells.

  16. Focus: Digital

    DEFF Research Database (Denmark)

    Technology has been an all-important and defining element within the arts throughout the 20th century, and it has fundamentally changed the ways in which we produce and consume music. With this Focus we investigate the latest developments in the digital domain – and their pervasiveness and rapid...... production and reception of contemporary music and sound art. With ‘Digital’ we present four composers' very different answers to how technology impact their work. To Juliana Hodkinson it has become an integral part of her sonic writing. Rudiger Meyer analyses the relationships between art and design and how...... pace, which demand a closer look at the relations between arts and technology. The composers’ understanding of her occupation is challenged – and alongside, mediatisation and changes in distribution mark an incursion on solid conceptions of the artwork. Together, this means changing conditions for both...

  17. Efficacy of the Combination of a PARP Inhibitor and UVC on Cancer Cells as Imaged by Focus Formation by the DNA Repair-related Protein 53BP1 Linked to Green Fluorescent Protein.

    Science.gov (United States)

    Tome, Yasunori; Uehara, Fuminari; Miwa, Shinji; Yano, Shuya; Mii, Sumiyuki; Efimova, Elena V; Bouvet, Michael; Kimura, Hiroaki; Tsuchiya, Hiroyuki; Kanaya, Fuminori; Hoffman, Robert M

    2016-08-01

    The ability to image DNA repair in cancer cells after irradiation, as well as its inhibition by potential therapeutic agents, is important for the further development of effective cancer therapy. 53BP1 is a DNA repair protein that is overexpressed and forms foci when double-stranded DNA breaks occur in DNA. The re-localization of green fluorescent protein (GFP) fused to the chromatin-binding domain of 53BP1 to form foci was imaged after UVC irradiation of breast and pancreatic cancer cells expressing 53BP1-GFP using confocal microscopy. During live-cell imaging, 53BP1-GFP focus formation was observed within 10 minutes after UVC irradiation. Most 53BP1 foci resolved by 100 minutes. To block UVC-induced double-strand break repair in cancer cells, poly(ADP-ribose) polymerase (PARP) was targeted with ABT-888 (veliparib). PARP inhibition markedly enhanced UVC-irradiation-induced persistence of 53BP1-foci, even beyond 100 minutes after UVC irradiation, and reduced proliferation of breast and pancreatic cancer cells. Confocal microscopy of 53BP1-GFP is a powerful method for imaging UVC-induced DNA damage and repair, as well as inhibition of repair. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  18. Analytical strategy for rapid identification and quantification of lubricant additives in mineral oil by high-performance thin-layer chromatography with UV absorption and fluorescence detection combined with mass spectrometry and infrared spectroscopy.

    Science.gov (United States)

    Dytkiewitz, Elisabeth; Morlock, Gertrud E

    2008-01-01

    A simple strategy for identification and quantification of lubricant additives in mineral oil was demonstrated by high-performance thin-layer chromatography with UV absorption and fluorescence detection using various coupling options, e.g., with attenuated total reflectance infrared (ATR-IR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and direct analysis in real-time mass spectrometry (DART-MS). For the additives zinc bis(O,O'-diisobutyl dithiophosphate), zinc bis(O,O'-didodecyl dithiophosphate), and Anglamol 99, 2 chromatographic systems were developed, i.e., a reversed-phase (RP) system on RP2 plates using an acetonitrile-based mobile phase and a normal-phase system on silica gel 60 plates using a toluene-based gradient. Densitometry was performed by absorption measurement at 220 nm. Repeatabilities (relative standard deviation, n = 6) between 2.2 and 5.5% and correlation coefficients >0.9973 were highly satisfactory for the analysis of these additives in the mineral oil. Primuline reagent was used to improve the detection limit of the lipophilic additives by a factor of 2, followed by fluorescence measurement at UV 366/>400 nm. For rapid identification by ATR-IR and FTIR, the respective additive zones on the plate were online extracted by an interface called ChromeXtract, concentrated, and directly applied for measurements in the wave number range of 4000-400 cm(-1). Identification was confirmed by online ESI-MS within a minute using ChromeXtract and by DART-MS within seconds.

  19. Ultrafast fluorescence of photosynthetic crystals and light-harvesting complexes

    NARCIS (Netherlands)

    Oort, van B.F.

    2008-01-01

    This thesis focuses on the study of photosynthetic pigment protein complexes using time resolved fluorescence techniques. Fluorescence spectroscopy often requires attaching fluorescent labels to the proteins under investigation. With photosynthetic proteins this is not necessary, because these

  20. Simultaneous separation of five major ribonucleic acids by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow: application to the rapid screening of 5S rRNA from ovarian cancer cells.

    Science.gov (United States)

    Shih, Ya-Chu; Liao, Ching-Ru; Chung, I-Che; Chang, Yu-Sun; Chang, Po-Ling

    2014-10-17

    RNA integrity is important in RNA studies because poor RNA quality may impact downstream methodologies. This study proposes a rapid and cost-effective method for the determination of RNA integrity based on CE-LIF in the presence of electroosmotic flow. The proposed method uses poly(ethylene) oxide (Mavg=4,000,000 Da) as a sieving matrix for total RNA separation. Ethidium bromide (μg mL(-1)) was dissolved in a polymer solution as an interchelating dye for on-column fluorescent labeling. The 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA from the total human RNA extracted from the cells were fully separated using the proposed method. The lowest detectable concentration of total RNA achieved was 100 pg μL(-1) with a 6 min sample injection followed by on-column concentration. In addition, the temperature-induced degradation of total RNA was observed by CE-LIF. The electropherograms revealed more fragmentation of 28S and 18S rRNAs by temperature-induced hydrolysis compared with the 5.8S rRNA, 5S rRNA and tRNA. Therefore, the results indicated that RNA degradation should be considered for long-term, high-temperature incubations in RNA-related experiments involving RNA hybridization. The proposed method is furthermore, applied to the determination of 5S rRNA overexpressed in ovarian cancer cells as compared to the cervical cancer cells. Overall, CE-LIF is highly promising for rapid screening of ovarian cancers without tedious pre-amplification steps. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Fluorescence Technology for Point of Care Wound Management.

    Science.gov (United States)

    Anghel, Ersilia L; Falola, Reuben A; Kim, Paul J

    2016-04-01

    As the prevalence of chronic wounds continues to rise, the need for point of care wound assessment has also increased. While a variety of technologies have been developed to improve diagnostic abilities and monitoring of wounds, none have proven completely effective in all settings. Further, many of the stalwart wound management techniques remain costly, time consuming, and technically challenging. The two key pivotal events of ischemia and infection can lead to limb loss. A relatively new crop of fluorescence-based technologies, including devices that measure pathogenic auto-fluorescence, fluorescence angiography, or map cutaneous oxygenation, are increasingly being utilized for adjunct wound assessment-both clinical and operative settings can address these events. These technologies offer rapid, efficient, visual, and quantitative data that can aid the wound provider in evaluating the viability of tissues, ensuring adequate perfusion, and optimizing wound bed preparation. In the following review, pathogenic auto-fluorescence is compared to gross evaluation of wound infection and culture based diagnostics, indocyanine green fluorescence angiography is compared to various methods of visual and physical assessments of tissue perfusion by the practitioner, and cutaneous oxygenation is compared to clinical signs of ischemia. We focus on the current applications of fluorescence technologies in wound management, with emphasis placed on the evidence for clinical and operative implementation, a safety analyses, procedural limitations, and the future direction of this growing field of wound assessment.

  2. Dielectrophoretic focusing integrated pulsed laser activated cell sorting

    Science.gov (United States)

    Zhu, Xiongfeng; Kung, Yu-Chun; Wu, Ting-Hsiang; Teitell, Michael A.; Chiou, Pei-Yu

    2017-08-01

    We present a pulsed laser activated cell sorter (PLACS) integrated with novel sheathless size-independent dielectrophoretic (DEP) focusing. Microfluidic fluorescence activated cell sorting (μFACS) systems aim to provide a fully enclosed environment for sterile cell sorting and integration with upstream and downstream microfluidic modules. Among them, PLACS has shown a great potential in achieving comparable performance to commercial aerosol-based FACS (>90% purity at 25,000 cells sec-1). However conventional sheath flow focusing method suffers a severe sample dilution issue. Here we demonstrate a novel dielectrophoresis-integrated pulsed laser activated cell sorter (DEP-PLACS). It consists of a microfluidic channel with 3D electrodes laid out to provide a tunnel-shaped electric field profile along a 4cmlong channel for sheathlessly focusing microparticles/cells into a single stream in high-speed microfluidic flows. All focused particles pass through the fluorescence detection zone along the same streamline regardless of their sizes and types. Upon detection of target fluorescent particles, a nanosecond laser pulse is triggered and focused in a neighboring channel to generate a rapidly expanding cavitation bubble for precise sorting. DEP-PLACS has achieved a sorting purity of 91% for polystyrene beads at a throughput of 1,500 particle/sec.

  3. Small acid soluble proteins for rapid spore identification.

    Energy Technology Data Exchange (ETDEWEB)

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  4. Fluorescence microscopy.

    Science.gov (United States)

    Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

    2014-10-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

  5. Teaching laser-induced fluorescence of plant leaves

    Science.gov (United States)

    Lenk, Sándor; Gádoros, Patrik; Kocsányi, László; Barócsi, Attila

    2016-11-01

    Plants convert carbon dioxide into sugars using the energy of sunlight. Absorbed light unused for conversion is dissipated primarily as heat with a small fraction re-emitted as fluorescence at longer wavelengths. One can use the latter to estimate photosynthetic activity. The illumination of intact leaves with strong light after keeping them in dark for tens of minutes results in a rapid increase followed by a slow decay of fluorescence emission from the fluorophore chlorophyll-a, called the Kautsky effect. This paper describes a laboratory practice that introduces students of physics or engineering into this research field. It begins with the spectral measurement of the fluorescence emitted by a plant leaf upon UV excitation. Then it focuses on the red and far-red components of the fluorescence emission spectrum characteristic to the chlorophyll-a molecule and presents an inexpensive demonstration of the Kautsky effect. As researchers use more complex measurement techniques and tools, the practice ends up with the demonstration of an intelligent fluorosensor, a compact tool developed for plant physiological research and horticulture applications together with a brief interpretation of some important fluorescence parameters.

  6. Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

    Science.gov (United States)

    Li, Wenyan; Houston, Kevin D.; Houston, Jessica P.

    2017-01-01

    Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

  7. Boronic acids for fluorescence imaging of carbohydrates.

    Science.gov (United States)

    Sun, Xiaolong; Zhai, Wenlei; Fossey, John S; James, Tony D

    2016-02-28

    "Fluorescence imaging" is a particularly exciting and rapidly developing area of research; the annual number of publications in the area has increased ten-fold over the last decade. The rapid increase of interest in fluorescence imaging will necessitate the development of an increasing number of molecular receptors and binding agents in order to meet the demand in this rapidly expanding area. Carbohydrate biomarkers are particularly important targets for fluorescence imaging given their pivotal role in numerous important biological events, including the development and progression of many diseases. Therefore, the development of new fluorescent receptors and binding agents for carbohydrates is and will be increasing in demand. This review highlights the development of fluorescence imaging agents based on boronic acids a particularly promising class of receptors given their strong and selective binding with carbohydrates in aqueous media.

  8. Beam loss caused by edge focusing of injection bump magnets and its mitigation in the 3-GeV rapid cycling synchrotron of the Japan Proton Accelerator Research Complex

    Directory of Open Access Journals (Sweden)

    H. Hotchi

    2016-01-01

    Full Text Available In the 3-GeV rapid cycling synchrotron of the Japan Proton Accelerator Research Complex, transverse injection painting is utilized not only to suppress space-charge induced beam loss in the low energy region but also to mitigate foil scattering beam loss during charge-exchange injection. The space-charge induced beam loss is well minimized by the combination of modest transverse painting and full longitudinal painting. But, for sufficiently mitigating the foil scattering part of beam loss, the transverse painting area has to be further expanded. However, such a wide-ranging transverse painting had not been realized until recently due to beta function beating caused by edge focusing of pulsed injection bump magnets during injection. This beta function beating additionally excites random betatron resonances through a distortion of the lattice superperiodicity, and its resultant deterioration of the betatron motion stability causes significant extra beam loss when expanding the transverse painting area. To solve this issue, we newly installed pulse-type quadrupole correctors to compensate the beta function beating. This paper presents recent experimental results on this correction scheme for suppressing the extra beam loss, while discussing the beam loss and its mitigation mechanisms with the corresponding numerical simulations.

  9. Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

    Science.gov (United States)

    Barizuddin, Syed; Balakrishnan, Baskar; Stringer, R Cody; Dweik, Majed

    2015-08-01

    A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-β-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases β-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and

  10. Genetically Encoded Fluorescent Redox Probes

    OpenAIRE

    Hui-Wang Ai; Wei Ren

    2013-01-01

    Redox processes are involved in almost every cell of the body as a consequence of aerobic life. In the past decades, redox biology has been increasingly recognized as one of the key themes in cell signaling. The progress has been accelerated by development of fluorescent probes that can monitor redox conditions and dynamics in cells and cell compartments. This short paper focuses on fluorescent redox probes that are genetically encoded, and discusses their properties, molecular mechanism, adv...

  11. Fluorescence lifetime based bioassays

    Science.gov (United States)

    Meyer-Almes, Franz-Josef

    2017-12-01

    Fluorescence lifetime (FLT) is a robust intrinsic property and material constant of fluorescent matter. Measuring this important physical indicator has evolved from a laboratory curiosity to a powerful and established technique for a variety of applications in drug discovery, medical diagnostics and basic biological research. This distinct trend was mainly driven by improved and meanwhile affordable laser and detection instrumentation on the one hand, and the development of suitable FLT probes and biological assays on the other. In this process two essential working approaches emerged. The first one is primarily focused on high throughput applications employing biochemical in vitro assays with no requirement for high spatial resolution. The second even more dynamic trend is the significant expansion of assay methods combining highly time and spatially resolved fluorescence data by fluorescence lifetime imaging. The latter approach is currently pursued to enable not only the investigation of immortal tumor cell lines, but also specific tissues or even organs in living animals. This review tries to give an actual overview about the current status of FLT based bioassays and the wide range of application opportunities in biomedical and life science areas. In addition, future trends of FLT technologies will be discussed.

  12. Quantitative approach of speleothems fluorescence

    Science.gov (United States)

    Quiers, Marine; Perrette, Yves; Poulenard, Jérôme; Chalmin, Emilie; Revol, Morgane

    2014-05-01

    In this study, we propose a framework to interpret quantitatively the fluorescence of speleothems organic matter (OM) by the way of a bank of water-extracted organic matter. Due to its efficiency to described dissolved organic matter (DOM) characteritics, fluorescence has been used to determined DOM signatures in natural systems, water circulations, OM transfer from soils, OM evolution in soils or recently, DOM changes in engineered treatment systems. Fluorescence has also been used in speleothems studies, mainly as a growth indicator. Only few studies interpret it as an environmental proxy. Indeed, the fluorescence of OM provides information on the type of organic molecules trapped in speleothems and their evolutions. But the most direct information given by fluorescence is the variation of OM quantities. Actually, increase of fluorescence intensity is generally related to an increase in OM quantity but may also be induced by calcite optical effect or qualitative change of OM. However, analytical technics used in water environments cannot be used for speleothem samples. In this study we propose to give a frame to interpret quantitatively the fluorescence signal of speleothems. 3 different samples of stalagmites from french northern Prealps were used. To allow the quantification of the fluorescence signal, we need to measure the fluorescence and the quantity of organic matter on the same sample. OM of speleothems was extracted by an acid digestion method and analysed with a spectrofluorimeter. However, it was not possible to quantify directly the OM, as the extract solvant was a high-concentrated acid. To solve this problem, a calibration using soil extracts was realised. Soils were chosen in order to represent the diversity of OM present in the environment above the caves. Attention was focused on soil and vegetation types, and landuse. Organic material was water extracted from soils and its fluorescence was also measured. Total organic carbon was performed on the

  13. Potential mercury emissions from fluorescent lamps production and obsolescence in mainland China.

    Science.gov (United States)

    Tan, Quanyin; Li, Jinhui

    2016-01-01

    The use of fluorescent lamps has expanded rapidly all over the world in recent years, because of their energy-saving capability. Consequently, however, mercury emissions from production, breakage, and discard of the lamps are drawing increasing concern from the public. This article focuses on evaluating the amount of mercury used for fluorescent lamp production, as well as the potential mercury emissions during production and breakage, in mainland China. It is expected to provide a comprehensive understanding about the risks present in the mercury from fluorescent lamps, and to know about the impacts of the policies on fluorescent lamps after their implementation. It is estimated that, in 2020, mercury consumption will be about 11.30-15.69 tonnes, a significant reduction of 34.9%-37.4% from that used in 2013, owing to improvement in mercury dosing dosage technology and tighter limitations on mercury content in fluorescent lamps. With these improvements, the amount of mercury remaining in fluorescent lamps and released during production is estimated to be 10.71-14.86 and 0.59-0.83 tonnes, respectively; the mercury released from waste fluorescent lamps is estimated to be about 5.37-7.59 tonnes. Also, a significant reduction to the mercury emission can be expected when a collection and treatment system is well established and conducted in the future. © The Author(s) 2015.

  14. Continuous-Flow Detector for Rapid Pathogen Identification

    Energy Technology Data Exchange (ETDEWEB)

    Barrett, Louise M. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics; Skulan, Andrew J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics; Singh, Anup K. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics; Cummings, Eric B. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics; Fiechtner, Gregory J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics

    2006-09-01

    This report describes the continued development of a low-power, portable detector for the rapid identification of pathogens such as B. anthracis and smallpox. Based on our successful demonstration of the continuous filter/concentrator inlet, we believe strongly that the inlet section will enable differentiation between viable and non-viable populations, between types of cells, and between pathogens and background contamination. Selective, continuous focusing of particles in a microstream enables highly selective and sensitive identification using fluorescently labeled antibodies and other receptors such as peptides, aptamers, or small ligands to minimize false positives. Processes such as mixing and lysing will also benefit from the highly localized particle streams. The concentrator is based on faceted prisms to contract microfluidic flows while maintaining uniform flowfields. The resulting interfaces, capable of high throughput, serve as high-, low-, and band-pass filters to direct selected bioparticles to a rapid, affinity-based detection system. The proposed device is superior to existing array-based detectors as antibody-pathogen binding can be accomplished in seconds rather than tens of minutes or even hours. The system is being designed to interface with aerosol collectors under development by the National Laboratories or commercial systems. The focused stream is designed to be interrogated using diode lasers to differentiate pathogens by light scattering. Identification of particles is done using fluorescently labeled antibodies to tag the particles, followed by multiplexed laser-induced fluorescence (LIF) detection (achieved by labeling each antibody with a different dye).

  15. Sensitive turn-on fluorescent detection of tartrazine based on fluorescence resonance energy transfer.

    Science.gov (United States)

    Huang, Sheng Tian; Shi, Yan; Li, Nian Bing; Luo, Hong Qun

    2012-01-18

    We introduce a sensitive, rapid, label-free and general fluorescent method for the determination of tartrazine by competitive binding to reduced graphene oxide (rGO) against fluorescein, and the fluorescence recovery upon fluorescein desorption from rGO provides a quantitative readout for tartrazine, giving a detection limit of 0.53 ng mL(-1).

  16. Introduction to fluorescence microscopy.

    Science.gov (United States)

    Ghiran, Ionita C

    2011-01-01

    This chapter is an overview of basic principles of fluorescence microscopy, including a brief history on the invention of this type of microscopy. The chapter highlights important points related to properties of fluorochromes, resolution in fluorescence microscopy, phase contrast and fluorescence, fluorescence filters, construction of a fluorescence microscope, and tips on the correct use of this equipment.

  17. Without 'Focus'

    Directory of Open Access Journals (Sweden)

    Aldo Sevi

    2010-12-01

    Full Text Available It is widely accepted that a notion of 'focus', more or less as conceived of in Jackendoff (1972, must be incorporated into our theory of grammar, as a means of accounting for certain observed correlations between prosodic facts and semantic/pragmatic facts. In this paper, we put forth the somewhat radical idea that the time has come to give up this customary view, and eliminate 'focus' from our theory of grammar. We argue that such a move is both economical and fruitful.Research over the years has revealed that the correlations between prosody, 'focus', and the alleged semantic/pragmatic effects of focus are much less clear and systematic than we may have initially hoped. First we argue that this state of affairs detracts significantly from the utility of our notion of 'focus', to the point of calling into question the very motivation for including it in the grammar. Then we look at some of the central data, and show how they might be analyzed without recourse to a notion of 'focus'. We concentrate on (i the effect of pitch accent placement on discourse congruence, and (ii the choice of 'associate' for the so-called 'focus sensitive' adverb only. We argue that our focus-free approach to the data improves empirical coverage, and begins to reveal patterns that have previously been obscured by preconceptions about 'focus'.ReferencesBeaver, D. & Clark, B. 2008. Sense and Sensitivity: How Focus Determines Meaning. Blackwell.Beaver, D., Clark, B., Flemming, E., Jaeger, T. F. & Wolters, M. 2007. ‘When semantics meets phonetics: Acoustical studies of second occurrence focus’. Language 83.2: 245–76.http://dx.doi.org/10.1353/lan.2007.0053Beckman, M. & Hirschberg, J. 1994. ‘The ToBI Annotation Conventions’. Ms.,http://www.cs.columbia.edu/~julia/files/conv.pdf.Bolinger, D. 1972. ‘Accent is predictable (if you are a mind-reader’. Language 48.3: 633–44.http://dx.doi.org/10.2307/412039Büring, D. 2006. ‘Focus projection and default

  18. Fluorescent nanoparticles for intracellular sensing: A review

    Energy Technology Data Exchange (ETDEWEB)

    Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  19. Fluorescence detection: SPIE volume 743

    Energy Technology Data Exchange (ETDEWEB)

    Menzel, E.R.

    1987-01-01

    This book contains proceedings arranged into four sessions. They are: Fluorescence spectroscopic techniques; Fluorescence in analysis and materials characterization; Fluorescence in medicine and biochemistry; and Fluorescence in criminalistics.

  20. Evaluation of a fluorescence-labelled oligonucleotide tide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears

    DEFF Research Database (Denmark)

    Nordentoft, Steen; Christensen, H.; Wegener, Henrik Caspar

    1997-01-01

    A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin-embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all...... with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin...

  1. Genetically Encoded Fluorescent Redox Probes

    Directory of Open Access Journals (Sweden)

    Hui-Wang Ai

    2013-11-01

    Full Text Available Redox processes are involved in almost every cell of the body as a consequence of aerobic life. In the past decades, redox biology has been increasingly recognized as one of the key themes in cell signaling. The progress has been accelerated by development of fluorescent probes that can monitor redox conditions and dynamics in cells and cell compartments. This short paper focuses on fluorescent redox probes that are genetically encoded, and discusses their properties, molecular mechanism, advantages and pitfalls. Our recent work on reaction-based encoded probes that are responsive to particular redox signaling molecules is also reviewed. Future challenges and directions are also commented.

  2. Genetically encoded fluorescent redox probes.

    Science.gov (United States)

    Ren, Wei; Ai, Hui-Wang

    2013-11-11

    Redox processes are involved in almost every cell of the body as a consequence of aerobic life. In the past decades, redox biology has been increasingly recognized as one of the key themes in cell signaling. The progress has been accelerated by development of fluorescent probes that can monitor redox conditions and dynamics in cells and cell compartments. This short paper focuses on fluorescent redox probes that are genetically encoded, and discusses their properties, molecular mechanism, advantages and pitfalls. Our recent work on reaction-based encoded probes that are responsive to particular redox signaling molecules is also reviewed. Future challenges and directions are also commented.

  3. Focusing horn

    CERN Multimedia

    1980-01-01

    This was the first magnetic horn developed by Simon Van der Meer to collect antiprotons in the AD complex. It was used for the AA (antiproton accumulator). Making an antiproton beam took a lot of time and effort. Firstly, protons were accelerated to an energy of 26 GeV/c (protons at 26GeV/c, antiprotons at 3.6GeV/c) in the PS and ejected onto a metal target. From the spray of emerging particles, a magnetic horn picked out 3.6 GeV antiprotons for injection into the AA through a wide-aperture focusing quadrupole magnet. For a million protons hitting the target, just one antiproton was captured, 'cooled' and accumulated. It took 3 days to make a beam of 3 x 10^11 -, three hundred thousand million - antiprotons. The development of this technology was a key step to the functioning of CERN's Super Proton Synchrotron as a proton - antiproton collider.

  4. Micro-focused ultrasonic solid-liquid extraction (muFUSLE) combined with HPLC and fluorescence detection for PAHs determination in sediments: optimization and linking with the analytical minimalism concept.

    Science.gov (United States)

    Capelo, J L; Galesio, M M; Felisberto, G M; Vaz, C; Pessoa, J Costa

    2005-06-15

    Analytical minimalism is a concept that deals with the optimization of all stages of an analytical procedure so that it becomes less time, cost, sample, reagent and energy consuming. The guide-lines provided in the USEPA extraction method 3550B recommend the use of focused ultrasound (FU), i.e., probe sonication, for the solid-liquid extraction of Polycyclic Aromatic Hydrocarbons, PAHs, but ignore the principle of analytical minimalism. The problems related with the dead sonication zones, often present when high volumes are sonicated with probe, are also not addressed. In this work, we demonstrate that successful extraction and quantification of PAHs from sediments can be done with low sample mass (0.125g), low reagent volume (4ml), short sonication time (3min) and low sonication amplitude (40%). Two variables are here particularly taken into account for total extraction: (i) the design of the extraction vessel and (ii) the solvent used to carry out the extraction. Results showed PAHs recoveries (EPA priority list) ranged between 77 and 101%, accounting for more than 95% for most of the PAHs here studied, as compared with the values obtained after soxhlet extraction. Taking into account the results reported in this work we recommend a revision of the EPA guidelines for PAHs extraction from solid matrices with focused ultrasound, so that these match the analytical minimalism concept.

  5. Laser Excited Fluorescence For Forensic Diagnostics

    Science.gov (United States)

    McKinney, Robert E.

    1986-07-01

    The application of laser excited fluorescence to the detection and identification of latent fingerprints was first accomplished ten years ago. The development of the technology has progressed rapidly with the introduction of commercial equipment by several manufacturers. Systems based on Argon-ion, Copper-vapor, and frequency-doubled Nd:YAG lasers are compared. The theoretical basis of detection by fluorescence is discussed along with the more useful techniques of dye staining. Other applications of the laser excited fluorescence in forensic investigation include gunshot residue analysis, serology, collection of trace evidence, and document examination.

  6. Highly fluorescent Ag nanoclusters: microwave-assisted green synthesis and Cr3+ sensing.

    Science.gov (United States)

    Liu, Shanhu; Lu, Feng; Zhu, Jun-Jie

    2011-03-07

    Highly fluorescent Ag nanoclusters were prepared in aqueous solution via a rapid microwave-assisted green approach and used as a novel fluorescence probe for the determination of Cr(3+) ions with high sensitivity and excellent selectivity.

  7. Laser Scanning Fluorescence Microscope

    Science.gov (United States)

    Hansen, Eric W.; Zelten, J. Peter; Wiseman, Benjamin A.

    1988-06-01

    We report on the development of a laser scanning fluorescence microscope possessing several features which facilitate its application to biological and biophysical analyses in living cells. It is built around a standard inverted microscope stand, enabling the use of standard optics, micromanipulation apparatus, and conventional (including video) microscopy in conjunction with laser scanning. The beam is scanned across the specimen by a pair of galvanometer-mounted mirrors, driven by a programmable controller which can operate in three modes: full raster scan, region of interest, and random-access. A full 512x512 pixel image can be acquired in one second. In region of interest mode, several subareas of the field can be selected for more rapid or detailed analysis. For those cases where the time scale of the observed phenomenon precludes full-field imaging, or where a full-field image is unnecessary, the random access mode enables an arbitrary pattern of isolated points to be selected and rapidly sequenced through. Via a graphical user interface implemented on the system's host computer, a user will be able to take a scout image either with video or a full-field laser scan, select regions or points on the scout image with a mouse, and set up experimental parameters such as detector integration times with a window-style menu. The instrument is designed to be a flexible testbed for investigating new techniques, without compromising its utility as a tool for biological research.

  8. Reviews in fluorescence 2010

    CERN Document Server

    Geddes, Chris D

    2011-01-01

    ""Reviews in Fluorescence 2010"", the seventh volume of the book serial from Springer, serves as a comprehensive collection of current trends and emerging hot topics in the field of fluorescence and closely related disciplines. It summarizes the year's progress in fluorescence and its applications, with authoritative analytical reviews specialized enough to be attractive to professional researchers, yet also appealing to the wider audience of scientists in related disciplines of fluorescence. ""Reviews in Fluorescence"" offers an essential reference material for any lab working in the fluoresc

  9. Principles of fluorescence techniques

    CERN Document Server

    2016-01-01

    Fluorescence techniques are being used and applied increasingly in academics and industry. The Principles of Fluorescence Techniques course will outline the basic concepts of fluorescence techniques and the successful utilization of the currently available commercial instrumentation. The course is designed for students who utilize fluorescence techniques and instrumentation and for researchers and industrial scientists who wish to deepen their knowledge of fluorescence applications. Key scientists in the field will deliver theoretical lectures. The lectures will be complemented by the direct utilization of steady-state and lifetime fluorescence instrumentation and confocal microscopy for FLIM and FRET applications provided by leading companies.

  10. Correlative fluorescence and electron microscopy.

    Science.gov (United States)

    Schirra, Randall T; Zhang, Peijun

    2014-10-01

    Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation--integration. Copyright © 2014 John Wiley & Sons, Inc.

  11. Rapid discrimination of Haemophilus influenzae, H. parainfluenzae, and H. haemolyticus by fluorescence in situ hybridization (FISH) and two matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) platforms.

    Science.gov (United States)

    Frickmann, Hagen; Christner, Martin; Donat, Martina; Berger, Anja; Essig, Andreas; Podbielski, Andreas; Hagen, Ralf Matthias; Poppert, Sven

    2013-01-01

    Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level. A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction. Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system's database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients.

  12. Noninferiority of glucose-6-phosphate dehydrogenase deficiency diagnosis by a point-of-care rapid test vs the laboratory fluorescent spot test demonstrated by copper inhibition in normal human red blood cells

    Science.gov (United States)

    Baird, J. Kevin; Dewi, Mewahyu; Subekti, Decy; Elyazar, Iqbal; Satyagraha, Ari W.

    2015-01-01

    Tens of millions of patients diagnosed with vivax malaria cannot safely receive primaquine therapy against repeated attacks caused by activation of dormant liver stages called hypnozoites. Most of these patients lack access to screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency, a highly prevalent disorder causing serious acute hemolytic anemia with primaquine therapy. We optimized CuCl inhibition of G6PD in normal red blood cells (RBCs) to assess G6PD diagnostic technologies suited to point of care in the impoverished rural tropics. The most widely applied technology for G6PD screening—the fluorescent spot test (FST)—is impractical in that setting. We evaluated a new point-of-care G6PD screening kit (CareStart G6PD, CSG) against FST using graded CuCl treatments to simulate variable hemizygous states, and varying proportions of CuCl-treated RBC suspensions to simulate variable heterozygous states of G6PD deficiency. In experiments double-blinded to CuCl treatment, technicians reading FST and CSG test (n = 269) classified results as positive or negative for deficiency. At G6PD activity ≤40% of normal (n = 112), CSG test was not inferior to FST in detecting G6PD deficiency (P = 0.003), with 96% vs 90% (P = 0.19) sensitivity and 75% and 87% (P = 0.01) specificity, respectively. The CSG test costs less, requires no specialized equipment, laboratory skills, or cold chain for successful application, and performs as well as the FST standard of care for G6PD screening. Such a device may vastly expand access to primaquine therapy and aid in mitigating the very substantial burden of morbidity and mortality imposed by the hypnozoite reservoir of vivax malaria. PMID:25312015

  13. Fabrication of Indocyanine Green and 2H, 3H-perfluoropentane loaded microbubbles for fluorescence and ultrasound imaging

    Science.gov (United States)

    He, Yutong; Wu, Qiang; Ma, Rong; Chang, Shufang; Shao, Pengfei; Xu, Ronald

    2016-03-01

    As a near-infrared (NIR) fluorescence dye, Indocyanine Green (ICG) has not gained broader clinical applications, owing to its multiple limitations such as concentration-dependent aggregation, low fluorescence quantum yield, poor physicochemical stability and rapid elimination from the body. In the meanwhile, 2H,3H-perfluoropentane (H-PFP) has been widely studied in ultrasound imaging as a vehicle for targeted delivery of contrast agents and drugs. We synthesized a novel dual-modal fluorescence and ultrasound contrast agent by encapsulating ICG and H-PFP in lipid microbubbles using a liquid-driven coaxial flow focusing (LDCFF) process. Uniform microbubbles with the sizes ranging from 1-10um and great ICG loading efficiency was achieved by this method. Our benchtop experiments showed that ICG/H-PFP microbubbles exhibited less aggregation, increased fluorescence intensity and more stable photostability compared to free ICG aqueous solution. Our phantom experiments demonstrated that ICG/H-PFP microbubbles enhanced the imaging contrasts in fluorescence imaging and ultrasonography. Our animal experiments indicated that ICG/H-PFP microbubbles extended the ICG life time and facilitated dual mode fluorescence and ultrasound imaging in vivo.

  14. Fluorescent Lamp Replacement Study

    Science.gov (United States)

    2017-07-01

    C -1 D FLUORESCENT LAMP SPECIFICATION SHEETS . . . . . . . . . . . . . . . . . . . . . . . . . . D -1 E LED WAVES’ LED ...friendly products, advances in efficiency, and lower production costs for lamps . The conversion of fluorescent bulbs to LED technology has many benefits...repeatedly turned on and off. (5) LEDs can be used in existing fluorescent lighting fixtures using LED retrofit kits or replacement lamps . (6

  15. A simple and sensitive fluorescent probe for specific detection of ...

    Indian Academy of Sciences (India)

    Yan-Fei Kang

    RAPID COMMUNICATION. A simple and sensitive fluorescent probe for specific detection ... strategy has attracted broad attention.17–24 Moreover, coumarin, a well-known fluorophore, exhibits low cyto- .... Urano Y 2010 New Strategies for Fluorescent Probe. Design in Medical Diagnostic Imaging Chem. Rev. 110. 2620.

  16. Novel disposable biochip platform employing supercritical angle fluorescence for enhanced fluorescence collection.

    Science.gov (United States)

    Hill, Duncan; McDonnell, Barry; Hearty, Stephen; Basabe-Desmonts, Lourdes; Blue, Robert; Trnavsky, Michal; McAtamney, Colm; O'Kennedy, Richard; MacCraith, Brian D

    2011-08-01

    This paper presents an overview of development of a novel disposable plastic biochip for multiplexed clinical diagnostic applications. The disposable biochip is manufactured using a low-cost, rapid turn- around injection moulding process and consists of nine parabolic elements on a planar substrate. The optical elements are based on supercritical angle fluorescence (SAF) which provides substantial enhancement of the fluorescence collection efficiency but also confines the fluorescence detection volume strictly to the immediate proximity of the biochip surface, thereby having the potential to discriminate against background fluorescence from the analyte solution. An optical reader is also described that enables interrogation and fluorescence collection from the nine optical elements on the chip. The sensitivity of the system was determined with a biotin-avidin assay while its clinical utility was demonstrated in an assay for C-reactive protein (CRP), an inflammation marker.

  17. Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters.

    Science.gov (United States)

    Mukherjee, Arnab; Walker, Joshua; Weyant, Kevin B; Schroeder, Charles M

    2013-01-01

    Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)-namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4-11), thermal stability (up to 60°C), and rapid maturation of fluorescence (fluorescence and slow kinetics of fluorescence maturation (10-40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable researchers to implement and further engineer improved FbFP-based reporters with enhanced brightness and tighter flavin binding, which will maximize their potential benefits.

  18. [Fluorescence microscopy detection of dermatophytes with Blankophor].

    Science.gov (United States)

    Haack, D; Zeller, R; Böhm, K H

    1987-01-01

    It is the first time that a fluorescent microscopic rapid-stain process is compared with the alkali method and with mycological culture examination. 810 skin scrapings primarily from horse, cat, and dog were available for analysis. The preparation of the samples for the fluorescent microscope test is easy and quick. The fluorescent stain solution keeps sufficiently long. When the slide preparations are looked at under the fluorescent microscope, the mycological elements light up and can easily be distinguished under survey enlargement. This leads to a considerable reduction of evaluation time. The microscopic proof of dermatophytes is considerably improved by the introduction of the fluorescent microscopic technique into mycological diagnosis, as this process is clearly superior to the alkali method. Dermatophytes are identified and finally evaluated after finishing the cultural analysis. Furthermore, the fluorescent microscopic proof of yeasts of the kind of Pityrosporum yeasts and of Demodex mites is possible. The not inconsiderable costs of the technical equipment may stand in the way of general and routine use of the fluorescent microscope for diagnosing dermatophytes. However, for laboratories with a large number of submitted skin scrapings, this process can be rated as a useful enrichment of their diagnostic potential.

  19. A novel endoscopic fluorescent band ligation method for tumor localization.

    Science.gov (United States)

    Hyun, Jong Hee; Kim, Seok-Ki; Kim, Kwang Gi; Kim, Hong Rae; Lee, Hyun Min; Park, Sunup; Kim, Sung Chun; Choi, Yongdoo; Sohn, Dae Kyung

    2016-10-01

    Accurate tumor localization is essential for minimally invasive surgery. This study describes the development of a novel endoscopic fluorescent band ligation method for the rapid and accurate identification of tumor sites during surgery. The method utilized a fluorescent rubber band, made of indocyanine green (ICG) and a liquid rubber solution mixture, as well as a near-infrared fluorescence laparoscopic system with a dual light source using a high-powered light-emitting diode (LED) and a 785-nm laser diode. The fluorescent rubber bands were endoscopically placed on the mucosae of porcine stomachs and colons. During subsequent conventional laparoscopic stomach and colon surgery, the fluorescent bands were assayed using the near-infrared fluorescence laparoscopy system. The locations of the fluorescent clips were clearly identified on the fluorescence images in real time. The system was able to distinguish the two or three bands marked on the mucosal surfaces of the stomach and colon. Resection margins around the fluorescent bands were sufficient in the resected specimens obtained during stomach and colon surgery. These novel endoscopic fluorescent bands could be rapidly and accurately localized during stomach and colon surgery. Use of these bands may make possible the excision of exact target sites during minimally invasive gastrointestinal surgery.

  20. Evanescent wave fluorescence biosensors: Advances of the last decade

    Science.gov (United States)

    Taitt, Chris Rowe; Anderson, George P.; Ligler, Frances S.

    2015-01-01

    Biosensor development has been a highly dynamic field of research and has progressed rapidly over the past two decades. The advances have accompanied the breakthroughs in molecular biology, nanomaterial sciences, and most importantly computers and electronics. The subfield of evanescent wave fluorescence biosensors has also matured dramatically during this time. Fundamentally, this review builds on our earlier 2005 review. While a brief mention of seminal early work will be included, this current review will focus on new technological developments as well as technology commercialized in just the last decade. Evanescent wave biosensors have found a wide array applications ranging from clinical diagnostics to biodefense to food testing; advances in those applications and more are described herein. PMID:26232145

  1. Rapid Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Perlin

    2005-08-14

    Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleic acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe development

  2. Fluorescence characterization of clinically-important bacteria.

    Science.gov (United States)

    Dartnell, Lewis R; Roberts, Tom A; Moore, Ginny; Ward, John M; Muller, Jan-Peter

    2013-01-01

    Healthcare-associated infections (HCAI/HAI) represent a substantial threat to patient health during hospitalization and incur billions of dollars additional cost for subsequent treatment. One promising method for the detection of bacterial contamination in a clinical setting before an HAI outbreak occurs is to exploit native fluorescence of cellular molecules for a hand-held, rapid-sweep surveillance instrument. Previous studies have shown fluorescence-based detection to be sensitive and effective for food-borne and environmental microorganisms, and even to be able to distinguish between cell types, but this powerful technique has not yet been deployed on the macroscale for the primary surveillance of contamination in healthcare facilities to prevent HAI. Here we report experimental data for the specification and design of such a fluorescence-based detection instrument. We have characterized the complete fluorescence response of eleven clinically-relevant bacteria by generating excitation-emission matrices (EEMs) over broad wavelength ranges. Furthermore, a number of surfaces and items of equipment commonly present on a ward, and potentially responsible for pathogen transfer, have been analyzed for potential issues of background fluorescence masking the signal from contaminant bacteria. These include bedside handrails, nurse call button, blood pressure cuff and ward computer keyboard, as well as disinfectant cleaning products and microfiber cloth. All examined bacterial strains exhibited a distinctive double-peak fluorescence feature associated with tryptophan with no other cellular fluorophore detected. Thus, this fluorescence survey found that an emission peak of 340nm, from an excitation source at 280nm, was the cellular fluorescence signal to target for detection of bacterial contamination. The majority of materials analysed offer a spectral window through which bacterial contamination could indeed be detected. A few instances were found of potential problems

  3. Fluorescence characterization of clinically-important bacteria.

    Directory of Open Access Journals (Sweden)

    Lewis R Dartnell

    Full Text Available Healthcare-associated infections (HCAI/HAI represent a substantial threat to patient health during hospitalization and incur billions of dollars additional cost for subsequent treatment. One promising method for the detection of bacterial contamination in a clinical setting before an HAI outbreak occurs is to exploit native fluorescence of cellular molecules for a hand-held, rapid-sweep surveillance instrument. Previous studies have shown fluorescence-based detection to be sensitive and effective for food-borne and environmental microorganisms, and even to be able to distinguish between cell types, but this powerful technique has not yet been deployed on the macroscale for the primary surveillance of contamination in healthcare facilities to prevent HAI. Here we report experimental data for the specification and design of such a fluorescence-based detection instrument. We have characterized the complete fluorescence response of eleven clinically-relevant bacteria by generating excitation-emission matrices (EEMs over broad wavelength ranges. Furthermore, a number of surfaces and items of equipment commonly present on a ward, and potentially responsible for pathogen transfer, have been analyzed for potential issues of background fluorescence masking the signal from contaminant bacteria. These include bedside handrails, nurse call button, blood pressure cuff and ward computer keyboard, as well as disinfectant cleaning products and microfiber cloth. All examined bacterial strains exhibited a distinctive double-peak fluorescence feature associated with tryptophan with no other cellular fluorophore detected. Thus, this fluorescence survey found that an emission peak of 340nm, from an excitation source at 280nm, was the cellular fluorescence signal to target for detection of bacterial contamination. The majority of materials analysed offer a spectral window through which bacterial contamination could indeed be detected. A few instances were found of

  4. Ribosomal genes in focus

    Science.gov (United States)

    Koberna, Karel; Malínský, Jan; Pliss, Artem; Mašata, Martin; Večeřová, Jaromíra; Fialová, Markéta; Bednár, Jan; Raška, Ivan

    2002-01-01

    T he organization of transcriptionally active ribosomal genes in animal cell nucleoli is investigated in this study in order to address the long-standing controversy with regard to the intranucleolar localization of these genes. Detailed analyses of HeLa cell nucleoli include direct localization of ribosomal genes by in situ hybridization and their indirect localization via nascent ribosomal transcript mappings. On the light microscopy (LM) level, ribosomal genes map in 10–40 fluorescence foci per nucleus, and transcription activity is associated with most foci. We demonstrate that each nucleolar focus observed by LM corresponds, on the EM level, to an individual fibrillar center (FC) and surrounding dense fibrillar components (DFCs). The EM data identify the DFC as the nucleolar subcompartment in which rRNA synthesis takes place, consistent with detection of rDNA within the DFC. The highly sensitive method for mapping nascent transcripts in permeabilized cells on ultrastructural level provides intense and unambiguous clustered immunogold signal over the DFC, whereas very little to no label is detected over the FC. This signal is strongly indicative of nascent “Christmas trees” of rRNA associated with individual rDNA genes, sampled on the surface of thin sections. Stereological analysis of the clustered transcription signal further suggests that these Christmas trees may be contorted in space and exhibit a DNA compaction ratio on the order of 4–5.5. PMID:12034768

  5. Fluorescence imaging agents in cancerology.

    Science.gov (United States)

    Paganin-Gioanni, Aurélie; Bellard, Elisabeth; Paquereau, Laurent; Ecochard, Vincent; Golzio, Muriel; Teissié, Justin

    2010-09-01

    One of the major challenges in cancer therapy is to improve early detection and prevention using novel targeted cancer diagnostics. Detection requests specific recognition. Tumor markers have to be ideally present on the surface of cancer cells. Their targeting with ligands coupled to imaging agents make them visible/detectable. Fluorescence imaging is a newly emerging technology which is becoming a complementary medical method for cancer diagnosis. It allows detection with a high spatio-temporal resolution of tumor markers in small animals and in clinical studies. In this review, we focus on the recent outcome of basic studies in the design of new approaches (probes and devices) used to detect tumor cells by fluorescence imaging.

  6. Pillararene-based fluorescent chemosensors: recent advances and perspectives.

    Science.gov (United States)

    Chen, Jin-Fa; Lin, Qi; Zhang, You-Ming; Yao, Hong; Wei, Tai-Bao

    2017-12-14

    In 2008, a new class of pillar-shaped supramolecular macrocyclic hosts was reported, known as "pillararenes". Their particular electron-rich cavity and the ease of their functionalization offer possibilities for the design and synthesis of novel fluorescent chemosensors. Subsequently, pillararene-based fluorescent sensors and probes have been rapidly developed. This feature article covers the most recent contributions from the pillararene-based fluorescent sensor field in terms of anion/cation sensing, small molecule recognition, biomolecule detection, fluorescent supramolecular aggregates, and biomedical imaging. Meanwhile, we hope that this feature article will inspire more effort to be devoted to this emerging field.

  7. Rapid Prototyping

    Science.gov (United States)

    1999-01-01

    Javelin, a Lone Peak Engineering Inc. Company has introduced the SteamRoller(TM) System as a commercial product. The system was designed by Javelin during a Phase II NASA funded small commercial product. The purpose of the invention was to allow automated-feed of flexible ceramic tapes to the Laminated Object Manufacturing rapid prototyping equipment. The ceramic material that Javelin was working with during the Phase II project is silicon nitride. This engineered ceramic material is of interest for space-based component.

  8. A topically-sprayable, activatable fluorescent and retaining probe, SPiDER-βGal for detecting cancer: Advantages of anchoring to cellular proteins after activation.

    Science.gov (United States)

    Nakamura, Yuko; Mochida, Ai; Nagaya, Tadanobu; Okuyama, Shuhei; Ogata, Fusa; Choyke, Peter L; Kobayashi, Hisataka

    2017-06-13

    SPiDER-βGal is a newly-developed probe that is activated by β-galactosidase and is then retained within cells by anchoring to intracellular proteins. Previous work has focused on gGlu-HMRG, a probe activated by γ-glutamyltranspeptidase, which demonstrated high sensitivity for the detection of peritoneal ovarian cancer metastases in an animal model. However, its fluorescence, after activation by γ-glutamyltranspeptidase, rapidly declines over time, limiting the actual imaging window and the ability to define the border of lesions. The purpose of this study is to compare the fluorescence signal kinetics of SPiDER-βGal with that of gGlu-HMRG using ovarian cancer cell lines in vitro and ex vivo tissue imaging. In vitro removal of gGlu-HMRG resulted in a rapid decrease of fluorescence intensity followed by a more gradual decrease up to 60 min while there was a gradual increase in fluorescence up to 60 min after removal of SPiDER-βGal. This is most likely due to internalization and retention of the dye within cells. This was also confirmed ex vivo tissue imaging using a red fluorescence protein (RFP)-labeled tumor model in which the intensity of fluorescence increased gradually after activation of SPiDER-βGal. Additionally, SPiDER-βGal resulted in intense enhancement within the tumor due to the high target-to-background ratio, which extended up to 60 min after activation. In contrast, gGlu-HMRG fluorescence resulted in decreasing fluorescence over time in extracted tumors. Thus, SPiDER-βGal has the advantages of higher signal with more signal retention, resulting in improved contrast of the tumor margin and suggesting it may be an alternative to existing activatable probes.

  9. Fluorescence Live Cell Imaging

    OpenAIRE

    Ettinger, Andreas; Wittmann, Torsten

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein (FP) tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio and to provide a suitable environment for ...

  10. Stink Bug Feeding Induces Fluorescence in Developing Cotton Bolls

    Directory of Open Access Journals (Sweden)

    Toews Michael D

    2011-08-01

    Full Text Available Abstract Background Stink bugs (Hemiptera: Pentatomidae comprise a critically important insect pest complex affecting 12 major crops worldwide including cotton. In the US, stink bug damage to developing cotton bolls causes boll abscission, lint staining, reduced fiber quality, and reduced yields with estimated losses ranging from 10 to 60 million dollars annually. Unfortunately, scouting for stink bug damage in the field is laborious and excessively time consuming. To improve scouting accuracy and efficiency, we investigated fluorescence changes in cotton boll tissues as a result of stink bug feeding. Results Fluorescent imaging under long-wave ultraviolet light showed that stink bug-damaged lint, the inner carpal wall, and the outside of the boll emitted strong blue-green fluorescence in a circular region near the puncture wound, whereas undamaged tissue emissions occurred at different wavelengths; the much weaker emission of undamaged tissue was dominated by chlorophyll fluorescence. We further characterized the optimum emission and excitation spectra to distinguish between stink bug damaged bolls from undamaged bolls. Conclusions The observed characteristic fluorescence peaks associated with stink bug damage give rise to a fluorescence-based method to rapidly distinguish between undamaged and stink bug damaged cotton bolls. Based on the fluorescent fingerprint, we envision a fluorescence reflectance imaging or a fluorescence ratiometric device to assist pest management professionals with rapidly determining the extent of stink bug damage in a cotton field.

  11. Stink bug feeding induces fluorescence in developing cotton bolls.

    Science.gov (United States)

    Xia, Jinjun; Mustafic, Adnan; Toews, Michael D; Haidekker, Mark A

    2011-08-04

    Stink bugs (Hemiptera: Pentatomidae) comprise a critically important insect pest complex affecting 12 major crops worldwide including cotton. In the US, stink bug damage to developing cotton bolls causes boll abscission, lint staining, reduced fiber quality, and reduced yields with estimated losses ranging from 10 to 60 million dollars annually. Unfortunately, scouting for stink bug damage in the field is laborious and excessively time consuming. To improve scouting accuracy and efficiency, we investigated fluorescence changes in cotton boll tissues as a result of stink bug feeding. Fluorescent imaging under long-wave ultraviolet light showed that stink bug-damaged lint, the inner carpal wall, and the outside of the boll emitted strong blue-green fluorescence in a circular region near the puncture wound, whereas undamaged tissue emissions occurred at different wavelengths; the much weaker emission of undamaged tissue was dominated by chlorophyll fluorescence. We further characterized the optimum emission and excitation spectra to distinguish between stink bug damaged bolls from undamaged bolls. The observed characteristic fluorescence peaks associated with stink bug damage give rise to a fluorescence-based method to rapidly distinguish between undamaged and stink bug damaged cotton bolls. Based on the fluorescent fingerprint, we envision a fluorescence reflectance imaging or a fluorescence ratiometric device to assist pest management professionals with rapidly determining the extent of stink bug damage in a cotton field.

  12. Fluorescent proteins: powerful tools in phagocyte biology.

    Science.gov (United States)

    Bajno, L; Grinstein, S

    1999-12-17

    Phagocyte functions such as chemotaxis and phagocytosis involve the rapid and transient development of cellular polarity. Study of this highly complex spatial and temporal cellular remodelling has been limited by the static nature of immunofluorescence and immunogold microscopy and because biochemical techniques are not vectorial. The recent introduction of fluorescent proteins (FPs) provides new approaches and opportunities to study phagocyte functions non-invasively, with excellent temporal and spatial resolution. This review summarizes the main properties and possible uses of green fluorescent protein (GFP) and its variants in phagocyte biology.

  13. Total internal reflection fluorescence (TIRF) microscopy.

    Science.gov (United States)

    Trache, Andreea; Meininger, Gerald A

    2008-08-01

    Total internal reflection fluorescence (TIRF) microscopy represents a method of exciting and visualizing fluorophores present in the near-membrane region of live or fixed cells grown on coverslips. TIRF microscopy is based on the total internal reflection phenomenon that occurs when light passes from a high-refractive medium (e.g., glass) into a low-refractive medium (e.g., cell, water). The evanescent field produced by total internally reflected light excites the fluorescent molecules at the cell-substrate interface and is accompanied by minimal exposure of the remaining cell volume. This technique provides high-contrast fluorescence images, with very low background and virtually no out-of-focus light, ideal for visualization and spectroscopy of single-molecule fluorescence near a surface. This unit presents, in a concise manner, the principle of operation, instrument diversity, and TIRF microscopy applications for the study of biological samples. Copyright 2008 by John Wiley & Sons, Inc.

  14. Palm kernel agar: An alternative culture medium for rapid detection ...

    African Journals Online (AJOL)

    Palm kernel agar: An alternative culture medium for rapid detection of aflatoxins in agricultural commodities. ... a pink background and blue or blue green fluorescence of palm kernel agar Under long wave UV light (366nm) as against the white background of DCA, which often interferes with fluorescence with corresponding ...

  15. Theoretical and experimental study of fiber-optic fluorescence immunosensors

    Science.gov (United States)

    Cao, He

    This dissertation investigates the optical detection of antigens (in this case, food pathogens such as Salmonella) with fiber-optic immunosensors. The major techniques used for this optical detection include: (1)Linking the antigens to some physical tracers that can be optically detected; (2)Collecting and transmitting the optical signal to an optical detector. From an optical point of view, the problem is a nonimaging-optics problem to collect a fluorescent signal from an extended Lambertian source and deliver it to an optical detection system with maximum energy transfer and distinct wavelength separation. A raytrace model of the optical detection system was used for numerical simulations to analyze and optimize the optical design. The result leads to an improvement of the optical detection. Related physical problems such as magnetic focusing effect, fluorescence detection, and wavelength separation have also been studied in detail. With the adoption of a single-step immunomagnetic assay, experimental studies have been conducted for the detection of Salmonella, with a dual- fiber optical probe and tapered tubular waveguide probes. The test results have shown that the detection system gives detection limit of approximately 106 CFU/ml with dual-fiber optical probes, and 105 CFU/ml with improved tubular waveguide probes. The system developed for this research project is designed as a cost-effective portable instrument that may be used for field-testing. Rapid and on-site detection, low cost instrumentation and a reusable optical probe have been emphasized throughout the study.

  16. Experiments with Fluorescent Lamps

    Science.gov (United States)

    Kraftmakher, Yaakov

    2010-10-01

    The experiments described below show the irradiance and illuminance spectra of two fluorescent lamps in relation to their color temperatures, and the efficacy in comparison to that of an incandescent lamp. Spectra of "warm white" and "cool daylight" fluorescent lamps are demonstrated.

  17. LEDs for fluorescence microscopy

    NARCIS (Netherlands)

    Young, I.T.; Garini, Y.; Dietrich, H.R.C.; Van Oel, W.; Liqui Lung, G.

    2004-01-01

    Traditional light sources for fluorescence microscopy have been mercury lamps, xenon lamps, and lasers. These sources have been essential in the development of fluorescence microscopy but each can have serious disadvantages: lack of near monochromaticity, heat generation, cost, lifetime of the light

  18. Membranes and Fluorescence microscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2009-01-01

    Fluorescence spectroscopy-based techniques using conventional fluorimeters have been extensively applied since the late 1960s to study different aspects of membrane-related phenomena, i.e., mainly relating to lipid-lipid and lipid-protein (peptide) interactions. Even though fluorescence...

  19. Multicolor, Fluorescent Supercapacitor Fiber.

    Science.gov (United States)

    Liao, Meng; Sun, Hao; Zhang, Jing; Wu, Jingxia; Xie, Songlin; Fu, Xuemei; Sun, Xuemei; Wang, Bingjie; Peng, Huisheng

    2017-10-05

    Fiber-shaped supercapacitors have attracted broad attentions from both academic and industrial communities due to the demonstrated potentials as next-generation power modules. However, it is important while remains challenging to develop dark-environment identifiable supercapacitor fibers for enhancement on operation convenience and security in nighttime applications. Herein, a novel family of colorful fluorescent supercapacitor fibers has been produced from aligned multi-walled carbon nanotube sheets. Fluorescent dye particles are introduced and stably anchored on the surfaces of aligned multi-walled carbon nanotubes to prepare hybrid fiber electrodes with a broad range of colors from red to purple. The fluorescent component in the dye introduces fluorescent indication capability to the fiber, which is particularly promising for flexible and wearable devices applied in dark environment. In addition, the colorful fluorescent supercapacitor fibers also maintain high electrochemical performance under cyclic bending and charge-discharge processes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Multispectral fluorescence imaging techniques for nondestructive food safety inspection

    Science.gov (United States)

    Kim, Moon S.; Lefcourt, Alan M.; Chen, Yud-Ren

    2004-03-01

    The use of spectral sensing has gained acceptance as a rapid means for nondestructive inspection of postharvest food produce. Current technologies generally use color or a single wavelength camera technology. The applicability and sensitivity of these techniques can be expanded through the use of multiple wavelengths. Reflectance in the Vis/NIR is the prevalent spectral technique. Fluorescence, compared to reflectance, is regarded as a more sensitive technique due to its dynamic responses to subtle changes in biological entities. Our laboratory has been exploring fluorescence as a potential means for detection of quality and wholesomeness of food products. Applications of fluorescence sensing require an understanding of the spectral characteristics emanating from constituents and potential contaminants. A number of factors affecting fluorescence emission characteristics are discussed. Because of relatively low fluorescence quantum yield from biological samples, a system with a powerful pulse light source such as a laser coupled with a gated detection device is used to harvest fluorescence, in the presence of ambient light. Several fluorescence sensor platforms developed in our laboratory, including hyperspectral imaging, and laser-induced fluorescence (LIF) and steady-state fluorescence imaging systems with multispectral capabilities are presented. We demonstrate the potential uses of recently developed fluorescence imaging platforms in food safety inspection of apples contaminated with animal feces.

  1. Antagonistic potential of fluorescent Pseudomonas and its impact on ...

    African Journals Online (AJOL)

    This study focused on the antagonistic potential of fluorescent Pseudomonas in vitro, and its inoculation effect on growth performance of Lycopersicon esculentum in Fusarium oxysporum and Rhizoctonia solani infested soil. Biochemical characteristics of fluorescent Pseudomonas showed that all ten isolates were positive ...

  2. Rapid mixing kinetic techniques.

    Science.gov (United States)

    Martin, Stephen R; Schilstra, Maria J

    2013-01-01

    Almost all of the elementary steps in a biochemical reaction scheme are either unimolecular or bimolecular processes that frequently occur on sub-second, often sub-millisecond, time scales. The traditional approach in kinetic studies is to mix two or more reagents and monitor the changes in concentrations with time. Conventional spectrophotometers cannot generally be used to study reactions that are complete within less than about 20 s, as it takes that amount of time to manually mix the reagents and activate the instrument. Rapid mixing techniques, which generally achieve mixing in less than 2 ms, overcome this limitation. This chapter is concerned with the use of these techniques in the study of reactions which reach equilibrium; the application of these methods to the study of enzyme kinetics is described in several excellent texts (Cornish-Bowden, Fundamentals of enzyme kinetics. Portland Press, 1995; Gutfreund, Kinetics for the life sciences. Receptors, transmitters and catalysis. Cambridge University Press, 1995).There are various ways to monitor changes in concentration of reactants, intermediates and products after mixing, but the most common way is to use changes in optical signals (absorbance or fluorescence) which often accompany reactions. Although absorbance can sometimes be used, fluorescence is often preferred because of its greater sensitivity, particularly in monitoring conformational changes. Such methods are continuous with good time resolution but they seldom permit the direct determination of the concentrations of individual species. Alternatively, samples may be taken from the reaction volume, mixed with a chemical quenching agent to stop the reaction, and their contents assessed by techniques such as HPLC. These methods can directly determine the concentrations of different species, but are discontinuous and have a limited time resolution.

  3. Dynamic fluorescence imaging with molecular agents for cancer detection

    Science.gov (United States)

    Kwon, Sun Kuk

    Non-invasive dynamic optical imaging of small animals requires the development of a novel fluorescence imaging modality. Herein, fluorescence imaging is demonstrated with sub-second camera integration times using agents specifically targeted to disease markers, enabling rapid detection of cancerous regions. The continuous-wave fluorescence imaging acquires data with an intensified or an electron-multiplying charge-coupled device. The work presented in this dissertation (i) assessed dose-dependent uptake using dynamic fluorescence imaging and pharmacokinetic (PK) models, (ii) evaluated disease marker availability in two different xenograft tumors, (iii) compared the impact of autofluorescence in fluorescence imaging of near-infrared (NIR) vs. red light excitable fluorescent contrast agents, (iv) demonstrated dual-wavelength fluorescence imaging of angiogenic vessels and lymphatics associated with a xenograft tumor model, and (v) examined dynamic multi-wavelength, whole-body fluorescence imaging with two different fluorescent contrast agents. PK analysis showed that the uptake of Cy5.5-c(KRGDf) in xenograft tumor regions linearly increased with doses of Cy5.5-c(KRGDf) up to 1.5 nmol/mouse. Above 1.5 nmol/mouse, the uptake did not increase with doses, suggesting receptor saturation. Target to background ratio (TBR) and PK analysis for two different tumor cell lines showed that while Kaposi's sarcoma (KS1767) exhibited early and rapid uptake of Cy5.5-c(KRGDf), human melanoma tumors (M21) had non-significant TBR differences and early uptake rates similar to the contralateral normal tissue regions. The differences may be due to different compartment location of the target. A comparison of fluorescence imaging with NIR vs. red light excitable fluorescent dyes demonstrates that NIR dyes are associated with less background signal, enabling rapid tumor detection. In contrast, animals injected with red light excitable fluorescent dyes showed high autofluorescence. Dual

  4. Fluorescence and Spectral Imaging

    Directory of Open Access Journals (Sweden)

    Ralph S. DaCosta

    2007-01-01

    Full Text Available Early identification of dysplasia remains a critical goal for diagnostic endoscopy since early discovery directly improves patient survival because it allows endoscopic or surgical intervention with disease localized without lymph node involvement. Clinical studies have successfully used tissue autofluorescence with conventional white light endoscopy and biopsy for detecting adenomatous colonic polyps, differentiating benign hyperplastic from adenomas with acceptable sensitivity and specificity. In Barrett's esophagus, the detection of dysplasia remains problematic because of background inflammation, whereas in the squamous esophagus, autofluorescence imaging appears to be more dependable. Point fluorescence spectroscopy, although playing a crucial role in the pioneering mechanistic development of fluorescence endoscopic imaging, does not seem to have a current function in endoscopy because of its nontargeted sampling and suboptimal sensitivity and specificity. Other point spectroscopic modalities, such as Raman spectroscopy and elastic light scattering, continue to be evaluated in clinical studies, but still suffer the significant disadvantages of being random and nonimaging. A recent addition to the fluorescence endoscopic imaging arsenal is the use of confocal fluorescence endomicroscopy, which provides real-time optical biopsy for the first time. To improve detection of dysplasia in the gastrointestinal tract, a new and exciting development has been the use of exogenous fluorescence contrast probes that specifically target a variety of disease-related cellular biomarkers using conventional fluorescent dyes and novel potent fluorescent nanocrystals (i.e., quantum dots. This is an area of great promise, but still in its infancy, and preclinical studies are currently under way.

  5. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  6. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  7. Fluorogen-based reporters for fluorescence imaging: a review

    Science.gov (United States)

    Jullien, Ludovic; Gautier, Arnaud

    2015-12-01

    Fluorescence bioimaging has recently jumped into a new area of spatiotemporal resolution and sensitivity thanks to synergistic advances in both optical physics and probe/biosensor design. This review focuses on the recent development of genetically encodable fluorescent reporters that bind endogenously present or exogenously applied fluorogenic chromophores (so-called fluorogens) and activate their fluorescence. We highlight the innovative engineering and design that gave rise to these new natural and synthetic fluorescent reporters, and describe some of the emerging applications in imaging and biosensing.

  8. A novel fluorescent assay for sucrose transporters

    Directory of Open Access Journals (Sweden)

    Gora Peter J

    2012-04-01

    Full Text Available Abstract Background We have developed a novel assay based on the ability of type I sucrose uptake transporters (SUTs to transport the fluorescent coumarin β-glucoside, esculin. Budding yeast (Saccharomyces cerevisiae is routinely used for the heterologous expression of SUTs and does not take up esculin. Results When type I sucrose transporters StSUT1 from potato or AtSUC2 from Arabidopsis were expressed in yeast, the cells were able to take up esculin and became brightly fluorescent. We tested a variety of incubation times, esculin concentrations, and buffer pH values and found that for these transporters, a 1 hr incubation at 0.1 to 1 mM esculin at pH 4.0 produced fluorescent cells that were easily distinguished from vector controls. Esculin uptake was assayed by several methods including fluorescence microscopy, spectrofluorometry and fluorescence-activiated cell sorting (FACS. Expression of the type II sucrose transporter OsSUT1 from rice did not result in increased esculin uptake under any conditions tested. Results were reproduced successfully in two distinct yeast strains, SEY6210 (an invertase mutant and BY4742. Conclusions The esculin uptake assay is rapid and sensitive and should be generally useful for preliminary tests of sucrose transporter function by heterologous expression in yeast. This assay is also suitable for selection of yeast showing esculin uptake activity using FACS.

  9. Fluorescent radiation converter

    Science.gov (United States)

    Viehmann, W. (Inventor)

    1981-01-01

    A fluorescence radiation converter is described which includes a substantially undoped optically transparent substrate and a waveshifter coating deposited on at least one portion of the substrate for absorption of radiation and conversion of fluorescent radiation. The coating is formed to substantially 1000 g/liter of a solvent, 70 to 200 g/liter of an organic polymer, and 0.2 to 25 g/liter of at least one organic fluorescent dye. The incoming incident radiation impinges on the coating. Radiation is absorbed by the fluorescent dye and is re-emitted as a longer wavelength radiation. Radiation is trapped within the substrate and is totally internally reflected by the boundary surface. Emitted radiation leaves the substrate ends to be detected.

  10. Fluorescent filtered electrophosphorescence

    Science.gov (United States)

    Forrest, Stephen R [Princeton, NJ; Sun, Yiru [Princeton, NJ; Giebink, Noel [Princeton, NJ; Thompson, Mark E [Anaheim Hills, CA

    2009-01-06

    The present invention relates to organic light emitting devices (OLEDs), and more specifically to OLEDS that emit light using a combination of fluorescent emitters and phosphorescent emitters for the efficient utilization of all of the electrically generated excitons.

  11. Introduction to fluorescence

    CERN Document Server

    Jameson, David M

    2014-01-01

    "An essential contribution to educating scientists in the principles of fluorescence. It will also be an important addition to the libraries of practitioners applying the principles of molecular fluorescence."-Ken Jacobson, Kenan Distinguished Professor of Cell Biology and Physiology, University of North Carolina at Chapel Hill"An exquisite compendium of fluorescence and its applications in biochemistry enriched by a very exciting historical perspective. This book will become a standard text for graduate students and other scientists."-Drs. Zygmunt (Karol) Gryczynski and Ignacy Gryczynski, University of North Texas Health Science Center"… truly a masterwork, combining clarity, precision, and good humor. The reader, novice or expert, will be pleased with the text and will not stop reading. It is a formidable account of the fluorescence field, which has impacted the life sciences so considerably in the last 60 years."-Jerson L. Silva, M.D., Ph.D., Professor and Director, National Institute of Science and Tech...

  12. Fluorescence (Multiwave) Confocal Microscopy.

    Science.gov (United States)

    Welzel, J; Kästle, Raphaela; Sattler, Elke C

    2016-10-01

    In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Fluorescent-protein-based probes: general principles and practices.

    Science.gov (United States)

    Ai, Hui-Wang

    2015-01-01

    An important application of fluorescent proteins is to derive genetically encoded fluorescent probes that can actively respond to cellular dynamics such as pH change, redox signaling, calcium oscillation, enzyme activities, and membrane potential. Despite the large diverse group of fluorescent-protein-based probes, a few basic principles have been established and are shared by most of these probes. In this article, the focus is on these general principles and strategies that guide the development of fluorescent-protein-based probes. A few examples are provided in each category to illustrate the corresponding principles. Since these principles are quite straightforward, others may adapt them to create fluorescent probes for their own interest. Hopefully, the development of the ever-growing family of fluorescent-protein-based probes will no longer be limited to a small number of laboratories specialized in senor development, leading to the situation that biological studies will be bettered assisted by genetically encoded sensors.

  14. Mercury dosing solutions for fluorescent lamps

    Science.gov (United States)

    Corazza, A.; Boffito, C.

    2008-07-01

    A review of the different technologies used to dose mercury in fluorescent lamps is presented. Conventional liquid mercury dosing is gradually being replaced with more reliable and environmentally friendly solutions that enable a significant reduction of the amount of mercury introduced in the lamp, so as to cope with more stringent regulations issued to minimize the environmental impact of exhausted lamps. This paper will review the most advanced novel methods to assure an accurate and fine dosing of mercury in fluorescent lamps, especially focusing on solutions based on the use of solid alloys.

  15. Mercury dosing solutions for fluorescent lamps

    Energy Technology Data Exchange (ETDEWEB)

    Corazza, A; Boffito, C [SAES Getters S.p.A., Viale Italia 77, Lainate (MI) 20020 (Italy)], E-mail: alessio_corazza@saes-group.com

    2008-07-21

    A review of the different technologies used to dose mercury in fluorescent lamps is presented. Conventional liquid mercury dosing is gradually being replaced with more reliable and environmentally friendly solutions that enable a significant reduction of the amount of mercury introduced in the lamp, so as to cope with more stringent regulations issued to minimize the environmental impact of exhausted lamps. This paper will review the most advanced novel methods to assure an accurate and fine dosing of mercury in fluorescent lamps, especially focusing on solutions based on the use of solid alloys.

  16. Fluorescence-based biosensors.

    Science.gov (United States)

    Strianese, Maria; Staiano, Maria; Ruggiero, Giuseppe; Labella, Tullio; Pellecchia, Claudio; D'Auria, Sabato

    2012-01-01

    The field of optical sensors has been a growing research area over the last three decades. A wide range of books and review articles has been published by experts in the field who have highlighted the advantages of optical sensing over other transduction methods. Fluorescence is by far the method most often applied and comes in a variety of schemes. Nowadays, one of the most common approaches in the field of optical biosensors is to combine the high sensitivity of fluorescence detection in combination with the high selectivity provided by ligand-binding proteins. In this chapter we deal with reviewing our recent results on the implementation of fluorescence-based sensors for monitoring environmentally hazardous gas molecules (e.g. nitric oxide, hydrogen sulfide). Reflectivity-based sensors, fluorescence correlation spectroscopy-based (FCS) systems, and sensors relying on the enhanced fluorescence emission on silver island films (SIFs) coupled to the total internal reflection fluorescence mode (TIRF) for the detection of gliadin and other prolamines considered toxic for celiac patients are also discussed herein.

  17. Maximizing the biochemical resolving power of fluorescence microscopy.

    Science.gov (United States)

    Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R

    2013-01-01

    Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems.

  18. Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters.

    Directory of Open Access Journals (Sweden)

    Arnab Mukherjee

    Full Text Available Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP-namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4-11, thermal stability (up to 60°C, and rapid maturation of fluorescence (<3 min.. In addition, the FbFP derived from Arabidopsis thaliana (iLOV emerged as a stable and nonperturbative reporter of promoter activity in Escherichia coli. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10-40 minutes for half maximal fluorescence recovery. From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to

  19. Focus group research.

    Science.gov (United States)

    Traynor, Michael

    2015-05-13

    A focus group is usually understood as a group of people brought together by a researcher to interact as a group. Focus group research explicitly uses interaction as part of its methodology. This article summarises the practice of running focus groups, explores the nature of focus group data and provides an example of focus group analysis.

  20. Rapid, single-molecule assays in nano/micro-fluidic chips with arrays of closely spaced parallel channels fabricated by femtosecond laser machining.

    Science.gov (United States)

    Canfield, Brian K; King, Jason K; Robinson, William N; Hofmeister, William H; Davis, Lloyd M

    2014-08-20

    Cost-effective pharmaceutical drug discovery depends on increasing assay throughput while reducing reagent needs. To this end, we are developing an ultrasensitive, fluorescence-based platform that incorporates a nano/micro-fluidic chip with an array of closely spaced channels for parallelized optical readout of single-molecule assays. Here we describe the use of direct femtosecond laser machining to fabricate several hundred closely spaced channels on the surfaces of fused silica substrates. The channels are sealed by bonding to a microscope cover slip spin-coated with a thin film of poly(dimethylsiloxane). Single-molecule detection experiments are conducted using a custom-built, wide-field microscope. The array of channels is epi-illuminated by a line-generating red diode laser, resulting in a line focus just a few microns thick across a 500 micron field of view. A dilute aqueous solution of fluorescently labeled biomolecules is loaded into the device and fluorescence is detected with an electron-multiplying CCD camera, allowing acquisition rates up to 7 kHz for each microchannel. Matched digital filtering based on experimental parameters is used to perform an initial, rapid assessment of detected fluorescence. More detailed analysis is obtained through fluorescence correlation spectroscopy. Simulated fluorescence data is shown to agree well with experimental values.

  1. Circular dichroism spectroscopy of fluorescent proteins

    NARCIS (Netherlands)

    Visser, N.V.; Hink, M.A.; Borst, J.W.; Krogt, van der G.N.M.; Visser, A.J.W.G.

    2002-01-01

    Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red

  2. Genetic Analysis of Digestive Physiology Using Fluorescent Phospholipid Reporters

    Science.gov (United States)

    Farber, Steven A.; Pack, Michael; Ho, Shiu-Ying; Johnson, Iain D.; Wagner, Daniel S.; Dosch, Roland; Mullins, Mary C.; Hendrickson, H. Stewart; Hendrickson, Elizabeth K.; Halpern, Marnie E.

    2001-05-01

    Zebrafish are a valuable model for mammalian lipid metabolism; larvae process lipids similarly through the intestine and hepatobiliary system and respond to drugs that block cholesterol synthesis in humans. After ingestion of fluorescently quenched phospholipids, endogenous lipase activity and rapid transport of cleavage products results in intense gall bladder fluorescence. Genetic screening identifies zebrafish mutants, such as fat free, that show normal digestive organ morphology but severely reduced phospholipid and cholesterol processing. Thus, fluorescent lipids provide a sensitive readout of lipid metabolism and are a powerful tool for identifying genes that mediate vertebrate digestive physiology.

  3. Neoplasm diagnostics based on fluorescence of polymethine dyes

    Science.gov (United States)

    Samtsov, Michael P.; Voropay, Eugene S.; Chalov, Vadim N.; Zhavrid, Edvard A.

    2002-05-01

    Investigated polymethine dye TICS has near IR bands of fluorescence and absorption within the transparency region of biological tissues. It can be detected up to 1.5 cm from the surface of the skin. The intensity of a fluorescence signal of TICS is linear for doses up to 2 mg/kg in both tumor and muscle tissue. The ratio of an intensity of light induced fluorescence in tumor tissue to one in muscle tissue is up to 3.6 for rapidly growing tumors. The retention time of TICS is 7 days in all tissues. TICS can be used in the detection of tumor boundaries and tumor internal structure.

  4. Synthesis and Sensing Applications of Fluorescent 3-Cinnamoyl Coumarins

    Directory of Open Access Journals (Sweden)

    Preeti Yadav

    2015-12-01

    Full Text Available We have synthesized two novel fluorescent 3-(4-diethylaminocinnamoyl coumarins that exhibit fluorescence quenching upon exposure to a nerve agent simulant, diethylchlorophosphate (DCP, providing a basis for rapid and sensitive DCP chemosensing. Furthermore, these coumarin derivatives display two-photon fluorescence upon illumination with near-infrared laser pulses and their two-photon (TP absorption cross-section was evaluated. The potential for TP bio-imaging of these compounds was investigated by their cellular uptake in HeLa cells by TP confocal microscopy.

  5. Fluorescence Microscopy as a Diagnostic Tool for Dermatophytosis.

    Science.gov (United States)

    Estela Cubells, Jose R; Victoria Martínez, Ana M; Martínez Leboráns, Lorena; Alegre de Miquel, Víctor

    2016-03-01

    Dermatophytosis is a superficial fungal infection of keratinized tissues. Dermatophytes can cause discomfort but are not usually life threatening. However, the infection can spread and may lead to systemic fungal infections in immunocompromised patients. Currently available diagnostic methods include potassium hydroxide (KOH) testing and periodic acid-Schiff (PAS) staining. However, most diagnostic techniques cannot be performed rapidly; days to weeks may be required for conclusive results. Certain dermatophytes autofluoresce and can be observed under fluorescence microscopy. The authors examined a series of 24 cases of hematoxylin and eosin-stained dermatophytoses using fluorescence microscopy and compared the results with those obtained using PAS staining. The diagnostic performance of fluorescence microscopy was better than that of PAS staining. Fluorescence microscopy allowed the detection of all the cases that were detected using PAS staining. In addition, fluorescence microscopy facilitated the detection of weak fluorescence in 2 cases with ambiguous PAS results. These results support the integration into clinical practice of fluorescence microscopy as a simple and rapid diagnostic tool for evaluating cases of suspected dermatophytosis.

  6. Fluorescein Derivatives in Intravital Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Michael S. Roberts

    2013-08-01

    Full Text Available Intravital fluorescence microscopy enables the direct imaging of fluorophores in vivo and advanced techniques such as fluorescence lifetime imaging (FLIM enable the simultaneous detection of multiple fluorophores. Consequently, it is now possible to record distribution and metabolism of a chemical in vivo and to optimise the delivery of fluorophores in vivo. Recent clinical applications with fluorescein and other intravital fluorescent stains have occurred in neurosurgery, dermatology [including photodynamic therapy (PDT] and endomicroscopy. Potential uses have been identified in periodontal disease, skin graft and cancer surgery. Animal studies have demonstrated that diseased tissue can be specifically stained with fluorophore conjugates. This review focuses on the fluorescein derived fluorophores in common clinical use and provides examples of novel applications from studies in tissue samples.

  7. Diversity and Evolution of Coral Fluorescent Proteins

    Science.gov (United States)

    Alieva, Naila O.; Konzen, Karen A.; Field, Steven F.; Meleshkevitch, Ella A.; Hunt, Marguerite E.; Beltran-Ramirez, Victor; Miller, David J.; Wiedenmann, Jörg; Salih, Anya; Matz, Mikhail V.

    2008-01-01

    GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a “chromo-red” color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural

  8. Diversity and evolution of coral fluorescent proteins.

    Directory of Open Access Journals (Sweden)

    Naila O Alieva

    2008-07-01

    Full Text Available GFP-like fluorescent proteins (FPs are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae and pink chromoprotein from Stylophora pistillata (Pocilloporidae, both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria. The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of

  9. Multigrade Teaching Rapid Appraisal Procedure.

    Science.gov (United States)

    Nielsen, Dean

    Multigrade classes have been recognized as part of elementary education for many years, but their special needs have been largely ignored. This manual focuses on the survey research that should predate the design of instructional management strategies in multigrade classrooms. It describes rapid and reliable ways to collect information about the…

  10. Multimodal fluorescence molecular imaging for in vivo characterization of skin cancer using endogenous and exogenous fluorophores

    Science.gov (United States)

    Miller, Jessica P.; Habimana-Griffin, LeMoyne; Edwards, Tracy S.; Achilefu, Samuel

    2017-06-01

    Similarity of skin cancer with many benign skin pathologies requires reliable methods to detect and differentiate the different types of these lesions. Previous studies have explored the use of disparate optical techniques to identify and estimate the invasive nature of melanoma and basal cell carcinoma with varying outcomes. Here, we used a concerted approach that provides complementary information for rapid screening and characterization of tumors, focusing on squamous cell carcinoma (SCC) of the skin. Assessment of in vivo autofluorescence lifetime (FLT) imaging of endogenous fluorophores that are excitable at longer wavelengths (480 nm) than conventional NADH and FAD revealed a decrease in the short FLT component for SCC compared to normal skin, with mean values of 0.57±0.026 ns and 0.61±0.021 ns, respectively (p=0.004). Subsequent systemic administration of a near-infrared fluorescent molecular probe in SCC bearing mice, followed by the implementation of image processing methods on data acquired from two-dimensional and three-dimensional fluorescence molecular imaging, allowed us to estimate the tumor volume and depth, as well as quantify the fluorescent probe in the tumor. The result suggests the involvement of lipofuscin-like lipopigments and riboflavin in SCC metabolism and serves as a model for staging SCC.

  11. The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae.

    Science.gov (United States)

    Rumin, Judith; Bonnefond, Hubert; Saint-Jean, Bruno; Rouxel, Catherine; Sciandra, Antoine; Bernard, Olivier; Cadoret, Jean-Paul; Bougaran, Gaël

    2015-01-01

    Microalgae are currently emerging as one of the most promising alternative sources for the next generation of food, feed, cosmetics and renewable energy in the form of biofuel. Microalgae constitute a diverse group of microorganisms with advantages like fast and efficient growth. In addition, they do not compete for arable land and offer very high lipid yield potential. Major challenges for the development of this resource are to select lipid-rich strains using high-throughput staining for neutral lipid content in microalgae species. For this purpose, the fluorescent dyes most commonly used to quantify lipids are Nile red and BODIPY 505/515. Their fluorescent staining for lipids offers a rapid and inexpensive analysis tool to measure neutral lipid content, avoiding time-consuming and costly gravimetric analysis. This review collates and presents recent advances in algal lipid staining and focuses on Nile red and BODIPY 505/515 staining characteristics. The available literature addresses the limitations of fluorescent dyes under certain conditions, such as spectral properties, dye concentrations, cell concentrations, temperature and incubation duration. Moreover, the overall conclusion of the present review study gives limitations on the use of fluorochrome for screening of lipid-rich microalgae species and suggests improved protocols for staining recalcitrant microalgae and recommendations for the staining quantification.

  12. BrightFocus Foundation

    Science.gov (United States)

    ... sooner. More science news Help us find a cure. Give to BrightFocus BrightFocus Updates BrightFocus Foundation Lauds Bill Gates Alzheimer’s Initiative “BrightFocus Foundation lauds today’s historic announcement by ...

  13. Concentrators using fluorescent substances

    Energy Technology Data Exchange (ETDEWEB)

    Hayashibara, M.; Tsukamoto, M. (Hitachi Seisakusho K.K., Tokyo (Japan))

    1990-01-01

    In luminescent concentrators - plates of polymethylmethacrylate (PMMA) or other transparent material with a fluorescent compound dispersed within them - incident light is trapped and concentrated by internal reflection, and shifted to a longer wavelength, as it interacts with fluorescent particles. Experience with the use of luminescent concentrators for electricity generation in conjunction with solar cells, in solar heaters, in amplifiers for light intensity, in long-wave converters and in display panels is discussed. Solar energy conversion efficiencies of 4-5% have been obtained in generating systems combining concentrators containing Fluorol 555 or Rhodamin 6G with GaAs solar cells. (author).

  14. Smartphone fluorescence spectroscopy

    Science.gov (United States)

    Yu, Hojoeng; Tan, Yafang; Cunningham, Brian T.

    2014-03-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

  15. Smartphone fluorescence spectroscopy.

    Science.gov (United States)

    Yu, Hojeong; Tan, Yafang; Cunningham, Brian T

    2014-09-02

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of specific nucleic acid sequences in a liquid test sample and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route toward portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

  16. Microflow Cytometers with Integrated Hydrodynamic Focusing

    Directory of Open Access Journals (Sweden)

    Martin Schmidt

    2013-04-01

    Full Text Available This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex principle. The sample—A suspension of micro particles or blood cells—is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood.

  17. Far-Field Fluorescence Nanoscopy

    Science.gov (United States)

    Hell, Stefan

    2009-03-01

    The resolution of a far-field optical microscopy is usually limited to d=λ/ λ( 2,α ) . - ( 2,α ) > 200 nm, with nα denoting the numerical aperture of the lens and λ the wavelength of light. While the diffraction barrier has prompted the invention of electron, scanning probe, and x-ray microscopy, the 3D-imaging of the interior of (live) cells requires the use of focused visible light. I will discuss new developments of optical microscopy that I anticipate to have a lasting impact on our understanding of living matter. Emphasis will be placed on physical concepts that have overcome the diffraction barrier in far-field fluorescence microscopy. To set the scene for future directions, I will show that all these concepts share a common strategy: exploiting selected states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. The first viable concept of this kind was Stimulated Emission Depletion (STED) microscopy where the spot diameter followsd λ/ λ( 2,α√1+I / I Is . - Is ) . - ( 2,α√1+I / I Is . - Is ); I / I Is . - Isis a measure of the strength with which the molecule is send from the fluorescent state to the dark ground state. For I / I Is . - Is->∞ it follows that d->0, meaning that the resolution that can, in principle, be molecular. The concept underlying STED microscopy can be expanded by employing other transitions that shuffle the molecule between a dark and a bright state, such as (i) shelving the fluorophore in a dark triplet state, and (ii) photoswitching between a `fluorescence activated' and a `fluorescence deactivated' conformational state. Examples for the latter include photochromic organic compounds, and fluorescent proteins which undergo a cis-trans photoisomerizations. Photoswitching provides ultrahigh resolution at ultralow light levels. Switching can be performed in an ensemble or individually in which case the image is assembled molecule by molecule at high resolution. By providing molecular

  18. Near-infrared fluorescent probe for detection of thiophenols in water samples and living cells.

    Science.gov (United States)

    Yu, Dehuan; Huang, Feihu; Ding, Shuangshuang; Feng, Guoqiang

    2014-09-02

    The development of probes for rapid, selective, and sensitive detection of the highly toxic thiophenols is of great importance in both environmental and biological science. Despite the appealing advantages of near-infrared (NIR) fluorescent detection, no NIR fluorescent probes have been reported for thiophenols to date. Using the chemical properties of thiophenols that are able to cleave sulfonamide selectively and efficiently under mild conditions, we herein report a dicyanomethylene-benzopyran (DCMB)-based NIR fluorescent probe for thiophenols. This probe features remarkable large Stokes shift and shows a rapid, highly selective, and sensitive detection process for thiophenols with significant NIR fluorescent turn-on responses. The potential applications of this new NIR fluorescent probe were demonstrated by the quantitative detection of thiophenol in real water samples and by fluorescent imaging of thiophenol in living cells.

  19. Rapid optical determination of β-lactamase and antibiotic activity

    Science.gov (United States)

    2014-01-01

    Background The absence of rapid tests evaluating antibiotic susceptibility results in the empirical prescription of antibiotics. This can lead to treatment failures due to escalating antibiotic resistance, and also furthers the emergence of drug-resistant bacteria. This study reports a rapid optical method to detect β-lactamase and thereby assess activity of β-lactam antibiotics, which could provide an approach for targeted prescription of antibiotics. The methodology is centred on a fluorescence quenching based probe (β-LEAF – β-Lactamase Enzyme Activated Fluorophore) that mimics the structure of β-lactam antibiotics. Results The β-LEAF assay was performed for rapid determination of β-lactamase production and activity of β-lactam antibiotic (cefazolin) on a panel of Staphylococcus aureus ATCC strains and clinical isolates. Four of the clinical isolates were determined to be lactamase producers, with the capacity to inactivate cefazolin, out of the twenty-five isolates tested. These results were compared against gold standard methods, nitrocefin disk test for β-lactamase detection and disk diffusion for antibiotic susceptibility, showing results to be largely consistent. Furthermore, in the sub-set of β-lactamase producers, it was demonstrated and validated that multiple antibiotics (cefazolin, cefoxitin, cefepime) could be assessed simultaneously to predict the antibiotic that would be most active for a given bacterial isolate. Conclusions The study establishes the rapid β-LEAF assay for β-lactamase detection and prediction of antibiotic activity using S. aureus clinical isolates. Although the focus in the current study is β-lactamase-based resistance, the overall approach represents a broad diagnostic platform. In the long-term, these studies form the basis for the development of assays utilizing a broader variety of targets, pathogens and drugs. PMID:24708478

  20. Fluorescent polymers from non-fluorescent photoreactive monomers.

    Science.gov (United States)

    Mueller, Jan O; Voll, Dominik; Schmidt, Friedrich G; Delaittre, Guillaume; Barner-Kowollik, Christopher

    2014-12-25

    A facile, fast and ambient-temperature avenue towards highly fluorescent polymers is introduced via polymerizing non-fluorescent photoreactive monomers based on light-induced NITEC chemistry, providing a platform technology for fluorescent polymers. The resulting polypyrazolines were analyzed in depth and the photo-triggered step-growth process was monitored in a detailed kinetic study.

  1. Recent Advances in Silicon Nanomaterial-Based Fluorescent Sensors.

    Science.gov (United States)

    Wang, Houyu; He, Yao

    2017-02-03

    During the past decades, owing to silicon nanomaterials' unique optical properties, benign biocompatibility, and abundant surface chemistry, different dimensional silicon nanostructures have been widely employed for rationally designing and fabricating high-performance fluorescent sensors for the detection of various chemical and biological species. Among of these, zero-dimensional silicon nanoparticles (SiNPs) and one-dimensional silicon nanowires (SiNWs) are of particular interest. Herein, we focus on reviewing recent advances in silicon nanomaterials-based fluorescent sensors from a broad perspective and discuss possible future directions. Firstly, we introduce the latest achievement of zero-dimensional SiNP-based fluorescent sensors. Next, we present recent advances of one-dimensional SiNW-based fluorescent sensors. Finally, we discuss the major challenges and prospects for the development of silicon-based fluorescent sensors.

  2. Fluorescence Microscopy of Single Molecules

    Science.gov (United States)

    Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

    2004-01-01

    The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

  3. Fluorescence spectroscopy in polymer science

    NARCIS (Netherlands)

    Raja, T.N.; Brouwer, A.M.; Demchenko, A.P.

    2011-01-01

    Polymer science is an interdisciplinary field, combining chemistry, physics, and in some cases biology. Structure, morphology, and dynamical phenomena in natural and synthetic polymers can be addressed using fluorescence spectroscopy. The most attractive aspect of fluorescent reporters is that their

  4. Who's who in fluorescence 2008

    CERN Document Server

    Geddes, Chris D

    2008-01-01

    The Journal of Fluorescence's sixth Who's Who directory publishes the names, contact details, specialty keywords, and a brief description of scientists employing fluorescence methodology and instrumentation in their working lives. This is a unique reference.

  5. Sputum direct fluorescent antibody (DFA)

    Science.gov (United States)

    ... ency/article/003553.htm Sputum direct fluorescent antibody (DFA) test To use the sharing features on this page, please enable JavaScript. Sputum direct fluorescent antibody (DFA) is a lab test that looks for micro- ...

  6. Focus Intonation in Bengali

    Science.gov (United States)

    Hasan, Md. Kamrul

    2015-01-01

    This work attempts to investigate the role of prosody in the syntax of focus in Bangla. The aim of this study is to show the intonation pattern of Bangla in emphasis and focus. In order to do that, the author has looked at the pattern of focus without-i/o as well as with the same. Do they really pose any different focus intonation pattern from…

  7. A fluorescence scanning electron microscope

    OpenAIRE

    Kanemaru, Takaaki; Hirata, Kazuho; Takasu, Shin-ichi; Isobe, Shin-Ichiro; Mizuki, Keiji; Mataka, Shuntaro; Nakamura, Kei-ichiro

    2010-01-01

    Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital c...

  8. Who's who in fluorescence 2005

    CERN Document Server

    Geddes, Chris D

    2006-01-01

    The Journal of Fluorescence's third Who's Who directory publishes the names, contact details, specialty keywords, photographs, and a brief description of scientists employing fluorescence methodology and instrumentation in their working livesThe directory provides company contact details with a brief list of fluorescence-related products.

  9. Plasmon assisted synthesis of highly fluorescing silver quantum cluster / polymer composites for biochemical sensing

    DEFF Research Database (Denmark)

    Bernard, S.; Kutter, J.P.; Mogensen, Klaus Bo

    2014-01-01

    , the polymer/oligomer grows from the AgNP film, while AgQCs are being embedded into the matrix. This can happen at a time scale of second and during photoactivation, the fluorescent signal emanating from AgQCs increases rapidly with time. The fluorescing composite was tested for detection of cyanide. Here, so...

  10. High throughput plasma N-glycome profiling using multiplexed labelling and UPLC with fluorescence detection

    DEFF Research Database (Denmark)

    Kneževic, Ana; Bones, Jonathan; Kracun, Stjepan Kresimir

    2011-01-01

    A rapid glycomic profiling method is described wherein N-glycans from plasma samples individually labelled with aniline, 2-aminobenzamide and 2-aminoacridone are mixed, co-injected and separated in the same HILIC-fluorescence run. Transfer of the multiplexed method to UPLC-fluorescence permits...

  11. Chlorophyll Fluorescence Analysis of Cyanobacterial Photosynthesis and Acclimation

    Science.gov (United States)

    Campbell, Douglas; Hurry, Vaughan; Clarke, Adrian K.; Gustafsson, Petter; Öquist, Gunnar

    1998-01-01

    Cyanobacteria are ecologically important photosynthetic prokaryotes that also serve as popular model organisms for studies of photosynthesis and gene regulation. Both molecular and ecological studies of cyanobacteria benefit from real-time information on photosynthesis and acclimation. Monitoring in vivo chlorophyll fluorescence can provide noninvasive measures of photosynthetic physiology in a wide range of cyanobacteria and cyanolichens and requires only small samples. Cyanobacterial fluorescence patterns are distinct from those of plants, because of key structural and functional properties of cyanobacteria. These include significant fluorescence emission from the light-harvesting phycobiliproteins; large and rapid changes in fluorescence yield (state transitions) which depend on metabolic and environmental conditions; and flexible, overlapping respiratory and photosynthetic electron transport chains. The fluorescence parameters FV/FM, FV′/FM′,qp,qN, NPQ, and φPS II were originally developed to extract information from the fluorescence signals of higher plants. In this review, we consider how the special properties of cyanobacteria can be accommodated and used to extract biologically useful information from cyanobacterial in vivo chlorophyll fluorescence signals. We describe how the pattern of fluorescence yield versus light intensity can be used to predict the acclimated light level for a cyanobacterial population, giving information valuable for both laboratory and field studies of acclimation processes. The size of the change in fluorescence yield during dark-to-light transitions can provide information on respiration and the iron status of the cyanobacteria. Finally, fluorescence parameters can be used to estimate the electron transport rate at the acclimated growth light intensity. PMID:9729605

  12. Prenatal detection of aneuploidies using fluorescence in situ ...

    Indian Academy of Sciences (India)

    Fluorescence in situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies in high risk pregnancies in an Indian set up.

  13. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  14. Influence of binding mechanism on labeling efficiency and luminous properties of fluorescent cellulose nanocrystals.

    Science.gov (United States)

    Ding, Qijun; Zeng, Jinsong; Wang, Bin; Gao, Wenhua; Chen, Kefu; Yuan, Zhe; Xu, Jun

    2017-11-01

    Cellulose nanocrystals (CNCs) have excellent properties, such as reproducibility, low biodegradability and a large amount of reactive hydroxyl groups on the surface. This study focused on the labeling efficiency and fluorescent properties of the fluorescent labeling of CNCs by means of electrostatic adsorption and covalent bonding. The CNCs in the sample were approximately 94.76% successfully labeled with dyes, and the number of dye molecules adsorbed by per CNC was approximately 208 by electrostatic adsorption. For the sample covalently linked, the efficiency of the fluorescent labeling was 95.51%, and the number of dye molecules attached to per CNC was 1038. The quenching mode of the fluorescent CNCs was dynamic quenching. The fluorescence lifetime and quantum yield of the fluorescent CNCs increased by 1-2 times compared to the free dye. A thorough investigation of the relation between the binding mechanism and the fluorescent properties in fluorescent CNCs was conducted. Copyright © 2017. Published by Elsevier Ltd.

  15. Timing and Operating Mode Design for Time-Gated Fluorescence Lifetime Imaging Microscopy

    Directory of Open Access Journals (Sweden)

    Chao Liu

    2013-01-01

    Full Text Available Steady-state fluorence imaging and time-resolved fluorescence imaging are two important areas in fluorescence imaging research. Fluorescence lifetime imaging is an absolute measurement method which is independent of excitation laser intensity, fluorophore concentration, and photobleaching compared to fluorescence intensity imaging techniques. Time-gated fluorescence lifetime imaging microscopy (FLIM can provide high resolution and high imaging frame during mature FLIM methods. An abstract time-gated FLIM model was given, and important temporal parameters are shown as well. Aiming at different applications of steady and transient fluorescence processes, two different operation modes, timing and lifetime computing algorithm are designed. High resolution and high frame can be achieved by one-excitation one-sampling mode and least square algorithm for steady imaging applications. Correspondingly, one-excitation two-sampling mode and rapid lifetime determination algorithm contribute to transient fluorescence situations.

  16. Fluorescent GPCR ligands as new tools in pharmacology-update, years 2008-early 2014.

    Science.gov (United States)

    Kuder, Kamil J; Kieć-Kononowicz, Katarzyna

    2014-01-01

    The robust of fluorescent techniques to study the ligand-receptor interaction followed by rapidly developing fluorescence imaging techniques resulted in a burst of the novel fluorescent ligands development. Taking into consideration not only ligand's high affinity to the receptor, but also their fluorescent properties to visualize the interaction even in the single cell level, gives the researchers a strong impulse to investigate that field of GPCR ligands. Moreover, paying attention to the "non pharmacological" advantages of these ligands, as well as the techniques to be used, fluorescent ligands become commonly used pharmacological tools to study GPCRs. Herein we report on the results described in the literature since late 2007 in the field of the fluorescent GPCR small, non-peptide ligands according the receptor affinity, fluorophores that has been used to tag the molecules and their fluorescent properties as well as their application in GPCR research.

  17. Single-Molecule Total Internal Reflection Fluorescence Microscopy

    OpenAIRE

    Kudalkar, Emily M.; Davis, Trisha N; Asbury, Charles L.

    2016-01-01

    The advent of total internal reflection fluorescence (TIRF) microscopy has permitted visualization of biological events on an unprecedented scale: the single molecule level. Using TIRF, it is now possible to view complex biological interactions such as cargo transport by a single molecular motor or DNA replication in real-time. TIRF allows for visualization of single molecules by eliminating out-of-focus fluorescence and enhancing the signal-to-noise ratio. TIRF has been instrumental for stud...

  18. Light Sheet Fluorescence Microscopy

    Science.gov (United States)

    Santi, Peter A.

    2011-01-01

    Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy. LSFMs produce well-registered serial sections that are suitable for three-dimensional reconstruction of tissue structures. Because of a lack of a commercial LSFM microscope, numerous versions of light sheet microscopes have been constructed by different investigators. This review describes development of the technology, reviews existing devices, provides details of one LSFM device, and shows examples of images and three-dimensional reconstructions of tissues that were produced by LSFM. PMID:21339178

  19. Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling.

    Directory of Open Access Journals (Sweden)

    Mariette Barbier

    Full Text Available Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications.

  20. Development of an automated procedure for fluorescent DNA sequencing.

    Science.gov (United States)

    Wilson, R K; Chen, C; Avdalovic, N; Burns, J; Hood, L

    1990-04-01

    We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.

  1. Magnetic fluorescent lamp

    Science.gov (United States)

    Berman, S. M.; Richardson, R. W.

    1983-12-01

    The radiant emission of a mercury argon discharge in a fluorescent lamp assembly is enhanced by providing means for establishing a magnetic field with lines of force along the path of electron flow through the bulb of the lamp assembly, to provide zeeman splitting of the ultraviolet spectral line. Optimum results are obtained when the magnetic field strength causes a zeeman splitting of approximately 1.7 times the thermal line width.

  2. Delayed fluorescence in photosynthesis.

    Science.gov (United States)

    Goltsev, Vasilij; Zaharieva, Ivelina; Chernev, Petko; Strasser, Reto J

    2009-01-01

    Photosynthesis is a very efficient photochemical process. Nevertheless, plants emit some of the absorbed energy as light quanta. This luminescence is emitted, predominantly, by excited chlorophyll a molecules in the light-harvesting antenna, associated with Photosystem II (PS II) reaction centers. The emission that occurs before the utilization of the excitation energy in the primary photochemical reaction is called prompt fluorescence. Light emission can also be observed from repopulated excited chlorophylls as a result of recombination of the charge pairs. In this case, some time-dependent redox reactions occur before the excitation of the chlorophyll. This delays the light emission and provides the name for this phenomenon-delayed fluorescence (DF), or delayed light emission (DLE). The DF intensity is a decreasing polyphasic function of the time after illumination, which reflects the kinetics of electron transport reactions both on the (electron) donor and the (electron) acceptor sides of PS II. Two main experimental approaches are used for DF measurements: (a) recording of the DF decay in the dark after a single turnover flash or after continuous light excitation and (b) recording of the DF intensity during light adaptation of the photosynthesizing samples (induction curves), following a period of darkness. In this paper we review historical data on DF research and recent advances in the understanding of the relation between the delayed fluorescence and specific reactions in PS II. An experimental method for simultaneous recording of the induction transients of prompt and delayed chlorophyll fluorescence and decay curves of DF in the millisecond time domain is discussed.

  3. Magnetic fluorescent lamp

    Science.gov (United States)

    Berman, S.M.; Richardson R.W.

    1983-12-29

    The radiant emission of a mercury-argon discharge in a fluorescent lamp assembly is enhanced by providing means for establishing a magnetic field with lines of force along the path of electron flow through the bulb of the lamp assembly, to provide Zeeman splitting of the ultraviolet spectral line. Optimum results are obtained when the magnetic field strength causes a Zeeman splitting of approximately 1.7 times the thermal line width.

  4. Fluorescent quantification of melanin

    OpenAIRE

    Fernandes, Bruno Pacheco; Matamá, Maria Teresa; Guimarães, Diana Isabel Pereira; Gomes, Andreia; Cavaco-Paulo, Artur

    2016-01-01

    Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non-melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Theref...

  5. Fluorescent Lamp Replacement Study

    Science.gov (United States)

    2017-07-01

    recycling , and can be disposed safely in a landfill. (2) LEDs offer reduced maintenance costs and fewer bulb replacements, significantly reducing...housings, plastic grates, old wiring) and the new LED technology (cardboard packaging) were broken down and separated into the appropriate container for... recycling . Several fixtures, ballasts and energy efficient fluorescent bulbs that were determined to be in pristine condition were returned to ATC

  6. Techniques for sperm evaluation using fluorescent probes

    Directory of Open Access Journals (Sweden)

    Andrielle Thainar Mendes Cunha

    2015-12-01

    Full Text Available  A variety of laboratory tests were developed to obtain more reliable results of sperm evaluation and increase the accuracy of sperm fertility predictions. These tests detected damage of sperm specific compartments or organelles, which cannot be detected in routine sperm analysis. The use of fluorescent probes and detection using fluorescent microscopy or flow cytometry is an important tool but a more precise and accurate laboratory test is needed. Propidium iodide and 6-carboxyfluorescein diacetate are used for evaluations of plasmatic membrane integrity. Fluorescein isothiocyanate, associated with conjugated lecithin Psium sativum or Arachis hypogaea, are used for evaluations of acrosome integrity. Two probes, MitoTracker or Rhodamine123, are generally used to measure the absence or presence of mitochondrial potential. However, a better option is 5,5’; 6,6’ - tetrachloro - 1,1’; 3,3’ -tetraetilbenzimidazolil-carbocyanine (JC-1 dye, which assesses not only the presence of mitochondrial potential and distinguished spermatozoa with poorly and highly functional mitochondria. Two techniques, TUNEL or COMETA, and the Acridine Orange Test (AOT dye are used to evaluate chromatin integrity. A fluorescence technique based on chlortetracycline (CTC or Merocyanine 540 is used to estimate whether sperm pass by or through the capacitation process. This review focuses on the fluorescent probes that are most widely used to evaluate plasma membrane integrity, capacitation, acrosome integrity, chromatin integrity and mitochondrial potential.

  7. Monitoring biological aerosols using UV fluorescence

    Science.gov (United States)

    Eversole, Jay D.; Roselle, Dominick; Seaver, Mark E.

    1999-01-01

    An apparatus has been designed and constructed to continuously monitor the number density, size, and fluorescent emission of ambient aerosol particles. The application of fluorescence to biological particles suspended in the atmosphere requires laser excitation in the UV spectral region. In this study, a Nd:YAG laser is quadrupled to provide a 266 nm wavelength to excite emission from single micrometer-sized particles in air. Fluorescent emission is used to continuously identify aerosol particles of biological origin. For calibration, biological samples of Bacillus subtilis spores and vegetative cells, Esherichia coli, Bacillus thuringiensis and Erwinia herbicola vegetative cells were prepared as suspensions in water and nebulized to produce aerosols. Detection of single aerosol particles, provides elastic scattering response as well as fluorescent emission in two spectral bands simultaneously. Our efforts have focuses on empirical characterization of the emission and scattering characteristics of various bacterial samples to determine the feasibility of optical discrimination between different cell types. Preliminary spectroscopic evidence suggest that different samples can be distinguished as separate bio-aerosol groups. In addition to controlled sample results, we will also discuss the most recent result on the effectiveness of detection outdoor releases and variations in environmental backgrounds.

  8. Charge transfer in green fluorescent protein.

    Science.gov (United States)

    van Thor, Jasper J; Sage, J Timothy

    2006-06-01

    Charge transfer reactions that contribute to the photoreactions of the wild type green fluorescent protein (GFP) do not occur in the isolated p-hydroxybenzylidene-imidazolidinone chromophore, demonstrating the role of the protein environment. The high quantum efficiency of the fluorescence photocycle that includes excited state proton transfer and the suppression of non-radiative pathways by the protein environment have been correlated with structural dynamics in the chromophore environment. A low quantum efficiency competing phototransformation reaction of GFP is accompanied by both proton and electron transfer, and closely mimics the charge redistribution that is occurring in the fluorescence photocycle. The protein response to this destabilising event has been demonstrated by cryo-trapping of early products in the reaction pathway and is found to be strong even at 100 K, including displacements of chromophore, protein, solvent and a photogenerated CO2 molecule derived from the decarboxylated Glu 222 side chain. We discuss the ramifications of the observation of strong conformational perturbations below the protein dynamical transition at approximately 200 K, in view of low temperature work on other light sensitive proteins such as myoglobin and bacteriorhodopsin. The proton and electron transfer in the phototransformation pathway mimics the proton and charge transfer which occurs during the fluorescence cycle, which leads to common structural responses in both photoreactions as shown by ultrafast spectroscopy. We review and discuss literature on light-induced and thermal charge transfer events, focusing on recent findings addressing conformational dynamics and implications for thermodynamic properties.

  9. Uptake of Fluorescent Gentamicin by Peripheral Vestibular Cells after Systemic Administration

    Science.gov (United States)

    Liu, Jianping; Kachelmeier, Allan; Dai, Chunfu; Li, Hongzhe; Steyger, Peter S.

    2015-01-01

    Objective In addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours. Results Low intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs’ sensory cells. Control vestibular tissues exposed to Dulbecco’s phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence. Conclusions All peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may

  10. Novel Application of Fluorescence Lifetime and Fluorescence Microscopy Enables Quantitative Access to Subcellular Dynamics in Plant Cells

    Science.gov (United States)

    Elgass, Kirstin; Caesar, Katharina; Schleifenbaum, Frank; Stierhof, York-Dieter; Meixner, Alfred J.; Harter, Klaus

    2009-01-01

    Background Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences. Principal Findings Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes. Significance One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy. PMID:19492078

  11. Blue fluorescent cGMP sensor for multiparameter fluorescence imaging.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Cyclic GMP (cGMP regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP and Ca(2+. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP. We cotransfected them and loaded a red fluorescent probe for Ca(2+ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca(2+, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks.

  12. New trends in fluorescence in situ hybridization for identification and functional analyses of microbes.

    Science.gov (United States)

    Wagner, Michael; Haider, Susanne

    2012-02-01

    Fluorescence in situ hybridization (FISH) has become an indispensable tool for rapid and direct single-cell identification of microbes by detecting signature regions in their rRNA molecules. Recent advances in this field include new web-based tools for assisting probe design and optimization of experimental conditions, easy-to-implement signal amplification strategies, innovative multiplexing approaches, and the combination of FISH with transmission electron microscopy or extracellular staining techniques. Further emerging developments focus on sorting FISH-identified cells for subsequent single-cell genomics and on the direct detection of specific genes within single microbial cells by advanced FISH techniques employing various strategies for massive signal amplification. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Rapid Active Sampling Package

    Science.gov (United States)

    Peters, Gregory

    2010-01-01

    A field-deployable, battery-powered Rapid Active Sampling Package (RASP), originally designed for sampling strong materials during lunar and planetary missions, shows strong utility for terrestrial geological use. The technology is proving to be simple and effective for sampling and processing materials of strength. Although this originally was intended for planetary and lunar applications, the RASP is very useful as a powered hand tool for geologists and the mining industry to quickly sample and process rocks in the field on Earth. The RASP allows geologists to surgically acquire samples of rock for later laboratory analysis. This tool, roughly the size of a wrench, allows the user to cut away swaths of weathering rinds, revealing pristine rock surfaces for observation and subsequent sampling with the same tool. RASPing deeper (.3.5 cm) exposes single rock strata in-situ. Where a geologist fs hammer can only expose unweathered layers of rock, the RASP can do the same, and then has the added ability to capture and process samples into powder with particle sizes less than 150 microns, making it easier for XRD/XRF (x-ray diffraction/x-ray fluorescence). The tool uses a rotating rasp bit (or two counter-rotating bits) that resides inside or above the catch container. The container has an open slot to allow the bit to extend outside the container and to allow cuttings to enter and be caught. When the slot and rasp bit are in contact with a substrate, the bit is plunged into it in a matter of seconds to reach pristine rock. A user in the field may sample a rock multiple times at multiple depths in minutes, instead of having to cut out huge, heavy rock samples for transport back to a lab for analysis. Because of the speed and accuracy of the RASP, hundreds of samples can be taken in one day. RASP-acquired samples are small and easily carried. A user can characterize more area in less time than by using conventional methods. The field-deployable RASP used a Ni

  14. Line scan--structured illumination microscopy super-resolution imaging in thick fluorescent samples.

    Science.gov (United States)

    Mandula, Ondrej; Kielhorn, Martin; Wicker, Kai; Krampert, Gerhard; Kleppe, Ingo; Heintzmann, Rainer

    2012-10-22

    Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of-focus fluorescence background deteriorates the illumination pattern and the reconstructed images suffer from influence of noise. We present a combination of structured illumination microscopy with line scanning. This technique reduces the out-of-focus fluorescence background, which improves the modulation and the quality of the illumination pattern and therefore facilitates the reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen's inner structure.

  15. Confocal reader for biochip screening and fluorescence microscopy.

    Science.gov (United States)

    Ruckstuhl, Thomas; Walser, Andreas; Verdes, Dorinel; Seeger, Stefan

    2005-03-15

    We developed a fluorescence reader for the sensitive detection of surface-generated fluorescence. The system is applicable for high resolution imaging as well as for the readout of large biochips. The surface of a microscope coverslip is scanned with a laser beam focused to a waist diameter of 500 nm (FWHM) by means of a single aspheric lens. Scanning large areas with a focused beam usually evokes the need of automatic control elements to adjust the laser spot to the designated position at the surface. Due to the special design of the reader, the focus keeps at the plane of the surface even when scanning large areas, obviating the requirement of any real time control. Thus the instrument is straightforward and inexpensive. Nevertheless it features a high sensitivity and high optical resolution. The versatility of the instrument is demonstrated by imaging cells and reading out a DNA-chip. The excellent sensitivity is shown by detecting single fluorescently labeled antibodies.

  16. FOCUS: Sustainable Mathematics Successes

    Science.gov (United States)

    Mireles, Selina V.; Acee, Taylor W.; Gerber, Lindsey N.

    2014-01-01

    The FOCUS (Fundamentals of Conceptual Understanding and Success) Co-Requisite Model Intervention (FOCUS Intervention) for College Algebra was developed as part of the Developmental Education Demonstration Projects (DEDP) in Texas. The program was designed to use multiple services, courses, and best practices to support student completion of a…

  17. Microfabricated particle focusing device

    Energy Technology Data Exchange (ETDEWEB)

    Ravula, Surendra K.; Arrington, Christian L.; Sigman, Jennifer K.; Branch, Darren W.; Brener, Igal; Clem, Paul G.; James, Conrad D.; Hill, Martyn; Boltryk, Rosemary June

    2013-04-23

    A microfabricated particle focusing device comprises an acoustic portion to preconcentrate particles over large spatial dimensions into particle streams and a dielectrophoretic portion for finer particle focusing into single-file columns. The device can be used for high throughput assays for which it is necessary to isolate and investigate small bundles of particles and single particles.

  18. Space Focus Lead Report

    Energy Technology Data Exchange (ETDEWEB)

    Reeves, Geoffrey D. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-08-10

    The Space Focus team is tasked with the definition of the Space Focused Science Topics, and with the review and ranking of the CSES proposals received in all the program areas. This is achieved by dedicated meetings or a series of informal discussions and/or e-mail reviews.

  19. Hyperspectral small animal fluorescence imaging: spectral selection imaging

    Science.gov (United States)

    Leavesley, Silas; Jiang, Yanan; Patsekin, Valery; Hall, Heidi; Vizard, Douglas; Robinson, J. Paul

    2008-02-01

    Molecular imaging is a rapidly growing area of research, fueled by needs in pharmaceutical drug-development for methods for high-throughput screening, pre-clinical and clinical screening for visualizing tumor growth and drug targeting, and a growing number of applications in the molecular biology fields. Small animal fluorescence imaging employs fluorescent probes to target molecular events in vivo, with a large number of molecular targeting probes readily available. The ease at which new targeting compounds can be developed, the short acquisition times, and the low cost (compared to microCT, MRI, or PET) makes fluorescence imaging attractive. However, small animal fluorescence imaging suffers from high optical scattering, absorption, and autofluorescence. Much of these problems can be overcome through multispectral imaging techniques, which collect images at different fluorescence emission wavelengths, followed by analysis, classification, and spectral deconvolution methods to isolate signals from fluorescence emission. We present an alternative to the current method, using hyperspectral excitation scanning (spectral selection imaging), a technique that allows excitation at any wavelength in the visible and near-infrared wavelength range. In many cases, excitation imaging may be more effective at identifying specific fluorescence signals because of the higher complexity of the fluorophore excitation spectrum. Because the excitation is filtered and not the emission, the resolution limit and image shift imposed by acousto-optic tunable filters have no effect on imager performance. We will discuss design of the imager, optimizing the imager for use in small animal fluorescence imaging, and application of spectral analysis and classification methods for identifying specific fluorescence signals.

  20. Structured illumination microscopy using photoswitchable fluorescent proteins

    Science.gov (United States)

    Hirvonen, Liisa; Mandula, Ondrej; Wicker, Kai; Heintzmann, Rainer

    2008-02-01

    In fluorescence microscopy the lateral resolution is limited to about 200 nm because of diffraction. Resolution improvement by a factor of two can be achieved using structured illumination, where a ine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Further resolution improvement can be achieved by saturating the transitions involved in fluorescence emission. Recently discovered photoswitchable proteins undergo transitions that are saturable at low illumination intensity. Combining this concept with structured illumination, theoretically unlimited resolution can be achieved, where the smallest resolvable distance will be determined by signal-to-noise ratio. This work focuses on the use of the photoswitchable protein Dronpa with structured illumination to achieve nanometre scale resolution in fixed cells.

  1. Intracellular magnesium detection by fluorescent indicators.

    Science.gov (United States)

    Trapani, Valentina; Schweigel-Röntgen, Monika; Cittadini, Achille; Wolf, Federica I

    2012-01-01

    Magnesium is essential for a wide variety of biochemical reactions and physiological functions, but its regulatory mechanisms (both at the cellular and at the systemic level) are still poorly characterized. Not least among the reasons for this gap are the technical difficulties in sensing minor changes occurring over a high background concentration. Specific fluorescent indicators are highly sensitive tools for dynamic evaluation of intracellular magnesium concentration. We herein discuss the main criteria to consider when choosing a magnesium-specific fluorescent indicator and provide examples among commercial as well as developmental sensors. We focus on spectrofluorimetric approaches to quantify Mg(2+) concentration in cell or mitochondria suspensions, and on imaging techniques to detect intracellular magnesium distribution and fluxes by live microscopy, reporting a detailed description of standard protocols for each method. The general guidelines we provide should be applicable to specific issues by any researcher in the field. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Fluorescent temperature sensor

    Science.gov (United States)

    Baker, Gary A [Los Alamos, NM; Baker, Sheila N [Los Alamos, NM; McCleskey, T Mark [Los Alamos, NM

    2009-03-03

    The present invention is a fluorescent temperature sensor or optical thermometer. The sensor includes a solution of 1,3-bis(1-pyrenyl)propane within a 1-butyl-1-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ionic liquid solvent. The 1,3-bis(1-pyrenyl)propane remains unassociated when in the ground state while in solution. When subjected to UV light, an excited state is produced that exists in equilibrium with an excimer. The position of the equilibrium between the two excited states is temperature dependent.

  3. Fluorescent Europium Chelate Stain

    Science.gov (United States)

    Scaff, W. L.; Dyer, D. L.; Mori, K.

    1969-01-01

    The europium chelate of 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (thenoyl-trifluoroacetone; TTA) is firmly bound to microorganisms. It fluoresces brightly at 613 nm with activation at 340 nm. Cells may be stained with 10−3m chelate in 50% ethyl alcohol, followed by washing with 50% ethyl alcohol. Equal or better stains are produced with 10−3m aqueous europium salt, water wash, and 10−2m aqueous TTA. A noncomplexing buffer should be used to maintain the pH at 6.5 to 6.8. Images PMID:4181107

  4. 10 CFR Appendix W to Subpart B of... - Uniform Test Method for Measuring the Energy Consumption of Medium Base Compact Fluorescent Lamps

    Science.gov (United States)

    2010-01-01

    ... of Medium Base Compact Fluorescent Lamps W Appendix W to Subpart B of Part 430 Energy DEPARTMENT OF... Consumption of Medium Base Compact Fluorescent Lamps 1. Scope: This appendix covers the test requirements used... rated life, rapid cycle stress, and lamp life of medium base compact fluorescent lamps. 2. Definitions...

  5. Development of a fluorescent cryocooler

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, B.C.; Buchwald, M.I.; Epstein, R.I.; Gosnell, T.R.; Mungan, C.E.

    1995-10-01

    Recent work at Los Alamos National Laboratory has demonstrated the physical principles for a new type of solid-state cryocooler based on anti-Stokes fluorescence. Design studies indicate that a vibration-free, low-mass ``fluorescent cryocooler`` could operate for years with efficiencies and cooling powers comparable to current commercial systems. This paper presents concepts for a fluorescent cryocooler, design considerations and expected performance.

  6. Final focus test beam

    Energy Technology Data Exchange (ETDEWEB)

    1991-03-01

    This report discusses the following: the Final Focus Test Beam Project; optical design; magnets; instrumentation; magnetic measurement and BPM calibration; mechanical alignment and stabilization; vacuum system; power supplies; control system; radiation shielding and personnel protection; infrastructure; and administration.

  7. Final focus nomenclature

    Energy Technology Data Exchange (ETDEWEB)

    Erickson, R.

    1986-08-08

    The formal names and common names for all devices in the final focus system of the SLC are listed. The formal names consist of a device type designator, microprocessor designator, and a four-digit unit number. (LEW)

  8. Focus group discussions

    CERN Document Server

    Hennink, Monique M

    2014-01-01

    The Understanding Research series focuses on the process of writing up social research. The series is broken down into three categories: Understanding Statistics, Understanding Measurement, and Understanding Qualitative Research. The books provide researchers with guides to understanding, writing, and evaluating social research. Each volume demonstrates how research should be represented, including how to write up the methodology as well as the research findings. Each volume also reviews how to appropriately evaluate published research. Focus Group Discussions addresses the challenges associated with conducting and writing focus group research. It provides detailed guidance on the practical and theoretical considerations in conducting focus group discussions including: designing the discussion guide, recruiting participants, training a field team, moderating techniques and ethical considerations. Monique Hennink describes how a methodology section is read and evaluated by others, such as journal reviewers or ...

  9. Rapidly rotating red giants

    Science.gov (United States)

    Gehan, Charlotte; Mosser, Benoît; Michel, Eric

    2017-10-01

    Stellar oscillations give seismic information on the internal properties of stars. Red giants are targets of interest since they present mixed modes, wich behave as pressure modes in the convective envelope and as gravity modes in the radiative core. Mixed modes thus directly probe red giant cores, and allow in particular the study of their mean core rotation. The high-quality data obtained by CoRoT and Kepler satellites represent an unprecedented perspective to obtain thousands of measurements of red giant core rotation, in order to improve our understanding of stellar physics in deep stellar interiors. We developed an automated method to obtain such core rotation measurements and validated it for stars on the red giant branch. In this work, we particularly focus on the specific application of this method to red giants having a rapid core rotation. They show complex spectra where it is tricky to disentangle rotational splittings from mixed-mode period spacings. We demonstrate that the method based on the identification of mode crossings is precise and efficient. The determination of the mean core rotation directly derives from the precise measurement of the asymptotic period spacing ΔΠ1 and of the frequency at which the crossing of the rotational components is observed.

  10. Simultaneous single molecule atomic force and fluorescence lifetime imaging

    Science.gov (United States)

    Schulz, Olaf; Koberling, Felix; Walters, Deron; Koenig, Marcelle; Viani, Jacob; Ros, Robert

    2010-02-01

    The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit, thus enabling the identification of different fluorescence labels within a macromolecular complex. We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope. The ability to move the sample and objective independently allows for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting (TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence lifetime imaging of YOYO-1 stained lambda-DNA samples and we showed silicon tip induced single molecule quenching on organic fluorophores.

  11. Scattered and Fluorescent Photon Track Reconstruction in a Biological Tissue

    Directory of Open Access Journals (Sweden)

    Maria N. Kholodtsova

    2014-01-01

    Full Text Available Appropriate analysis of biological tissue deep regions is important for tumor targeting. This paper is concentrated on photons’ paths analysis in such biotissue as brain, because optical probing depth of fluorescent and excitation radiation differs. A method for photon track reconstruction was developed. Images were captured focusing on the transparent wall close and parallel to the source fibres, placed in brain tissue phantoms. The images were processed to reconstruct the photons most probable paths between two fibres. Results were compared with Monte Carlo simulations and diffusion approximation of the radiative transfer equation. It was shown that the excitation radiation optical probing depth is twice more than for the fluorescent photons. The way of fluorescent radiation spreading was discussed. Because of fluorescent and excitation radiation spreads in different ways, and the effective anisotropy factor, geff, was proposed for fluorescent radiation. For the brain tissue phantoms it were found to be 0.62±0.05 and 0.66±0.05 for the irradiation wavelengths 532 nm and 632.8 nm, respectively. These calculations give more accurate information about the tumor location in biotissue. Reconstruction of photon paths allows fluorescent and excitation probing depths determination. The geff can be used as simplified parameter for calculations of fluorescence probing depth.

  12. Recommendations for fluorescence instrument qualification: the new ASTM Standard Guide.

    Science.gov (United States)

    DeRose, Paul C; Resch-Genger, Ute

    2010-03-01

    Aimed at improving quality assurance and quantitation for modern fluorescence techniques, ASTM International (ASTM) is about to release a Standard Guide for Fluorescence, reviewed here. The guide's main focus is on steady state fluorometry, for which available standards and instrument characterization procedures are discussed along with their purpose, suitability, and general instructions for use. These include the most relevant instrument properties needing qualification, such as linearity and spectral responsivity of the detection system, spectral irradiance reaching the sample, wavelength accuracy, sensitivity or limit of detection for an analyte, and day-to-day performance verification. With proper consideration of method-inherent requirements and limitations, many of these procedures and standards can be adapted to other fluorescence techniques. In addition, procedures for the determination of other relevant fluorometric quantities including fluorescence quantum yields and fluorescence lifetimes are briefly introduced. The guide is a clear and concise reference geared for users of fluorescence instrumentation at all levels of experience and is intended to aid in the ongoing standardization of fluorescence measurements.

  13. Focus Group Guide

    Science.gov (United States)

    2017-07-01

    clarification.  Determine demographics and number needed for focus groups (all male, all female, mixed gender , by rank, by section, etc.; 8–15...members (based on section, rank, race, gender , etc.) view an issue? Are the concerns focused in a single section or do they seem to be unit-wide...Sexual Harassment (C) Sex Harassment Retaliation (D) Discrimination - Sex (E) Discrimination - Race (F) Discrimination - Disability (G

  14. High harmonics focusing undulator

    Energy Technology Data Exchange (ETDEWEB)

    Varfolomeev, A.A.; Hairetdinov, A.H.; Smirnov, A.V.; Khlebnikov, A.S. [Kurchatov Institute, Moscow (Russian Federation)

    1995-12-31

    It was shown in our previous work that there exist a possibility to enhance significantly the {open_quote}natural{close_quote} focusing properties of the hybrid undulator. Here we analyze the actual undulator configurations which could provide such field structure. Numerical simulations using 2D code PANDIRA were carried out and the enhanced focusing properties of the undulator were demonstrated. The obtained results provide the solution for the beam transport in a very long (short wavelength) undulator schemes.

  15. Fluorescent nanodiamonds for ultrasensitive detection

    Science.gov (United States)

    Kimball, Joseph; Shumilov, Dmytro; Maliwa, Badri; Zerda, T. W.; Rout, Bibhu; Fudala, Rafal; Raut, Sangram; Gryczynski, Ignacy; Simanek, Eric; Borejdo, Julian; Rich, Ryan; Akopova, Irina; Gryczynski, Zygmunt

    2014-03-01

    Fluorescent nanodiamonds (NDs) are new and emerging nanomaterials that have potential to be used as fluorescence imaging agents and also as a highly versatile platform for the controlled functionalization and delivery of a wide spectrum of therapeutic agents. We will utilize two experimental methods, TIRF, a relatively simple method based on total internal reflection fluorescence and SPRF, fluorescence enhanced by resonance coupling with surface plasmons. We estimate that the SPRF method will be 100 times sensitive than currently available similar detectors based on detectors. The ultimate goal of this research is to develop microarray platforms that could be used for sensitive, fast and inexpensive gene sequencing and protein detection.

  16. Focused critical care echocardiography.

    Science.gov (United States)

    Oren-Grinberg, Achikam; Talmor, Daniel; Brown, Samuel M

    2013-11-01

    Portable ultrasound is now used routinely in many ICUs for various clinical applications. Echocardiography performed by noncardiologists, both transesophageal and transthoracic, has evolved to broad applications in diagnosis, monitoring, and management of critically ill patients. This review provides a current update on focused critical care echocardiography for the management of critically ill patients. Source data were obtained from a PubMed search of the medical literature, including the PubMed "related articles" search methodology. Although studies demonstrating improved clinical outcomes for critically ill patients managed by focused critical care echocardiography are generally lacking, there is evidence to suggest that some intermediate outcomes are improved. Furthermore, noncardiologists can learn focused critical care echocardiography and adequately interpret the information obtained. Noncardiologists can also successfully incorporate focused critical care echocardiography into advanced cardiopulmonary life support. Formal training and proctoring are important for safe application of focused critical care echocardiography in clinical practice. Further outcomes-based research is urgently needed to evaluate the efficacy of focused critical care echocardiography.

  17. Plutonium focus area

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-08-01

    To ensure research and development programs focus on the most pressing environmental restoration and waste management problems at the U.S. Department of Energy (DOE), the Assistant Secretary for the Office of Environmental Management (EM) established a working group in August 1993 to implement a new approach to research and technology development. As part of this new approach, EM developed a management structure and principles that led to the creation of specific Focus Areas. These organizations were designed to focus the scientific and technical talent throughout DOE and the national scientific community on the major environmental restoration and waste management problems facing DOE. The Focus Area approach provides the framework for intersite cooperation and leveraging of resources on common problems. After the original establishment of five major Focus Areas within the Office of Technology Development (EM-50, now called the Office of Science and Technology), the Nuclear Materials Stabilization Task Group (EM-66) followed the structure already in place in EM-50 and chartered the Plutonium Focus Area (PFA). The following information outlines the scope and mission of the EM, EM-60, and EM-66 organizations as related to the PFA organizational structure.

  18. Wide Field-of-View Fluorescence Imaging of Coral Reefs

    Science.gov (United States)

    Treibitz, Tali; Neal, Benjamin P.; Kline, David I.; Beijbom, Oscar; Roberts, Paul L. D.; Mitchell, B. Greg; Kriegman, David

    2015-01-01

    Coral reefs globally are declining rapidly because of both local and global stressors. Improved monitoring tools are urgently needed to understand the changes that are occurring at appropriate temporal and spatial scales. Coral fluorescence imaging tools have the potential to improve both ecological and physiological assessments. Although fluorescence imaging is regularly used for laboratory studies of corals, it has not yet been used for large-scale in situ assessments. Current obstacles to effective underwater fluorescence surveying include limited field-of-view due to low camera sensitivity, the need for nighttime deployment because of ambient light contamination, and the need for custom multispectral narrow band imaging systems to separate the signal into meaningful fluorescence bands. Here we describe the Fluorescence Imaging System (FluorIS), based on a consumer camera modified for greatly increased sensitivity to chlorophyll-a fluorescence, and we show high spectral correlation between acquired images and in situ spectrometer measurements. This system greatly facilitates underwater wide field-of-view fluorophore surveying during both night and day, and potentially enables improvements in semi-automated segmentation of live corals in coral reef photographs and juvenile coral surveys. PMID:25582836

  19. Replication-competent fluorescent-expressing influenza B virus.

    Science.gov (United States)

    Nogales, Aitor; Rodríguez-Sánchez, Irene; Monte, Kristen; Lenschow, Deborah J; Perez, Daniel R; Martínez-Sobrido, Luis

    2016-02-02

    Influenza B viruses (IBVs) cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated morbidity and mortality. Studying influenza viruses requires the use of secondary methodologies to identify virus-infected cells. To this end, replication-competent influenza A viruses (IAVs) expressing easily traceable fluorescent proteins have been recently developed. In contrast, similar approaches for IBV are mostly lacking. In this report, we describe the generation and characterization of replication-competent influenza B/Brisbane/60/2008 viruses expressing fluorescent mCherry or GFP fused to the C-terminal of the viral non-structural 1 (NS1) protein. Fluorescent-expressing IBVs display similar growth kinetics and plaque phenotype to wild-type IBV, while fluorescent protein expression allows for the easy identification of virus-infected cells. Without the need of secondary approaches to monitor viral infection, fluorescent-expressing IBVs represent an ideal approach to study the biology of IBV and an excellent platform for the rapid identification and characterization of antiviral therapeutics or neutralizing antibodies using high-throughput screening approaches. Lastly, fluorescent-expressing IBVs can be combined with the recently described reporter-expressing IAVs for the identification of novel therapeutics to combat these two important human respiratory pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Fundamentals of fluorescence microscopy exploring life with light

    CERN Document Server

    Mondal, Partha Pratim

    2014-01-01

    This book starts at an introductory level and leads reader to the most advanced developments in fluorescence imaging and super-resolution techniques that have enabled the emergence of new disciplines such as nanobioimaging, multiphoton microscopy, photodynamic therapy, nanometrology and nanosensors. The interdisciplinary subject of fluorescence microscopy and imaging requires complete knowledge of imaging optics and molecular physics. So, this book approaches the subject by introducing optical imaging concepts before going deep into the advanced imaging systems and their applications. Molecular orbital theory forms the basis for understanding fluorescent molecules and thereby facilitates complete explanation of light-matter interaction at the geometrical focus. The two disciplines have some overlap since light controls the states of molecules and conversely, molecular states control the emitted light. These two mechanisms together determine essential fluorescence  factors and phenomena such as, molecular cro...

  1. Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

    Directory of Open Access Journals (Sweden)

    Angel Orte

    2012-07-01

    Full Text Available Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS, an advanced modification of conventional fluorescence correlation spectroscopy (FCS that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.

  2. Processes and Technologies for the Recycling of Spent Fluorescent Lamps

    Directory of Open Access Journals (Sweden)

    Kujawski Wojciech

    2014-09-01

    Full Text Available The growing industrial application of rare earth metals led to great interest in the new technologies for the recycling and recovery of REEs from diverse sources. This work reviews the various methods for the recycling of spent fluorescent lamps. The spent fluorescent lamps are potential source of important rare earth elements (REEs such as: yttrium, terbium, europium, lanthanum and cerium. The characteristics of REEs properties and construction of typical fl uorescent lamps is described. The work compares also current technologies which can be utilized for an efficient recovery of REEs from phosphors powders coming from spent fluorescent lamps. The work is especially focused on the hydrometallurgical and pyrometallurgical processes. It was concluded that hydrometallurgical processes are especially useful for the recovery of REEs from spent fluorescent lamps. Moreover, the methods used for recycling of REEs are identical or very similar to those utilized for the raw ores processing.

  3. Global analysis of fluorescence decays to probe the internal dynamics of fluorescently labeled macromolecules.

    Science.gov (United States)

    Duhamel, Jean

    2014-03-11

    The aim of this review is to introduce the reader first to the mathematical complexity associated with the analysis of fluorescence decays acquired with solutions of macromolecules labeled with a fluorophore and its quencher that are capable of interacting with each other via photophysical processes within the macromolecular volume, second to the experimental and mathematical approaches that have been proposed over the years to handle this mathematical complexity, and third to the information that one can expect to retrieve with respect to the internal dynamics of such fluorescently labeled macromolecules. In my view, the ideal fluorophore-quencher pair to use in studying the internal dynamics of fluorescently labeled macromolecules would involve a long-lived fluorophore, a fluorophore and a quencher that do not undergo energy migration, and a photophysical process that results in a change in fluorophore emission upon contact between the excited fluorophore and quencher. Pyrene, with its ability to form an excimer on contact between excited-state and ground-state species, happens to possess all of these properties. Although the concepts described in this review apply to any fluorophore and quencher pair sharing pyrene's exceptional photophysical properties, this review focuses on the study of pyrene-labeled macromolecules that have been characterized in great detail over the past 40 years and presents the main models that are being used today to analyze the fluorescence decays of pyrene-labeled macromolecules reliably. These models are based on Birks' scheme, the DMD model, the fluorescence blob model, and the model free analysis. The review also provides a step-by-step protocol that should enable the noneducated user to achieve a successful decay analysis exempt of artifacts. Finally, some examples of studies of pyrene-labeled macromolecules are also presented to illustrate the different types of information that can be retrieved from these fluorescence decay

  4. Fluorescent Biosensors Based on Single-Molecule Counting.

    Science.gov (United States)

    Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

    2016-09-20

    Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the

  5. Synthesis, characterisation and spectroscopic analysis of fluorescent dyes for applications-based chemistry

    OpenAIRE

    Higginbotham, Heather Fay

    2017-01-01

    This research project investigates the synthesis and spectroscopic analysis of organic and inorganic fluorescent and luminescent materials for future use in applications-based chemistry. This work specifically focuses upon fluorescent dyes with tunable emission properties designed for the investigation of applications less commonly explored in literature. The use of advanced spectroscopic and single particle fluorescence techniques is also an underlying theme of each chapter, used to elucidat...

  6. Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry

    Science.gov (United States)

    Houston, Jessica P.; Naivar, Mark A.; Freyer, James P.

    2010-01-01

    Frequency-domain flow cytometry techniques are combined with modifications to the digital signal processing capabilities of the Open Reconfigurable Cytometric Acquisition System (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency modulated detector signals, implementing Fourier analysis programming with ORCAS’ digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5–25 ns simulated lifetime), pulse widths ranging from 2 to 15 µs, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142° to 1.6°. The lowest coefficients of variation (flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a radiofrequency modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to ~98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will both allow better dissemination of this technology and better exploit the traditionally underutilized parameter of fluorescence lifetime

  7. Antiproton focusing horn

    CERN Multimedia

    1992-01-01

    This focusing horn was developed in 1992 by Remo Maccaferri, Jean Claude Schnuriger and Lubrano di Scampamorte and is still operating in the AD complex at CERN (as of 2017). This device could pulse at 400 KA (160 KA for the previous version). This enabled an antiproton collection ten times better than the old one. Firstly, protons were accelerated to an energy of 26 GeV/c and ejected onto a metal target. From the spray of emerging particles, the magnetic horn picked out 3.6 GeV antiprotons for injection into the AA through a wide-aperture focusing quadrupole magnet. For a million protons hitting the target, ten antiprotons were captured, 'cooled' and accumulated. It took 3 days to make a beam of 3 x 10^11 - three hundred thousand million - antiprotons. Originally magnetic focusing horns were developed by Simon van der Meer - see for example object AC-022 in this database.

  8. Decontamination & decommissioning focus area

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-08-01

    In January 1994, the US Department of Energy Office of Environmental Management (DOE EM) formally introduced its new approach to managing DOE`s environmental research and technology development activities. The goal of the new approach is to conduct research and development in critical areas of interest to DOE, utilizing the best talent in the Department and in the national science community. To facilitate this solutions-oriented approach, the Office of Science and Technology (EM-50, formerly the Office of Technology Development) formed five Focus AReas to stimulate the required basic research, development, and demonstration efforts to seek new, innovative cleanup methods. In February 1995, EM-50 selected the DOE Morgantown Energy Technology Center (METC) to lead implementation of one of these Focus Areas: the Decontamination and Decommissioning (D & D) Focus Area.

  9. The focus factor

    DEFF Research Database (Denmark)

    Nicolaisen, Jeppe; Frandsen, Tove Faber

    2015-01-01

    Introduction. We present a new bibliometric indicator to measure journal specialisation over time, named the focus factor. This new indicator is based on bibliographic coupling and counts the percentage of re-citations given in subsequent years. Method. The applicability of the new indicator....... To validate re-citations as caused by specialisation, other possible causes were measured and correlated (obsolescence, journal self-citations and number of references). Results. The results indicate that the focus factor is capable of distinguishing between general and specialised journals and thus...... effectively measures the intended phenomenon (i.e., journal specialisation). Only weak correlations were found between journal re-citations and obsolescence, journal self-citations, and number of references. Conclusions. The focus factor successfully measures journal specialisation over time. Measures based...

  10. Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells

    Science.gov (United States)

    Scott, Brandon L.; Hoppe, Adam D.

    2015-01-01

    Powerful new methods have extended FRET microscopy to the imaging of three or more interacting proteins inside living cells. Here, we compared widely available fluorescent proteins to find the best trio for 3-Way FRET imaging. We focused on readily available cyan, yellow, and red proteins that have high quantum yields, large extinction coefficients and good photostability, which defined these candidate proteins: CyPet/mTFP1/mTurqoise2, mCitrine/YPet, and TagRFP/TagRFPt/mRuby2/mCherry. By taking advantage of the high structural similarity across the fluorescent proteins, we generated structurally similar, but photophysically distinct donor/acceptor and triple fluorophore fusion proteins and measured their FRET efficiencies inside living cells. Surprisingly, their published photophysical parameters and calculated Förster distances did not predict the best combinations of FPs. Using cycloheximide to inhibit protein synthesis, we found that the different FP maturation rates had a strong effect on the FRET efficiency. This effect was pronounced when comparing rapidly maturing yellow and slowly maturing red FPs. We found that red FPs with inferior photophysics gave superior FRET efficiencies because of faster maturation rates. Based on combined metrics for the FRET efficiency, fluorophore photophysics and fluorophore maturation we determined that Turqoise2, YPet and Cherry were the best available FPs for live cell 3-Way FRET measurements. PMID:26130463

  11. Deliberative Discussion Focus Groups.

    Science.gov (United States)

    Rothwell, Erin; Anderson, Rebecca; Botkin, Jeffrey R

    2016-05-01

    This article discusses a new approach for the conduct of focus groups in health research. Identifying ways to educate and inform participants about the topic of interest prior to the focus group discussion can promote more quality data from informed opinions. Data on this deliberative discussion approach are provided from research within three federally funded studies. As healthcare continues to improve from scientific and technological advancements, educating the research participants prior to data collection about these complexities is essential to gather quality data. © The Author(s) 2015.

  12. Assessing Photosynthesis by Fluorescence Imaging

    Science.gov (United States)

    Saura, Pedro; Quiles, Maria Jose

    2011-01-01

    This practical paper describes a novel fluorescence imaging experiment to study the three processes of photochemistry, fluorescence and thermal energy dissipation, which compete during the dissipation of excitation energy in photosynthesis. The technique represents a non-invasive tool for revealing and understanding the spatial heterogeneity in…

  13. Visible fluorescent proteins for FRET

    NARCIS (Netherlands)

    Kremers, G.J.; Goedhart, J.; Gadella, T.W.J.

    2009-01-01

    This chapter discusses the use of Visible fluorescent proteins (VFPs) for FRET studies, a comprehensive table with Förster radii of VFP pairs is presented and recommendations for choosing the right pairs are made. The chapter discusses VFPs that are used for studies that use fluorescence resonance

  14. Fluorescent Proteins for Flow Cytometry.

    Science.gov (United States)

    Hawley, Teresa S; Hawley, Robert G; Telford, William G

    2017-04-03

    Fluorescent proteins have become standard tools for cell and molecular biologists. The color palette of fluorescent proteins spans the ultraviolet, visible, and near-infrared spectrum. Utility of fluorescent proteins has been greatly facilitated by the availability of compact and affordable solid state lasers capable of providing various excitation wavelengths. In theory, the plethora of fluorescent proteins and lasers make it easy to detect multiple fluorescent proteins simultaneously. However, in practice, heavy spectral overlap due to broad excitation and emission spectra presents a challenge. In conventional flow cytometry, careful selection of excitation wavelengths and detection filters is necessary. Spectral flow cytometry, an emerging methodology that is not confined by the "one color, one detector" paradigm, shows promise in the facile detection of multiple fluorescent proteins. This chapter provides a synopsis of fluorescent protein development, a list of commonly used fluorescent proteins, some practical considerations and strategies for detection, and examples of applications. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  15. Fluorescence Spectra of Highlighter Inks

    Science.gov (United States)

    Birriel, Jennifer J.; King, Damon

    2018-01-01

    Fluorescence spectra excited by laser pointers have been the subject of several papers in "TPT". These papers all describe a fluorescence phenomenon in which the reflected laser light undergoes a change in color: this color change results from the combination of some partially reflected laser light and additional colors generated by…

  16. Time-Resolved Fluorescence in Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Shu-Chi Allison Yeh

    2014-12-01

    Full Text Available Photodynamic therapy (PDT has been used clinically for treating various diseases including malignant tumors. The main advantages of PDT over traditional cancer treatments are attributed to the localized effects of the photochemical reactions by selective illumination, which then generate reactive oxygen species and singlet oxygen molecules that lead to cell death. To date, over- or under-treatment still remains one of the major challenges in PDT due to the lack of robust real-time dose monitoring techniques. Time-resolved fluorescence (TRF provides fluorescence lifetime profiles of the targeted fluorophores. It has been demonstrated that TRF offers supplementary information in drug-molecular interactions and cell responses compared to steady-state intensity acquisition. Moreover, fluorescence lifetime itself is independent of the light path; thus it overcomes the artifacts given by diffused light propagation and detection geometries. TRF in PDT is an emerging approach, and relevant studies to date are scattered. Therefore, this review mainly focuses on summarizing up-to-date TRF studies in PDT, and the effects of PDT dosimetric factors on the measured TRF parameters. From there, potential gaps for clinical translation are also discussed.

  17. High resolution UV spectroscopy and laser-focused nanofabrication

    NARCIS (Netherlands)

    Myszkiewicz, G.

    2005-01-01

    This thesis combines two at first glance different techniques: High Resolution Laser Induced Fluorescence Spectroscopy (LIF) of small aromatic molecules and Laser Focusing of atoms for Nanofabrication. The thesis starts with the introduction to the high resolution LIF technique of small aromatic

  18. Detection of Hg2+ in water environment by fluorescence spectroscopic methods

    Science.gov (United States)

    Li, Zhen; Zhang, Jinsong; Hu, Hong; Wan, Ruyi; Yao, Youwei

    2015-08-01

    Inorganic mercury (Hg2+) produces toxic effects even at very low concentration. High sensitive fluorescent probes for Hg2+ detection has been researched and synthesized. A fluorescence detection system is built for Hg2+ detection in water environment with fluorescent probes as the detection reagent. Fiber coupled LED with high brightness is developed and used as excitation light source. And the optimized excitation wavelength is about 520 nm. The measurements of fluorescence spectra is obtained by means of optical fiber spectroscopic techniques. Fluorescence detection experiments are carried out for a range of different concentrations of Hg2+ in aqueous solutions. The center wavelength of the fluorescence spectra is about 580 nm which is unchanged in the experiments. Relationship between Hg2+ concentrations and the fluorescence intensity is studied. A positive correlation exists between the intensity of fluorescence spectrum and the concentrations of Hg2+. The fluorescence intensity grows with increasing the concentration of Hg2+ for the same excitation light. When the concentration of Hg2+ is high enough, the fluorescence intensity increases slowly. And a numerical model is built for the concentration calculating. The detection limit is 0.005 μmol/L in the experiments. The Hg2+ detection system reported has many advantages such as small size, rapid response, high-sensitivity, and can be used for on-site testing of the water quality.

  19. Direct solid surface fluorescence spectroscopy of standard chemicals and humic acid in ternary system.

    Science.gov (United States)

    Mounier, S; Nicolodelli, G; Redon, R; Milori, D M B P

    2017-04-15

    The front face fluorescence spectroscopy is often used to quantify chemicals in well-known matrices as it is a rapid and powerful technique, with no sample preparation. However it was not used to investigate extracted organic matter like humic substances. This work aims to fully investigate for the first time front face fluorescence spectroscopy response of a ternary system including boric acid, tryptophan and humic substances, and two binaries system containing quinine sulfate or humic substance in boric acid. Pure chemicals, boric acid, tryptophan, quinine sulfate and humic acid were mixed together in solid pellet at different contents from 0 to 100% in mass. The measurement of excitation emission matrix of fluorescence (3D fluorescence) and laser induced fluorescence were then done in the front face mode. Fluorescence matrices were decomposed using the CP/PARAFAC tools after scattering treatments. Results show that for 3D fluorescence there is no specific component for tryptophan and quinine sulfate, and that humic substances lead to a strong extinction effect for mixture containing quinine sulfate. Laser induced fluorescence gives a very good but non-specific related response for both quinine sulfate and tryptophan. No humic substances fluorescence response was found, but extinction effect is observed as for 3D fluorescence. This effect is stronger for quinine sulfate than for tryptophan. These responses were modeled using a simple absorbance versus emission model. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Direct solid surface fluorescence spectroscopy of standard chemicals and humic acid in ternary system

    Science.gov (United States)

    Mounier, S.; Nicolodelli, G.; Redon, R.; Milori, D. M. B. P.

    2017-04-01

    The front face fluorescence spectroscopy is often used to quantify chemicals in well-known matrices as it is a rapid and powerful technique, with no sample preparation. However it was not used to investigate extracted organic matter like humic substances. This work aims to fully investigate for the first time front face fluorescence spectroscopy response of a ternary system including boric acid, tryptophan and humic substances, and two binaries system containing quinine sulfate or humic substance in boric acid. Pure chemicals, boric acid, tryptophan, quinine sulfate and humic acid were mixed together in solid pellet at different contents from 0 to 100% in mass. The measurement of excitation emission matrix of fluorescence (3D fluorescence) and laser induced fluorescence were then done in the front face mode. Fluorescence matrices were decomposed using the CP/PARAFAC tools after scattering treatments. Results show that for 3D fluorescence there is no specific component for tryptophan and quinine sulfate, and that humic substances lead to a strong extinction effect for mixture containing quinine sulfate. Laser induced fluorescence gives a very good but non-specific related response for both quinine sulfate and tryptophan. No humic substances fluorescence response was found, but extinction effect is observed as for 3D fluorescence. This effect is stronger for quinine sulfate than for tryptophan. These responses were modeled using a simple absorbance versus emission model.

  1. Focus on Delivery

    DEFF Research Database (Denmark)

    Hasman, Kirsten; Barfoed, Anne

    Background: Compared to other Nordic countries, Denmark has a high incidence of anal sphincter injury. Recent studies indicate that a strict focus on prevention of severe perineal trauma has decreased the incidence (1). This has resulted in changed clinical procedures in several Danish labour wards...

  2. Homework. Focus On

    Science.gov (United States)

    Rahal, Michelle Layer

    2010-01-01

    Homework has been an integral part of the educational system for over 100 years. What likely began as simple memorization tasks has evolved into complex projects and sparked an increasingly heated debate over the purpose and value of homework assignments. This "Focus On" examines the purpose of homework, how to create homework that has value,…

  3. Focusing on the Invisible

    Science.gov (United States)

    Haley, Tim R.

    2008-01-01

    This article seeks to answer the question of whether or not the design and development of an educational laboratory really changes when the focus is on nanotechnology. It explores current laboratory building trends and the added considerations for building a nanotechnology laboratory. The author leaves the reader with additional points to consider…

  4. Compassion-Focused EMDR

    National Research Council Canada - National Science Library

    Kennedy, Angela

    2014-01-01

    ... and reprocessing (EMDR), particularly where shame or attachment trauma is involved or for those traumas that have impacted on the structure of the self, for example, dissociation. A structured compassion-focused EMDR (CF-EMDR) seems likely to be particularly useful for therapists wishing to pay positive attention to strengths and well-being. The primary t...

  5. Fluorescence calibration method for single-particle aerosol fluorescence instruments

    Science.gov (United States)

    Shipley Robinson, Ellis; Gao, Ru-Shan; Schwarz, Joshua P.; Fahey, David W.; Perring, Anne E.

    2017-05-01

    Real-time, single-particle fluorescence instruments used to detect atmospheric bioaerosol particles are increasingly common, yet no standard fluorescence calibration method exists for this technique. This gap limits the utility of these instruments as quantitative tools and complicates comparisons between different measurement campaigns. To address this need, we have developed a method to produce size-selected particles with a known mass of fluorophore, which we use to calibrate the fluorescence detection of a Wideband Integrated Bioaerosol Sensor (WIBS-4A). We use mixed tryptophan-ammonium sulfate particles to calibrate one detector (FL1; excitation = 280 nm, emission = 310-400 nm) and pure quinine particles to calibrate the other (FL2; excitation = 280 nm, emission = 420-650 nm). The relationship between fluorescence and mass for the mixed tryptophan-ammonium sulfate particles is linear, while that for the pure quinine particles is nonlinear, likely indicating that not all of the quinine mass contributes to the observed fluorescence. Nonetheless, both materials produce a repeatable response between observed fluorescence and particle mass. This procedure allows users to set the detector gains to achieve a known absolute response, calculate the limits of detection for a given instrument, improve the repeatability of the instrumental setup, and facilitate intercomparisons between different instruments. We recommend calibration of single-particle fluorescence instruments using these methods.

  6. Water-soluble conjugated polymers for fluorescent-enzyme assays.

    Science.gov (United States)

    Feng, Fude; Liu, Libing; Yang, Qiong; Wang, Shu

    2010-08-17

    Enzyme assays are receiving more and more research and application interest because of the rapidly increasing demands of clinical diagnosis, environmental analysis, drug discovery, and molecular biology. Water-soluble light-harvesting conjugated polymers (CPs) coordinate the action of a large number of absorbing units to afford an amplified fluorescence signal, which makes them useful as optical platforms in highly sensitive chemical and biological sensors. This Feature Article highlights recent developments of water-soluble CPs for fluorescent assays of enzymes. Different signal transduction mechanisms, such as electron transfer, fluorescence resonance energy transfer (FRET), and aggregation or conformation changes of CPs, are employed in these assays according to the dissimilar nature of enzymes. Potential challenges and future research directions in these approaches based on CPs are also discussed. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Effects of ozone and relative humidity on fluorescence spectra of octapeptide bioaerosol particles

    Science.gov (United States)

    Pan, Yong-Le; Santarpia, Joshua L.; Ratnesar-Shumate, Shanna; Corson, Elizabeth; Eshbaugh, Jonathan; Hill, Steven C.; Williamson, Chatt C.; Coleman, Mark; Bare, Christopher; Kinahan, Sean

    2014-01-01

    The effects of ozone and relative humidity (RH) at common atmospheric levels on the properties of single octapeptide bioaerosol particles were studied using an improved rotating reaction chamber, an aerosol generator, an ultraviolet aerodynamic particle sizer (UVAPS), an improved single particle fluorescence spectrometer (SPFS), and equipments to generate, monitor and control the ozone and RH. Aerosol particles (mean diameter 2 μm) were generated from a slurry of octapeptide in phosphate buffered saline, injected into the rotating chamber, and kept airborne for hours. Bioaerosols were sampled from the chamber hourly for the measurements of particle-size distribution, concentration, total fluorescence excited at 355-nm, and single particle fluorescence spectra excited at 266-nm and 351-nm under different controlled RH (20%, 50%, or 80%) and ozone concentration (0 or 150 ppb). The results show that: (1) Particle size, concentration, and the 263-nm-excited fluorescence intensity decrease at different rates under different combinations of the RH and ozone concentrations used. (2) The 263-nm-excited UV fluorescence (280-400 nm) decreased more rapidly than the 263-nm-excited visible fluorescence (400-560 nm), and decreased most rapidly when ozone is present and RH is high. (3) The UV fluorescence peak near 340 nm slightly shifts to the shorter wavelength (blue-shift), consistent with a more rapid oxidation of tryptophan than tyrosine. (4) The 351/355-nm-excited fluorescence (430-580 nm/380-700 nm) increases when ozone is present, especially when the RH is high. (5) The 351/355-nm-excited fluorescence increase that occurs as the tryptophan emission in the UV decreases, and the observation that these changes occur more rapidly at higher RH with the present of ozone, are consistent with the oxidation of tryptophan by ozone and the conversion of the resulting ozonides to N-formyl kynurenine and kynurenine.

  8. Rapid Prototyping Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — The ARDEC Rapid Prototyping (RP) Laboratory was established in December 1992 to provide low cost RP capabilities to the ARDEC engineering community. The Stratasys,...

  9. Planetary Surface Analysis Using Fast Laser Spectroscopic Techniques: Combined Microscopic Raman, LIBS, and Fluorescence Spectroscopy

    Science.gov (United States)

    Blacksberg, J.; Rossman, G. R.; Maruyama, Y.; Charbon, E.

    2011-12-01

    In situ exploration of planetary surfaces has to date required multiple techniques that, when used together, yield important information about their formation histories and evolution. We present a time-resolved laser spectroscopic technique that could potentially collect complementary sets of data providing information on mineral structure, composition, and hydration state. Using a picosecond-scale pulsed laser and a fast time-resolved detector we can simultaneously collect spectra from Raman, Laser Induced Breakdown Spectroscopy (LIBS), and fluorescence emissions that are separated in time due to the unique decay times of each process. The use of a laser with high rep rate (40 KHz) and low pulse energy (1 μJ/pulse) allows us to rapidly collect high signal to noise Raman spectra while minimizing sample damage. Increasing the pulse energy by about an order of magnitude creates a microscopic plasma near the surface and enables the collection of LIBS spectra at an unusually high rep rate and low pulse energy. Simultaneously, broader fluorescence peaks can be detected with lifetimes varying from nanosecond to microsecond. We will present Raman, LIBS, and fluorescence spectra obtained on natural mineral samples such as sulfates, clays, pyroxenes and carbonates that are of interest for Mars mineralogy. We demonstrate this technique using a photocathode-based streak camera detector as well as a newly-developed solid state Single Photon Avalanche Diode (SPAD) sensor array based on Complementary Metal-Oxide Semiconductor (CMOS) technology. We will discuss the impact of system design and detector choice on science return of a potential planetary surface mission, with a specific focus on size, weight, power, and complexity. The research described here was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (NASA).

  10. Basic theory and methods of X-ray fluorescence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Duimakaev, S.I.; Shpolyansky, A.Y.; Duimakaeva, T.G.; Losev, V.N. (Rostovskij-na-Donu Gosudarstvennyj Univ. (USSR)); Vershinina, N.V. (Rostov Inst. of Railway Transport Engineers, Rostov-na-Don (USSR)); Tsvetyansky, A.L. (NPO ' Soyuztsvetmetavtomatika' , Rostov-na-Don (USSR))

    1989-09-01

    The Soviet analysts' major advances in both theory and application of X-ray fluorescence (XRF) analysis are reviewed. The focus is on issues concerning XRF analysis of heterogeneous samples, a theoretical account of interelement effects, and employment of scattered radiation as the internal standard. (orig.).

  11. Optical probing of single fluorescent molecules and proteins

    NARCIS (Netherlands)

    Garcia Parajo, M.F.; Veerman, J.A.; Bouwhuis, R.; Bouwhuis, Rudo; van Hulst, N.F.; Vallée, R.A.L.

    2001-01-01

    Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis o­n the underlying principles methodology and the application of single-molecule analysis at room temperature. This Minireview is mainly focused o­n the application of confocal and

  12. Atomic Absorption, Atomic Fluorescence, and Flame Emission Spectrometry.

    Science.gov (United States)

    Horlick, Gary

    1984-01-01

    This review is presented in six sections. Sections focus on literature related to: (1) developments in instrumentation, measurement techniques, and procedures; (2) performance studies of flames and electrothermal atomizers; (3) applications of atomic absorption spectrometry; (4) analytical comparisons; (5) atomic fluorescence spectrometry; and (6)…

  13. Development of a Green Fluorescent Protein-Based Laboratory Curriculum

    Science.gov (United States)

    Larkin, Patrick D.; Hartberg, Yasha

    2005-01-01

    A laboratory curriculum has been designed for an undergraduate biochemistry course that focuses on the investigation of the green fluorescent protein (GFP). The sequence of procedures extends from analysis of the DNA sequence through PCR amplification, recombinant plasmid DNA synthesis, bacterial transformation, expression, isolation, and…

  14. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  15. Single-molecule spectroscopy of fluorescent proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  16. Experimental design and quality assurance: in situ fluorescence instrumentation

    Science.gov (United States)

    Conmy, Robyn N.; Del Castillo, Carlos E.; Downing, Bryan D.; Chen, Robert F.

    2014-01-01

    Both instrument design and capabilities of fluorescence spectroscopy have greatly advanced over the last several decades. Advancements include solid-state excitation sources, integration of fiber optic technology, highly sensitive multichannel detectors, rapid-scan monochromators, sensitive spectral correction techniques, and improve data manipulation software (Christian et al., 1981, Lochmuller and Saavedra, 1986; Cabniss and Shuman, 1987; Lakowicz, 2006; Hudson et al., 2007). The cumulative effect of these improvements have pushed the limits and expanded the application of fluorescence techniques to numerous scientific research fields. One of the more powerful advancements is the ability to obtain in situ fluorescence measurements of natural waters (Moore, 1994). The development of submersible fluorescence instruments has been made possible by component miniaturization and power reduction including advances in light sources technologies (light-emitting diodes, xenon lamps, ultraviolet [UV] lasers) and the compatible integration of new optical instruments with various sampling platforms (Twardowski et at., 2005 and references therein). The development of robust field sensors skirt the need for cumbersome and or time-consuming filtration techniques, the potential artifacts associated with sample storage, and coarse sampling designs by increasing spatiotemporal resolution (Chen, 1999; Robinson and Glenn, 1999). The ability to obtain rapid, high-quality, highly sensitive measurements over steep gradients has revolutionized investigations of dissolved organic matter (DOM) optical properties, thereby enabling researchers to address novel biogeochemical questions regarding colored or chromophoric DOM (CDOM). This chapter is dedicated to the origin, design, calibration, and use of in situ field fluorometers. It will serve as a review of considerations to be accounted for during the operation of fluorescence field sensors and call attention to areas of concern when making

  17. Clinical application of fluorescence in situ hybridization for prenatal diagnosis

    Directory of Open Access Journals (Sweden)

    Shu-fang JIANG

    2012-07-01

    Full Text Available Objective To establish and optimize the procedures of fluorescence in situ hybridization(FISH), and evaluate its clinical value in rapid prenatal diagnosis of fetal numerical abnormality of chromosomes 21, 18, 13, X, Y. Methods Amniotic fluid or fetal blood was sampled by routine invasive procedures. After the amniotic fluid cells or fetal blood cells were separated and sequentially processed with hypotonic solution, fixation solution, smear and high temperature, they were hybridized in situ with two panels of specific fluorescence probes to detect numerical abnormality of chromosomes 21, 18, 13, X, Y. All the samples were also cultured and analyzed for their karyotype by conventional methods. Results When it was used as a diagnostic criterion of chromosomal number that the fluorescence signals were observed in ≥90% cells, GLP 13/GLP 21 probe panel showed 2 green/2 red fluorescence signals and CSP18/CSP X/CSP Y probe panel showed 2 blue/2 yellow (female or 2 blue/1 yellow/1 red fluorescence signals (male under normal condition. The test reports of all 196 cases were sent out in 72-96 hours, and 7 cases of Down syndrome, 2 cases of trisomy 18 and 1 case of sex chromosomal numerical abnormality were detected, which were accordant with karyotype analysis results reported one month later. Conclusions FISH has potential for clinical application, and is applicable to rapid prenatal diagnosis of fetal numerical abnormality of chromosomes 21, 18, 13, X, Y. The rapid FISH, together with conventional karyotyping, offer a valuable means for prenatal diagnosis of fetal aneuploidies.

  18. Reliability of acridine orange fluorescence microscopy in oral cytodiagnosis

    Directory of Open Access Journals (Sweden)

    Nilima Prakash

    2011-01-01

    Full Text Available Context and Aims: The oral cavity is the most predominant location in the head and neck region for primary malignant epithelial tumors. Oral cancer is estimated to be the sixth most common malignancy. Early recognition is imperative for successful treatment and good prognosis. Exfoliative cytology is a simple and reasonably effective technique for rapid initial evaluation of a suspicious oral lesion. The present study was conducted to determine the reliability of acridine orange fluorescence microscopy for cytodiagnosis as a more rapid and easier method for the final evaluation of the cytological specimen. Materials and Methods: Smears were collected from 20 individuals with oral lesions suspicious of malignancy, oral lesions not suggestive of malignancy and normal buccal mucosa. One smear was stained with Papanicolaou stain and another one with acridine orange stain. The differences in the study group and control group were compared by means of the χ2 (Chi-square test. The results were considered statistically significant whenever P was <0.05. Results: The acridine orange fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence - a cytochemical criterion. The efficacy of the stain was higher than the conventional Papanicolaou stain in screening of oral lesions suspicious of malignancy. However, the acridine orange fluorescence stain did not differentiate effectively between malignant cells and rapidly proliferating cells, as the technique is based on the nucleic acid content. Conclusion: The fluorescent acridine orange method can be used reliably for the screening of carcinomas and it is especially helpful in the follow-up detection of recurrent carcinoma in previously treated cases.

  19. Detection of vapor phase mercury species by laser fluorescence methods

    Science.gov (United States)

    Tong, Xiaomei

    Several mercury species emissions have been identified in off-gases from industrial processes. At present, there is no commercial continuous emission monitoring (CEM) technique or instrumentation to reliably monitor volatile mercury species emissions from industrial stacks. Conventional measurement methods, such as cold vapor trap based techniques for elemental mercury, have difficulty in achieving both high sensitivity and the fast time resolution required for real-time monitoring. This doctoral research work gives a systematic study of potential methods for real-time trace detection of volatile elemental mercury and mercury compounds in industrial stack gases. It is based on laser-induced fluorescence techniques; photofragment fluorescence spectroscopy for detection of volatile mercury compounds, and resonance fluorescence for detection of elemental mercury. The capabilities and limitations of these detection techniques are investigated in this dissertation. Detection of mercury compounds is a challenge since they are non-fluorescent. With photofragment fluorescence spectroscopy, target compound concentrations are related to the fluorescence intensity from an excited fragment. In this doctoral research work, low concentrations of mercuric bromide vapor in an atmospheric pressure flow cell are irradiated by a focused laser beam at 222nm. Photofragment fluorescence is monitored at 253.7nm. Two detection schemes, Charge Coupled Device (CCD) and photomultiplier tube (PMT), are applied for the measurement of photofragment fluorescence. The performances of these two systems are compared in the dissertation. A supersonic jet is combined with resonance fluorescence for detection of elemental mercury vapor. With test gas expanded into a vacuum, fluorescence quenching and spectral broadening are reduced. In the experiment, the gas jet is crossed with a laser beam at 253.7nm to excite atomic fluorescence, which is distinguished from the elastic background by time gating

  20. Intraoperative fluorescence imaging for personalized brain tumor resection: Current state and future directions

    Directory of Open Access Journals (Sweden)

    Evgenii Belykh

    2016-10-01

    Full Text Available Introduction: Fluorescence-guided surgery is one of the rapidly emerging methods of surgical theranostics. In this review, we summarize current fluorescence techniques used in neurosurgical practice for brain tumor patients, as well as future applications of recent laboratory and translational studies.Methods: Review of the literature.Results: A wide spectrum of fluorophores that have been tested for brain surgery is reviewed. Beginning with a fluorescein sodium application in 1948 by Moore, fluorescence guided brain tumor surgery is either routinely applied in some centers or is under active study in clinical trials. Besides the trinity of commonly used drugs (fluorescein sodium, 5-ALA and ICG, less studied fluorescent stains, such as tetracyclines, cancer-selective alkylphosphocholine analogs, cresyl violet, acridine orange, and acriflavine can be used for rapid tumor detection and pathological tissue examination. Other emerging agents such as activity-based probes and targeted molecular probes that can provide biomolecular specificity for surgical visualization and treatment are reviewed. Furthermore, we review available engineering and optical solutions for fluorescent surgical visualization. Instruments for fluorescent-guided surgery are divided into wide-field imaging systems and hand-held probes. Recent advancements in quantitative fluorescence-guided surgery are discussed.Conclusion: We are standing on the doorstep of the era of marker-assisted tumor management. Innovations in the fields of surgical optics, computer image analysis, and molecular bioengineering are advancing fluorescence-guided tumor resection paradigms, leading to cell-level approaches to visualization and resection of brain tumors.

  1. Magnetic Focusing Horn

    CERN Multimedia

    1974-01-01

    This magnetic focusing horn was used for the AA (antiproton accumulator). Its development was an important step towards using CERN's Super Proton Synchrotron as a proton - antiproton collider. This eventually led to the discovery of the W and Z particles in 1983. Making an antiproton beam took a lot of time and effort. Firstly, protons were accelerated to an energy of 26 GeV in the PS and ejected onto a metal target. From the spray of emerging particles, a magnetic horn picked out 3.6 GeV antiprotons for injection into the AA through a wide-aperture focusing quadrupole magnet. For a million protons hitting the target, just one antiproton was captured, 'cooled' and accumulated. It took 3 days to make a beam of 3 x 10^11 -, three hundred thousand million - antiprotons.

  2. Focusing of electromagnetic waves

    Energy Technology Data Exchange (ETDEWEB)

    Dhayalan, V.

    1996-12-31

    The focusing of electromagnetic waves inside a slab has been examined together with two special cases in which the slab is reduced to a single interface or a single medium. To that end the exact solutions for the fields inside a layered medium have been used, given in terms of the outside current source in order to obtain the solutions for the focused electric field inside a slab. Both exact and asymptotic solutions of the problem have been considered, and the validity of the latter has been discussed. The author has developed a numerical algorithm for evaluation of the diffraction integral with special emphasis on reducing the computing time. The numerical techniques in the paper can be readily applied to evaluate similar diffraction integrals occurring e.g. in microstrip antennas. 46 refs.

  3. Toward fluorescence nanoscopy.

    Science.gov (United States)

    Hell, Stefan W

    2003-11-01

    For more than a century, the resolution of focusing light microscopy has been limited by diffraction to 180 nm in the focal plane and to 500 nm along the optic axis. Recently, microscopes have been reported that provide three- to sevenfold improved axial resolution in live cells. Moreover, a family of concepts has emerged that overcomes the diffraction barrier altogether. Its first exponent, stimulated emission depletion microscopy, has so far displayed a resolution down to 28 nm. Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level. As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever.

  4. Dense Plasma Focus Modeling

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hui [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Li, Shengtai [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Jungman, Gerard [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Hayes-Sterbenz, Anna Catherine [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-08-31

    The mechanisms for pinch formation in Dense Plasma Focus (DPF) devices, with the generation of high-energy ions beams and subsequent neutron production over a relatively short distance, are not fully understood. Here we report on high-fidelity 2D and 3D numerical magnetohydrodynamic (MHD) simulations using the LA-COMPASS code to study the pinch formation dynamics and its associated instabilities and neutron production.

  5. A Material Focus

    DEFF Research Database (Denmark)

    Vallgårda, Anna K. A.; Sokoler, Tomas

    2009-01-01

    In this paper we build on the notion of computational composites, which hold a material perspective on computational technology. We argue that a focus on the material aspects of the technology could be a fruitful approach to achieve new expressions and to gain a new view on the technology's role...... in design. We study two of the computer's material properties: computed causality and connectability and through developing two computational composites that utilize these properties we begin to explore their potential expressions....

  6. Subsurface contaminants focus area

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-08-01

    The US Department of Enregy (DOE) Subsurface Contaminants Focus Area is developing technologies to address environmental problems associated with hazardous and radioactive contaminants in soil and groundwater that exist throughout the DOE complex, including radionuclides, heavy metals; and dense non-aqueous phase liquids (DNAPLs). More than 5,700 known DOE groundwater plumes have contaminated over 600 billion gallons of water and 200 million cubic meters of soil. Migration of these plumes threatens local and regional water sources, and in some cases has already adversely impacted off-site rsources. In addition, the Subsurface Contaminants Focus Area is responsible for supplying technologies for the remediation of numerous landfills at DOE facilities. These landfills are estimated to contain over 3 million cubic meters of radioactive and hazardous buried Technology developed within this specialty area will provide efective methods to contain contaminant plumes and new or alternative technologies for development of in situ technologies to minimize waste disposal costs and potential worker exposure by treating plumes in place. While addressing contaminant plumes emanating from DOE landfills, the Subsurface Contaminants Focus Area is also working to develop new or alternative technologies for the in situ stabilization, and nonintrusive characterization of these disposal sites.

  7. Particle Accelerator Focus Automation

    Science.gov (United States)

    Lopes, José; Rocha, Jorge; Redondo, Luís; Cruz, João

    2017-08-01

    The Laboratório de Aceleradores e Tecnologias de Radiação (LATR) at the Campus Tecnológico e Nuclear, of Instituto Superior Técnico (IST) has a horizontal electrostatic particle accelerator based on the Van de Graaff machine which is used for research in the area of material characterization. This machine produces alfa (He+) and proton (H+) beams of some μA currents up to 2 MeV/q energies. Beam focusing is obtained using a cylindrical lens of the Einzel type, assembled near the high voltage terminal. This paper describes the developed system that automatically focuses the ion beam, using a personal computer running the LabVIEW software, a multifunction input/output board and signal conditioning circuits. The focusing procedure consists of a scanning method to find the lens bias voltage which maximizes the beam current measured on a beam stopper target, which is used as feedback for the scanning cycle. This system, as part of a wider start up and shut down automation system built for this particle accelerator, brings great advantages to the operation of the accelerator by turning it faster and easier to operate, requiring less human presence, and adding the possibility of total remote control in safe conditions.

  8. Particle Accelerator Focus Automation

    Directory of Open Access Journals (Sweden)

    Lopes José

    2017-08-01

    Full Text Available The Laboratório de Aceleradores e Tecnologias de Radiação (LATR at the Campus Tecnológico e Nuclear, of Instituto Superior Técnico (IST has a horizontal electrostatic particle accelerator based on the Van de Graaff machine which is used for research in the area of material characterization. This machine produces alfa (He+ and proton (H+ beams of some μA currents up to 2 MeV/q energies. Beam focusing is obtained using a cylindrical lens of the Einzel type, assembled near the high voltage terminal. This paper describes the developed system that automatically focuses the ion beam, using a personal computer running the LabVIEW software, a multifunction input/output board and signal conditioning circuits. The focusing procedure consists of a scanning method to find the lens bias voltage which maximizes the beam current measured on a beam stopper target, which is used as feedback for the scanning cycle. This system, as part of a wider start up and shut down automation system built for this particle accelerator, brings great advantages to the operation of the accelerator by turning it faster and easier to operate, requiring less human presence, and adding the possibility of total remote control in safe conditions.

  9. Following protein association in vivo with fluorescence fluctuation spectroscopy

    Science.gov (United States)

    Muller, Joachim D.

    2003-07-01

    The combination of fluorescence correlation spectroscopy and two-photon excitation provides us with a powerful spectroscopic technique. Its submicron resolution and single molecule sensitivity make it an attractive technique for in vivo applications. Experiments have demonstrated that quantitative in vivo fluorescence fluctuation measurements are feasible, despite the presence of autofluorescence and the heterogeneity of the cellular environment. I will demonstrate that molecular brightness of proteins tagged with green fluorescent protein (GFP) is a useful and robust parameter for in vivo studies. Knowledge of photon statistics is crucial for the interpretation of fluorescence fluctuation experiments. I will describe photon counting histogram (PCH) analysis, which determines the molecular brightness and complements autocorrelation analysis. Non-ideal detector effects and their influence on the photon statistics will be discussed. The goal of in vivo fluorescence fluctuation experiments is to address functional properties of biomolecules. We will focus on retinoid X receptor (RXR), a nuclear receptor, which is crucial for the regulation of gene expression. The fluorescence brightness of RXR tagged with EGFP will be used to probe the oligomerization state of RXR.

  10. Probing protein interactions in cells by fluorescence fluctuation spectroscopy

    Science.gov (United States)

    Mueller, Joachim

    2003-03-01

    The combination of fluorescence correlation spectroscopy and two-photon excitation provides us with a powerful spectroscopic technique. Its submicron resolution and single molecule sensitivity make it an attractive technique for in vivo applications. Experiments have demonstrated that quantitative in vivo fluorescence fluctuation measurements are feasible, despite the presence of autofluorescence and the heterogeneity of the cellular environment. I will demonstrate that molecular brightness of proteins tagged with green fluorescent protein (GFP) is a useful and robust parameter for in vivo studies. Knowledge of photon statistics is crucial for the interpretation of fluorescence fluctuation experiments. I will describe photon counting histogram (PCH) analysis, which determines the molecular brightness and complements autocorrelation analysis. Non-ideal detector effects and their influence on the photon statistics will be discussed. The goal of in vivo fluorescence fluctuation experiments is to address functional properties of biomolecules. We will focus on retinoid X receptor (RXR), a nuclear receptor, which is crucial for the regulation of gene expression. The fluorescence brightness of RXR tagged with EGFP will be used to probe the oligomerization state of RXR.

  11. Sources and dynamics of fluorescent particles in hospitals.

    Science.gov (United States)

    Pereira, M L; Knibbs, L D; He, C; Grzybowski, P; Johnson, G R; Huffman, J A; Bell, S C; Wainwright, C E; Matte, D L; Dominski, F H; Andrade, A; Morawska, L

    2017-09-01

    Fluorescent particles can be markers of bioaerosols and are therefore relevant to nosocomial infections. To date, little research has focused on fluorescent particles in occupied indoor environments, particularly hospitals. In this study, we aimed to determine the spatial and temporal variation of fluorescent particles in two large hospitals in Brisbane, Australia (one for adults and one for children). We used an Ultraviolet Aerodynamic Particle Sizer (UVAPS) to identify fluorescent particle sources, as well as their contribution to total particle concentrations. We found that the average concentrations of both fluorescent and non-fluorescent particles were higher in the adults' hospital (0.06×106 and 1.20×106  particles/m3 , respectively) than in the children's hospital (0.03×106 and 0.33×106  particles/m3 , respectively) (Pfluorescent particles was higher in the children's hospital. Based on the concentration results and using activity diaries, we were able to identify sources of particle production within the two hospitals. We demonstrated that particles can be easily generated by a variety of everyday activities, which are potential sources of exposure to pathogens. Future studies to further investigate their role in nosocomial infection are warranted. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Fluorescent labelling in living cells.

    Science.gov (United States)

    Schneider, Anselm Fabian Lowell; Hackenberger, Christian Peter Richard

    2017-12-01

    The labelling of proteins with green fluorescent protein enabled the visualization of proteins in living cells for the first time. Since then, much progress has been made in the field. Modern strategies allow the labelling of proteins in live cells through a range of specialized methods with sophisticated chemical probes that show enhanced photophysical properties compared to fluorescent proteins. This review briefly summarizes recent advances in the field of fluorescent chemical protein labelling inside living cells and illustrates key aspects on the requirements and advantages of each given method. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

    Science.gov (United States)

    Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

    2012-01-01

    The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

  14. Two-Photon Excited Fluorescence from Biological Aerosol Particles

    Science.gov (United States)

    2010-09-29

    induced fluorescence (LIF) to provide an initial rapid indication of the presence of biological aerosol particles. Examples of recent ultraviolet (UV...pp.4960-4965, 2007. 10. J. W. Lou, M. Currie, V. Sivaprakasam, and J. D. Eversole, “Green and Ultraviolet Pulse Generation with a Compact, Fiber...solutions,” Journal of photochemistry and photobiology 65, 6, 931-936 (1997). 18. S. Dad, R.H. Bisby, I.P. Clark and A.W. Parker, “Identification

  15. Interactions between natural organic ligands and trace metals studied by fluorescence lifetime and fluorescence quenching

    Science.gov (United States)

    Nouhi, Ayoub; Hajjoul, Houssam; Redon, Roland; Gagné, Jean-Pierre; Mounier, Stéphane

    2017-04-01

    order to calibrate our assays and compare our results with literature. Several studies have shown that static quenching occurs in that case (Brun and Schröder, 1975; Lavrik and Mulloev, 2010; Ventry et al., 1991; Babko, 1968). Indeed, after processing the EEFMs and TRLFS data, we found a fluorescence intensity decay by about 50% and a constant lifetime for the fluorophore suggesting a static quenching, in agreement with the literature. In the second step, we have studied the interactions between metal and different types of natural organic matters. In this case, EEMFs and TRLFS experiments were done on samples prepared by dissolving copper in four different fractions of organic matter extracted from estuarine water (St. Lawrence Estuary, Canada). Organic matter was obtained using DAX-8 and XAD-4 resins in series. Humic and fulvic acids are obtained following the IHSS protocol. The results of interaction between humic substances and copper gathered after processing data on PROGMEEF have shown a fluorescence intensity decay by about 57% for the first component and 88% for the second component. The fluorescence lifetime for both components were close to 2 ns and 6 ns respectively and the pH range was stable and close to 6. This means that a static quenching takes place in this case in agreement with the literature. Our study also focused on the investigation of complexation of organic matter by other metals in particular Aluminum, Arsenic, Europium and Uranium.

  16. Highly sensitive synchronous fluorescence measurement of danofloxacin in pharmaceutical and milk samples using aluminium (III) enhanced fluorescence.

    Science.gov (United States)

    Kaur, Kuldeep; Saini, Shivender; Singh, Baldev; Malik, Ashok Kumar

    2012-09-01

    A simple, rapid and sensitive constant wavelength synchronous fluorescence method is developed for the determination of danofloxacin (DAN) in pharmaceutical formulations and its residue in milk based on Al(III) enhanced fluorescence. The synchronous fluorescence intensity of the system is measured at 435 nm using ∆ λ = 80 nm and an excitation wavelength of 280 nm. A good linear relationship between enhanced fluorescence intensity and DAN concentration is obtained in the range of 3-100 ng mL(-1)(r (2) = 0.9991). The limit of detection (LOD, S/N = 3) of the present method is 0.9 ng mL(-1). The proposed method can be successfully applied to the determination of DAN in pharmaceutical formulations and in milk without serious interferences from common excipients, metal ions and other co-existing substances. The method can be used as a rapid screening to judge whether the DAN residues in milk exceed Maximum Residue Limits (MRLs) or not.

  17. Detection of Salmonella typhi utilizing bioconjugated fluorescent polymeric nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Swati, E-mail: swatijain.iitd@gmail.com; Chattopadhyay, Sruti, E-mail: sruticiitd@gmail.com; Jackeray, Richa; Abid, Zainul; Singh, Harpal, E-mail: harpal2000@yahoo.com [Centre for Biomedical Engineering, Indian Institute of Technology-Delhi (India)

    2016-05-15

    Present work demonstrates effective utilization of functionalized polymeric fluorescent nanoparticles as biosensing probe for the detection of Salmonella typhi bacteria on modified polycarbonate (PC) filters in about 3 h. Antibody modified-PC membranes were incubated with contaminated bacterial water for selective capturing which were detected by synthesized novel bioconjugate probe. Core–shell architecture of polymeric nanoparticles endows them with aqueous stabilization and keto-enolic functionalities making them usable for covalently linking S. typhi antibodies without any crosslinker or activator. Bradford analysis revealed that one nanoparticle has an average of 3.51 × 10{sup −19} g or 21 × 10{sup 4} bound S. typhi Ab molecules. Analysis of the regions of interest (ROI) in fluorescent micrographs of modified fluoroimmunoassay showed higher detection sensitivity of 5 × 10{sup 2} cells/mL due to signal amplification unlike conventional naked dye FITC-Ab conjugate. Fluorescence of pyrene dye remained same on immobilization of biomolecules and nanoparticles showed stable fluorescent intensity under prolong exposure to laser owing to protective polymeric layer allowing accurate identification of bacteria. Surface-functionalized PC matrix and fluorescent label NPs permit covalent interactions among biomolecules enhancing signal acquisitions showing higher detection efficiency as compared to conventional microtiter plate-based system. Our novel immunoassay has the potential to be explored as rapid detection method for identifying S. typhi contaminations in water.Graphical Abstract.

  18. Detection of Salmonella typhi utilizing bioconjugated fluorescent polymeric nanoparticles

    Science.gov (United States)

    Jain, Swati; Chattopadhyay, Sruti; Jackeray, Richa; Abid, Zainul; Singh, Harpal

    2016-05-01

    Present work demonstrates effective utilization of functionalized polymeric fluorescent nanoparticles as biosensing probe for the detection of Salmonella typhi bacteria on modified polycarbonate (PC) filters in about 3 h. Antibody modified-PC membranes were incubated with contaminated bacterial water for selective capturing which were detected by synthesized novel bioconjugate probe. Core-shell architecture of polymeric nanoparticles endows them with aqueous stabilization and keto-enolic functionalities making them usable for covalently linking S. typhi antibodies without any crosslinker or activator. Bradford analysis revealed that one nanoparticle has an average of 3.51 × 10-19 g or 21 × 104 bound S. typhi Ab molecules. Analysis of the regions of interest (ROI) in fluorescent micrographs of modified fluoroimmunoassay showed higher detection sensitivity of 5 × 102 cells/mL due to signal amplification unlike conventional naked dye FITC-Ab conjugate. Fluorescence of pyrene dye remained same on immobilization of biomolecules and nanoparticles showed stable fluorescent intensity under prolong exposure to laser owing to protective polymeric layer allowing accurate identification of bacteria. Surface-functionalized PC matrix and fluorescent label NPs permit covalent interactions among biomolecules enhancing signal acquisitions showing higher detection efficiency as compared to conventional microtiter plate-based system. Our novel immunoassay has the potential to be explored as rapid detection method for identifying S. typhi contaminations in water.

  19. Understanding microbial/DOM interactions using fluorescence and flow cytometry

    Science.gov (United States)

    Fox, Bethany; Rushworth, Cathy; Attridge, John; Anesio, Alexandre; Cox, Tim; Reynolds, Darren

    2015-04-01

    The transformation and movement of dissolved organic carbon (DOC) within freshwater aquatic systems is an important factor in the global cycling of carbon. DOC within aquatic systems is known to underpin the microbial food web and therefore plays an essential role in supporting and maintaining the aquatic ecosystem. Despite this the interactions between bacteria and dissolved organic matter (DOM) are not well understood, although the literature indicates that the microbial processing of bioavailable DOM is essential during the production of autochthonous, labile, DOM. DOM can be broadly characterised by its fluorescing properties and Coble et al. (2014) define terrestrially derived DOM as exhibiting "peak C" fluorescence, whilst labile microbially derived DOM is defined as showing "peak T" fluorescence. Our work explores the microbial/DOM interactions by analysing aquatic samples using fluorescence excitation and emission matrices (EEMs) in conjunction with microbial consumption of dissolved oxygen. Environmental and synthetic water samples were subjected to fluorescence characterisation using both fluorescence spectroscopy and in situ fluorescence sensors (Chelsea Technologies Group Ltd.). PARAFAC analysis and peak picking were performed on EEMs and compared with flow cytometry data, used to quantify bacterial numbers present within samples. Synthetic samples were created using glucose, glutamic acid, nutrient-rich water and a standard bacterial seed. Synthetic samples were provided with terrestrially derived DOM via the addition of an aliquot of environmental water. Using a closed system approach, samples were incubated over time (up to a maximum of 20 days) and analysed at pre-defined intervals. The main focus of our work is to improve our understanding of microbial/DOM interactions and how these interactions affect both the DOM characteristics and microbial food web in freshwater aquatic systems. The information gained, in relation to the origin, microbial

  20. Advanced in X-ray fluorescence holography

    CERN Document Server

    Hayashi, K

    2002-01-01

    X-ray fluorescence holography (XFH) can resolve 'phase problem' in crystal diffraction and therefore it provides 3D atomic images around specific elements. Since first demonstration of the XFH in 1996, view of atoms has been improved rapidly with the refinement of the hologram data collection method. The present performance of the XFH makes it possible to apply to impurity, thin film and quasicrystal, and opens a way to practical tool for determination of local structure. In this paper, theory including solutions for twin image problem, advanced experimental systems and application to Si sub 0 sub . sub 9 sub 9 sub 9 Ge sub 0 sub . sub 0 sub 0 sub 1 are discussed. (author)

  1. Interest of fluorescence derivatization and fluorescence probe assisted post-column detection of phospholipids: a short review.

    Science.gov (United States)

    Ibrahim, Hanadi; Caudron, Eric; Kasselouri, Athena; Prognon, Pratrice

    2010-01-18

    Phospholipids are essential constituents of all living cell membranes. There are many analytical methods available for the quantitative and qualitative determination of phospholipids, but since these molecules lack chromophores, common absorbance based methods are of limited use. Beside mass spectrometry, some less specific approaches that are routinely used are evaporative light scattering detection or fluorescence, which exhibit sufficient sensitivity. Here, we focus on fluorescence, which remains an interesting way to quantify phospholipids. Two ways of detecting phospholipids by fluorescence are possible coupled with separation techniques such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE): firstly, pre-column derivatization procedures and secondly, probe assisted post-column detection with suitable fluorescence reagents. In both cases, the common purpose is to increase the detection sensitivity. It is shown that, whereas pre-column derivatization is characterized by selectivity due to the chemical functionality of the analyte involved in the derivatization process, in supramolecular post-column derivatization, the selectivity only proceeds from the capacity of the lipid to involve supramolecular assemblies with a fluorescence probe. The aim of this review is to summarize available experiments concerning fluorescence detection of phospholipids. The interest and limitation of such detection approaches are discussed.

  2. Light-induced fluorescence for pulpal diagnosis

    Science.gov (United States)

    Ebihara, Arata; Liaw, Lih-Huei L.; Krasieva, Tatiana B.; Wilder-Smith, Petra B. B.

    2001-04-01

    A direct non-histological means of pulpal diagnosis remains elusive to clinical practice. Clinical vitality testing remains limited to electric, thermal criteria, or laser Doppler flowmetry. The goal of these investigations was to determine the feasibility of using light-induced fluorescence as a non-invasive modality for pulpal evaluation. Such a capability would, for example, permit expanded use of pulpotomy/pulpectomy techniques. Clinically healthy and diseased human extirpated pulpal tissues were used in this study. After excision, they were rapidly frozen and standard cryosections prepared. Measurement of tissue excitation/emission characteristics was performed using spectrographic analysis. A low-light level fluorescence microscopy system was then used to image autofluorescence localization and intensity at optimal excitation/detection parameters. Excitation/detection parameters used in this study included 405/605, 405/635, 405/670, 440/550, and 440/635. Autofluorescence intensities in healthy tissues were significantly stronger than those in diseased tissues at optimal parameters. It is postulated that autofluorescence characteristics are related to pathology- related structural changes in the pulp. This work provides the basis for further investigation into the relation between autofluorescence, histology and clinical symptoms.

  3. Fluorescence imaging to quantify crop residue cover

    Science.gov (United States)

    Daughtry, C. S. T.; Mcmurtrey, J. E., III; Chappelle, E. W.

    1994-01-01

    Crop residues, the portion of the crop left in the field after harvest, can be an important management factor in controlling soil erosion. Methods to quantify residue cover are needed that are rapid, accurate, and objective. Scenes with known amounts of crop residue were illuminated with long wave ultraviolet (UV) radiation and fluorescence images were recorded with an intensified video camera fitted with a 453 to 488 nm band pass filter. A light colored soil and a dark colored soil were used as background for the weathered soybean stems. Residue cover was determined by counting the proportion of the pixels in the image with fluorescence values greater than a threshold. Soil pixels had the lowest gray levels in the images. The values of the soybean residue pixels spanned nearly the full range of the 8-bit video data. Classification accuracies typically were within 3(absolute units) of measured cover values. Video imaging can provide an intuitive understanding of the fraction of the soil covered by residue.

  4. Rheological phenomena in focus

    CERN Document Server

    Boger, DV

    1993-01-01

    More than possibly any other scientific discipline, rheology is easily visualized and the relevant literature contains many excellent photographs of unusual and often bizarre phenomena. The present book brings together these photographs for the first time. They are supported by a full explanatory text. Rheological Phenomena in Focus will be an indispensable support manual to all those who teach rheology or have to convince colleagues of the practical relevance of the subject within an industrial setting. For those who teach fluid mechanics, the book clearly illustrates the difference be

  5. Focus on Organic Conductors

    Directory of Open Access Journals (Sweden)

    Shinya Uji, Takehiko Mori and Toshihiro Takahashi

    2009-01-01

    Full Text Available Organic materials are usually thought of as electrical insulators. Progress in chemical synthesis, however, has brought us a rich variety of conducting organic materials, which can be classified into conducting polymers and molecular crystals. Researchers can realize highly conducting molecular crystals in charge-transfer complexes, where suitable combinations of organic electron donor or acceptor molecules with counter ions or other organic molecules provide charge carriers. By means of a kind of chemical doping, the charge-transfer complexes exhibit high electrical conductivity and, thanks to their highly crystalline nature, even superconductivity has been observed. This focus issue of Science and Technology of Advanced Materials is devoted to the research into such 'organic conductors'The first organic metal was (TTF(TCNQ, which was found in 1973 to have high conductivity at room temperature and a metal–insulator transition at low temperatures. The first organic superconductor was (TMTSF2PF6, whose superconductivity under high pressures was reported by J´erome in 1980. After these findings, the research on organic conductors exploded. Hundreds of organic conductors have been reported, among which more than one hundred exhibit superconductivity. Recently, a single-component organic conductor has been found with metallic conductivity down to low temperatures.In these organic conductors, in spite of their simple electronic structures, much new physics has arisen from the low dimensionality. Examples are charge and spin density waves, characteristic metal–insulator transitions, charge order, unconventional superconductivity, superconductor–insulator transitions, and zero-gap conductors with Dirac cones. The discovery of this new physics is undoubtedly derived from the development of many intriguing novel organic conductors. High quality single crystals are indispensable to the precise measurement of electronic states.This focus issue

  6. Ultrasensitive fluorescence immunoassay for detection of ochratoxin A using catalase-mediated fluorescence quenching of CdTe QDs

    Science.gov (United States)

    Huang, Xiaolin; Zhan, Shengnan; Xu, Hengyi; Meng, Xianwei; Xiong, Yonghua; Chen, Xiaoyuan

    2016-04-01

    Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to perform a dynamic linear detection of OTA ranging from 0.05 pg mL-1 to 10 pg mL-1. The half maximal inhibitory concentration was 0.53 pg mL-1 and the limit of detection was 0.05 pg mL-1. These values were approximately 283- and 300-folds lower than those of horseradish peroxidase (HRP)-based conventional ELISA, respectively. The reported method is accurate, highly reproducible, and specific against other mycotoxins in agricultural products as well. In summary, the developed fluorescence immunoassay based on H2O2-induced fluorescence quenching of CdTe QDs can be used for the rapid and highly sensitive detection of mycotoxins or haptens in food safety monitoring.Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to

  7. Fluorescence lifetime imaging microscopy (FLIM).

    NARCIS (Netherlands)

    van Munster, E.B.; Gadella, Th.W.J.; Rietdorf, J.

    2005-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is a technique to map the spatial distribution of nanosecond excited state lifetimes within microscopic images. FLIM systems have been implemented both in the frequency domain, using sinusoidally intensity-modulated excitation light and modulated

  8. Advanced Methods in Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Luke Fritzky

    2013-01-01

    Full Text Available It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

  9. Indocyanine green fluorescence angiography for quantitative evaluation of in situ parathyroid gland perfusion and function after total thyroidectomy.

    Science.gov (United States)

    Lang, Brian Hung-Hin; Wong, Carlos K H; Hung, Hing Tsun; Wong, Kai Pun; Mak, Ka Lun; Au, Kin Bun

    2017-01-01

    Because the fluorescent light intensity on an indocyanine green fluorescence angiography reflects the blood perfusion within a focused area, the fluorescent light intensity in the remaining in situ parathyroid glands may predict postoperative hypocalcemia risk after total thyroidectomy. Seventy patients underwent intraoperative indocyanine green fluorescence angiography after total thyroidectomy. Any parathyroid glands with a vascular pedicle was left in situ while any parathyroid glands without pedicle or inadvertently removed was autotransplanted. After total thyroidectomy, an intravenous 2.5 mg indocyanine green fluorescence angiography was given and real-time fluorescent images of the thyroid bed were recorded using the SPY imaging system (Novadaq, Ontario, Canada). The fluorescent light intensity of each indocyanine green fluorescence angiography as well as the average and greatest fluorescent light intensity in each patient were calculated. Postoperative hypocalcemia was defined as adjusted calcium 150% developed postoperative hypocalcemia while 9 (81.8%) patients with a greatest fluorescent light intensity ≤150% did. Similarly, no patients with an average fluorescent light intensity >109% developed PH while 9 (30%) with an average fluorescent light intensity ≤109% did. The greatest fluorescent light intensity was more predictive than day-0 postoperative hypocalcemia (P = .027) and % PTH drop day-0 to 1 (P parathyroid glands function and predicting postoperative hypocalcemia risk after total thyroidectomy. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. CERN In Focus

    CERN Multimedia

    CERN audiovisual service

    2008-01-01

    First edition 2008 of Cern in Focus. On behalf of the audiovisual team, a selection of the latest videos filmed at CERN. Every six weeks, we will bring you the latest in CERN's activities, from LHC start up to the Computing Grid, featuring the experiments and many other goings-on at CERN. The agenda of this first edition of CERN in Focus features the visit of the prime minister of Malta, Lawrence Gonzi... CMS and the final descent of the YE-1 end cap... The departure of UA1 magnets to Japan... The start up of sectors 4 and 5... And finally, in our sports round up... We'll talk about football. New in brief this month... The final bolt is in place : On 7th November, in the bowels of the LHC tunnel, CERN's Director General Robert Aymar tightened a gold-plated bolt for the last arc interconnection of sector 1-2. This symbolic gesture marks the completion of all the arc interconnections of the LHC. Last welding work: it was never going to be an easy task. On this day last year just one sector had been completed,...

  11. A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe.

    Science.gov (United States)

    Liu, Hua; Na, Weidan; Liu, Ziping; Chen, Xueqian; Su, Xingguang

    2017-06-15

    In this paper, a facile and rapid fluorescence turn-on assay for fluorescent detection of ascorbic acid (AA) was developed by using the orange emission graphene quantum dots (GQDs). In the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2), catechol can be oxidized by hydroxyl radicals and converted to o-benzoquinone, which can significantly quench the fluorescence of GQDs. However, when AA present in the system, it can consume part of H2O2 and hydroxyl radicals to inhibit the generation of o-benzoquinone, resulting in fluorescence recovery. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of H2O2 in the range of 3.33-500µM with a detection limit of 1.2µM. The linear detection for AA was in the range from 1.11 to 300µM with a detection limit of 0.32µM. The proposed method was applied to the determination of AA in human serum samples with satisfactory results. Copyright © 2017. Published by Elsevier B.V.

  12. Fluorescence axial nanotomography with plasmonics.

    Science.gov (United States)

    Cade, Nicholas I; Fruhwirth, Gilbert O; Krasavin, Alexey V; Ng, Tony; Richards, David

    2015-01-01

    We present a novel imaging technique with super-resolution axial sensitivity, exploiting the changes in fluorescence lifetime above a plasmonic substrate. Using conventional confocal fluorescence lifetime imaging, we show that it is possible to deliver down to 6 nm axial position sensitivity of fluorophores in whole biological cell imaging. We employ this technique to map the topography of the cellular membrane, and demonstrate its application in an investigation of receptor-mediated endocytosis in carcinoma cells.

  13. X-ray fluorescence holography

    CERN Document Server

    Hayashi, K; Takahashi, Y

    2003-01-01

    X-ray fluorescence holography (XFH) is a new structural analysis method of determining a 3D atomic arrangement around fluorescing atoms. We developed an XFH apparatus using advanced X-ray techniques and succeeded in obtaining high-quality hologram data. Furthermore, we introduced applications to the structural analysis of a thin film and the environment around dopants and, discussed the quantitative analysis of local lattice distortion. (author)

  14. Light Sheet Fluorescence Microscopy (LSFM)

    OpenAIRE

    Adams, Michael W.; Loftus, Andrew F.; Dunn, Sarah E.; Joens, Matthew S.; Fitzpatrick, James A. J.

    2015-01-01

    The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light Sheet Fluorescent Mi...

  15. Radiation Dosimetry via Automated Fluorescence Microscopy

    Science.gov (United States)

    Castleman, Kenneth R.; Schulze, Mark

    2005-01-01

    A developmental instrument for assessment of radiation-induced damage in human lymphocytes includes an automated fluorescence microscope equipped with a one or more chargecoupled- device (CCD) video camera(s) and circuitry to digitize the video output. The microscope is also equipped with a three-axis translation stage that includes a rotation stage, and a rotary tray that holds as many as thirty specimen slides. The figure depicts one version of the instrument. Once the slides have been prepared and loaded into the tray, the instrument can operate unattended. A computer controls the operation of the stage, tray, and microscope, and processes the digital fluorescence-image data to recognize and count chromosomes that have been broken, presumably by radiation. The design and method of operation of the instrument exploit fluorescence in situ hybridization (FISH) of metaphase chromosome spreads, which is a technique that has been found to be valuable for monitoring the radiation dose to circulating lymphocytes. In the specific FISH protocol used to prepare specimens for this instrument, metaphase lymphocyte cultures are chosen for high mitotic index and highly condensed chromosomes, then several of the largest chromosomes are labeled with three of four differently colored whole-chromosome-staining dyes. The three dyes, which are used both individually and in various combinations, are fluorescein isothiocyanate (FITC), Texas Red (or equivalent), and Cy5 (or equivalent); The fourth dye 4',6-diamidino- 2-phenylindole (DAPI) is used as a counterstain. Under control by the computer, the microscope is automatically focused on the cells and each slide is scanned while the computer analyzes the DAPI-fluorescence images to find the metaphases. Each metaphase field is recentered in the field of view and refocused. Then a four-color image (more precisely, a set of images of the same view in the fluorescent colors of the four dyes) is acquired. By use of pattern

  16. Effects of Anaerobiosis on Chlorophyll Fluorescence Yield in Spinach (Spinacia oleracea) Leaf Discs.

    Science.gov (United States)

    Harris, G. C.; Heber, U.

    1993-01-01

    When spinach (Spinacia oleracea) leaf discs were incubated in a dark anaerobic environment, the chlorophyll fluorescence yield was much increased relative to the aerobic control. Occasionally, the fluorescence yield of the darkened anaerobic samples approached 80% of the maximum fluorescence. The anaerobic incubation period also induced in a leaf disc the capacity to exhibit a low light-mediated chlorophyll fluorescence induction phenomenon. This involved a rapid and slow increase in fluorescence yield, followed by a slow quenching. This could be induced by light levels as low as 400 [mu]W m-2. The anaerobic-dependent increase in chlorophyll fluorescence yield could be relaxed by either far-red light, O2, or a saturating pulse of white light. It was concluded that the anaerobic-dependent increase in chlorophyll fluorescence yield was due to a dark reduction of the plastoquinone pool and its relaxation by reoxidation. Darkened isolated chloroplasts did not exhibit a fluorescence yield increase under anaerobic conditions. Fluorescence slowly increased only when dithiothreitol or dithionite was added. PMID:12231769

  17. Measurement of protein-like fluorescence in river and waste water using a handheld spectrophotometer.

    Science.gov (United States)

    Baker, Andy; Ward, David; Lieten, Shakti H; Periera, Ryan; Simpson, Ellie C; Slater, Malcolm

    2004-07-01

    Protein-like fluorescence intensity in rivers increases with increasing anthropogenic DOM inputs from sewerage and farm wastes. Here, a portable luminescence spectrophotometer was used to investigate if this technology could be used to provide both field scientists with a rapid pollution monitoring tool and process control engineers with a portable waste water monitoring device, through the measurement of river and waste water tryptophan-like fluorescence from a range of rivers in NE England and from effluents from within two waste water treatment plants. The portable spectrophotometer determined that waste waters and sewerage effluents had the highest tryptophan-like fluorescence intensity, urban streams had an intermediate tryptophan-like fluorescence intensity, and the upstream river samples of good water quality the lowest tryptophan-like fluorescence intensity. Replicate samples demonstrated that fluorescence intensity is reproducible to +/- 20% for low fluorescence, 'clean' river water samples and +/- 5% for urban water and waste waters. Correlations between fluorescence measured by the portable spectrophotometer with a conventional bench machine were 0.91; (Spearman's rho, n = 143), demonstrating that the portable spectrophotometer does correlate with tryptophan-like fluorescence intensity measured using the bench spectrophotometer.

  18. Theoretical Investigations of Optical Origins of Fluorescent Graphene Quantum Dots

    Science.gov (United States)

    Wang, Jingang; Cao, Shuo; Ding, Yong; Ma, Fengcai; Lu, Wengang; Sun, Mengtao

    2016-04-01

    The optical properties of graphene quantum dots (GQDs) were investigated theoretically. We focused on the photoinduced charge transfer and electron-hole coherence of single-layer graphene in the electronic transitions in the visible regions. Surface functionalization with donor or acceptor groups produced a red shift in the absorption spectrum, and electrons and holes were highly delocalized. The recombination of excited, well-separated electron-hole (e-h) pairs can result in enhanced fluorescence. This fluorescence enhancement by surface functionalization occurs because of the decreased symmetry of the graphene resulting from the roughened structure of the surface-functionalized GQDs.

  19. Theoretical Investigations of Optical Origins of Fluorescent Graphene Quantum Dots

    Science.gov (United States)

    Wang, Jingang; Cao, Shuo; Ding, Yong; Ma, Fengcai; Lu, Wengang; Sun, Mengtao

    2016-01-01

    The optical properties of graphene quantum dots (GQDs) were investigated theoretically. We focused on the photoinduced charge transfer and electron-hole coherence of single-layer graphene in the electronic transitions in the visible regions. Surface functionalization with donor or acceptor groups produced a red shift in the absorption spectrum, and electrons and holes were highly delocalized. The recombination of excited, well-separated electron-hole (e–h) pairs can result in enhanced fluorescence. This fluorescence enhancement by surface functionalization occurs because of the decreased symmetry of the graphene resulting from the roughened structure of the surface-functionalized GQDs. PMID:27094439

  20. Rapid Airplane Parametric Input Design (RAPID)

    Science.gov (United States)

    Smith, Robert E.

    1995-01-01

    RAPID is a methodology and software system to define a class of airplane configurations and directly evaluate surface grids, volume grids, and grid sensitivity on and about the configurations. A distinguishing characteristic which separates RAPID from other airplane surface modellers is that the output grids and grid sensitivity are directly applicable in CFD analysis. A small set of design parameters and grid control parameters govern the process which is incorporated into interactive software for 'real time' visual analysis and into batch software for the application of optimization technology. The computed surface grids and volume grids are suitable for a wide range of Computational Fluid Dynamics (CFD) simulation. The general airplane configuration has wing, fuselage, horizontal tail, and vertical tail components. The double-delta wing and tail components are manifested by solving a fourth order partial differential equation (PDE) subject to Dirichlet and Neumann boundary conditions. The design parameters are incorporated into the boundary conditions and therefore govern the shapes of the surfaces. The PDE solution yields a smooth transition between boundaries. Surface grids suitable for CFD calculation are created by establishing an H-type topology about the configuration and incorporating grid spacing functions in the PDE equation for the lifting components and the fuselage definition equations. User specified grid parameters govern the location and degree of grid concentration. A two-block volume grid about a configuration is calculated using the Control Point Form (CPF) technique. The interactive software, which runs on Silicon Graphics IRIS workstations, allows design parameters to be continuously varied and the resulting surface grid to be observed in real time. The batch software computes both the surface and volume grids and also computes the sensitivity of the output grid with respect to the input design parameters by applying the precompiler tool

  1. Comparison of Xpert Flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza A and B.

    Science.gov (United States)

    DiMaio, Michael A; Sahoo, Malaya K; Waggoner, Jesse; Pinsky, Benjamin A

    2012-12-01

    Influenza infections are associated with thousands of hospital admissions and deaths each year. Rapid detection of influenza is important for prompt initiation of antiviral therapy and appropriate patient triage. In this study the Cepheid Xpert Flu assay was compared with two rapid antigen tests, BinaxNOW Influenza A & B and BD Directigen EZ Flu A+B, as well as direct fluorescent antibody testing for the rapid detection of influenza A and B. Using real-time, hydrolysis probe-based, reverse transcriptase PCR as the reference method, influenza A sensitivity was 97.3% for Xpert Flu, 95.9% for direct fluorescent antibody testing, 62.2% for BinaxNOW, and 71.6% for BD Directigen. Influenza B sensitivity was 100% for Xpert Flu and direct fluorescent antibody testing, 54.5% for BinaxNOW, and 48.5% for BD Directigen. Specificity for influenza A was 100% for Xpert Flu, BinaxNOW, and BD Directigen, and 99.2% for direct fluorescent antibody testing. All methods demonstrated 100% specificity for influenza B. These findings support the use of the Xpert Flu assay in settings requiring urgent diagnosis of influenza A and B. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Single aflatoxin contaminated corn kernel analysis with fluorescence hyperspectral image

    Science.gov (United States)

    Yao, Haibo; Hruska, Zuzana; Kincaid, Russell; Ononye, Ambrose; Brown, Robert L.; Cleveland, Thomas E.

    2010-04-01

    Aflatoxins are toxic secondary metabolites of the fungi Aspergillus flavus and Aspergillus parasiticus, among others. Aflatoxin contaminated corn is toxic to domestic animals when ingested in feed and is a known carcinogen associated with liver and lung cancer in humans. Consequently, aflatoxin levels in food and feed are regulated by the Food and Drug Administration (FDA) in the US, allowing 20 ppb (parts per billion) limits in food and 100 ppb in feed for interstate commerce. Currently, aflatoxin detection and quantification methods are based on analytical tests including thin-layer chromatography (TCL) and high performance liquid chromatography (HPLC). These analytical tests require the destruction of samples, and are costly and time consuming. Thus, the ability to detect aflatoxin in a rapid, nondestructive way is crucial to the grain industry, particularly to corn industry. Hyperspectral imaging technology offers a non-invasive approach toward screening for food safety inspection and quality control based on its spectral signature. The focus of this paper is to classify aflatoxin contaminated single corn kernels using fluorescence hyperspectral imagery. Field inoculated corn kernels were used in the study. Contaminated and control kernels under long wavelength ultraviolet excitation were imaged using a visible near-infrared (VNIR) hyperspectral camera. The imaged kernels were chemically analyzed to provide reference information for image analysis. This paper describes a procedure to process corn kernels located in different images for statistical training and classification. Two classification algorithms, Maximum Likelihood and Binary Encoding, were used to classify each corn kernel into "control" or "contaminated" through pixel classification. The Binary Encoding approach had a slightly better performance with accuracy equals to 87% or 88% when 20 ppb or 100 ppb was used as classification threshold, respectively.

  3. CERN in Focus

    CERN Multimedia

    CERN audiovisual service

    2008-01-01

    CERN in Focus 1ère édition. Le Service Audiovisuel vous propose un panorama des dernières vidéos tournées au CERN. En effet, toutes les six semaines, nous vous présenterons l'ensemble des activités, du démarrage du LHC à la grille de calcul, en passant par les différentes expériences. Au sommaire de cette édition, la visite du premier ministre de Malte, CMS et la derniere descente du YE-1 Le depart des aimants UA1 pour le Japon La mise en marche des secteurs 4 et 5 Et enfin, ATLAS et la descente... BREVES Dernieres soudures LHC Derniers boulonnages LHC College leman Appel a candidature Open Day

  4. Doing focus group research

    DEFF Research Database (Denmark)

    Lindegaard, Laura Bang

    2014-01-01

    that interview data can be of some use if the distinction between natural and contrived data is given up and replaced with a distinction between interview data as topic or as resource. In greater detail, such scholars argue that interview data are perfectly adequate if the researcher wants to study the topic......Scholars of ethnomethodologically informed discourse studies are often sceptical of the use of interview data such as focus group data. Some scholars quite simply reject interview data with reference to a general preference for so-called naturally occurring data. Other scholars acknowledge...... of interview interaction, but inadequate as data for studying phenomena that go beyond the phenomenon of interview interaction. Neither of these more and less sceptical positions are, on the face of it, surprising due to the ethnomethodological commitment to study social order as accomplished in situ...

  5. Regadenoson: a focused update.

    Science.gov (United States)

    Ghimire, Gopal; Hage, Fadi G; Heo, Jaekyeong; Iskandrian, Ami E

    2013-04-01

    Since its approval by the Food and Drug Administration in 2008, regadenoson has become the most commonly used vasodilator in the United States. Previous reviews have summarized the pre-clinical and clinical data on the use of regadenoson for myocardial perfusion imaging (MPI). Since then, data have emerged on the safety of this agent in special groups of patients such as those with chronic kidney disease, airway disease (asthma and chronic obstructive pulmonary disease), and liver disease. There has also been recent interest in the use of regadenoson in hybrid protocols with exercise as a way to improve patient tolerance and image quality. Finally, although regadenoson was approved for clinical use based on the agreement rate of regadenoson MPI and adenosine MPI with regards to perfusion abnormalities, data are now available on the prognostic data derived from regadenoson MPI. We will briefly summarize these recent reports here in a focused update on the use of regadenoson for MPI.

  6. Focused Ultrasound and Lithotripsy.

    Science.gov (United States)

    Ikeda, Teiichiro; Yoshizawa, Shin; Koizumi, Norihiro; Mitsuishi, Mamoru; Matsumoto, Yoichiro

    2016-01-01

    Shock wave lithotripsy has generally been a first choice for kidney stone removal. The shock wave lithotripter uses an order of microsecond pulse durations and up to a 100 MPa pressure spike triggered at approximately 0.5-2 Hz to fragment kidney stones through mechanical mechanisms. One important mechanism is cavitation. We proposed an alternative type of lithotripsy method that maximizes cavitation activity to disintegrate kidney stones using high-intensity focused ultrasound (HIFU). Here we outline the method according to the previously published literature (Matsumoto et al., Dynamics of bubble cloud in focused ultrasound. Proceedings of the second international symposium on therapeutic ultrasound, pp 290-299, 2002; Ikeda et al., Ultrasound Med Biol 32:1383-1397, 2006; Yoshizawa et al., Med Biol Eng Comput 47:851-860, 2009; Koizumi et al., A control framework for the non-invasive ultrasound the ragnostic system. Proceedings of 2009 IEEE/RSJ International Conference on Intelligent Robotics and Systems (IROS), pp 4511-4516, 2009; Koizumi et al., IEEE Trans Robot 25:522-538, 2009). Cavitation activity is highly unpredictable; thus, a precise control system is needed. The proposed method comprises three steps of control in kidney stone treatment. The first step is control of localized high pressure fluctuation on the stone. The second step is monitoring of cavitation activity and giving feedback on the optimized ultrasound conditions. The third step is stone tracking and precise ultrasound focusing on the stone. For the high pressure control we designed a two-frequency wave (cavitation control (C-C) waveform); a high frequency ultrasound pulse (1-4 MHz) to create a cavitation cloud, and a low frequency trailing pulse (0.5 MHz) following the high frequency pulse to force the cloud into collapse. High speed photography showed cavitation collapse on a kidney stone and shock wave emission from the cloud. We also conducted in-vitro erosion tests of model and natural

  7. The FOCUS trial

    DEFF Research Database (Denmark)

    Glenthøj, Louise B; Fagerlund, Birgitte; Randers, Lasse

    2015-01-01

    of the cognitive training to their everyday lives. Follow-up assessments will be conducted at 6 and 12 months after randomisation. The primary outcome is the composite score on the Brief Assessment of Cognition in Schizophrenia at cessation of treatment after 6 months. Secondary outcomes are social and daily...... versus standard treatment. The cognitive remediation consists of 24 weekly group-based and manualised sessions targeting neurocognition and social cognition. In addition to the group sessions, the patients will be offered 12 individual sessions aiming at maximising the transfer of the effects......: This is the first trial to evaluate the effects of neurocognitive and social cognitive remediation in UHR patients. The FOCUS trial results will provide evidence on the effect of targeted and comprehensive cognitive rehabilitation on cognition, daily living, and symptomatology as well as long-term outcome...

  8. Focusing on customer service.

    Science.gov (United States)

    1996-01-01

    This booklet is devoted to a consideration of how good customer service in family planning programs can generate demand for products and services, bring customers back, and reduce costs. Customer service is defined as increasing client satisfaction through continuous concern for client preferences, staff accountability to clients, and respect for the rights of clients. Issues discussed include the introduction of a customer service approach and gaining staff commitment. The experience of PROSALUD in Bolivia in recruiting appropriate staff, supervising staff, soliciting client feedback, and marketing services is offered as an example of a successful customer service approach. The key customer service functions are described as 1) establishing a welcoming atmosphere, 2) streamlining client flow, 3) personalizing client services, and 4) organizing and providing clear information to clients. The role of the manager in developing procedures is explored, and the COPE (Client-Oriented Provider-Efficient) process is presented as a good way to begin to make improvements. Techniques in staff training in customer service include brainstorming, role playing, using case studies (examples of which are provided), and engaging in practice sessions. Training also leads to the development of effective customer service attitudes, and the differences between these and organizational/staff-focused attitudes are illustrated in a chart. The use of communication skills (asking open-ended questions, helping clients express their concerns, engaging in active listening, and handling difficult situations) is considered. Good recovery skills are important when things go wrong. Gathering and using client feedback is the next topic considered. This involves identifying, recording, and discussing customer service issues as well as taking action on these issues and evaluating the results. The booklet ends by providing a sample of customer service indicators, considering the maintenance of a

  9. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Miotke, Laura; Maity, Arindam; Ji, Hanlee

    2015-01-01

    of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence......) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base...... pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least...

  10. Fluorescence spectroscopy as a tool for determination of organic matter removal efficiency at water treatment works

    Directory of Open Access Journals (Sweden)

    M. Z. Bieroza

    2010-04-01

    Full Text Available Organic matter (OM in drinking water treatment is a common impediment responsible for increased coagulant and disinfectant dosages, formation of carcinogenic disinfection-by products, and microbial re-growth in distribution system. The inherent heterogeneity of OM implies the utilization of advanced analytical techniques for its characterization and assessment of removal efficiency. Here, the application of simple fluorescence excitation-emission technique to OM characterization in drinking water treatment is presented. The fluorescence data of raw and clarified water was obtained from 16 drinking water treatment works. The reduction in fulvic-like fluorescence was found to significantly correlate with OM removal measured with total organic carbon (TOC. Fluorescence properties, fulvic- and tryptophan-like regions, were found to discriminate OM fractions of different removal efficiencies. The results obtained in the study show that fluorescence spectroscopy provides a rapid and accurate characterization and quantification of OM fractions and indication of their treatability in conventional water treatment.

  11. Stochastic Micro-Pattern for Automated Correlative Fluorescence - Scanning Electron Microscopy

    Science.gov (United States)

    Begemann, Isabell; Viplav, Abhiyan; Rasch, Christiane; Galic, Milos

    2015-01-01

    Studies of cellular surface features gain from correlative approaches, where live cell information acquired by fluorescence light microscopy is complemented by ultrastructural information from scanning electron micrographs. Current approaches to spatially align fluorescence images with scanning electron micrographs are technically challenging and often cost or time-intensive. Relying exclusively on open-source software and equipment available in a standard lab, we have developed a method for rapid, software-assisted alignment of fluorescence images with the corresponding scanning electron micrographs via a stochastic gold micro-pattern. Here, we provide detailed instructions for micro-pattern production and image processing, troubleshooting for critical intermediate steps, and examples of membrane ultra-structures aligned with the fluorescence signal of proteins enriched at such sites. Together, the presented method for correlative fluorescence – scanning electron microscopy is versatile, robust and easily integrated into existing workflows, permitting image alignment with accuracy comparable to existing approaches with negligible investment of time or capital. PMID:26647824

  12. Rapid shallow breathing

    Science.gov (United States)

    ... the smallest air passages of the lungs in children ( bronchiolitis ) Pneumonia or other lung infection Transient tachypnea of the newborn Anxiety and panic Other serious lung disease Home Care Rapid, shallow breathing should not be treated at home. It is ...

  13. Rapid Strep Test

    Science.gov (United States)

    ... worse than normal. Your first thoughts turn to strep throat. A rapid strep test in your doctor’s office ... your suspicions.Viruses cause most sore throats. However, strep throat is an infection caused by the Group A ...

  14. Design Strategies for Fluorescent Biodegradable Polymeric Biomaterials.

    Science.gov (United States)

    Zhang, Yi; Yang, Jian

    2013-01-14

    The marriage of biodegradable polymer and fluorescent imaging has resulted in an important area of polymeric biomaterials: biodegradable fluorescent polymers. Researchers have put significant efforts on developing versatile fluorescent biomaterials due to their promising in biological/biomedical labeling, tracking, monitoring, imaging, and diagnostic applications, especially in drug delivery, tissue engineering, and cancer imaging applications. Biodegradable fluorescent polymers can function not only as implant biomaterials but also as imaging probes. Currently, there are two major classes of biodegradable polymers used as fluorescent materials. The first class is the combination of non-fluorescent biodegradable polymers and fluorescent agents such as organic dyes and quantum dots. Another class of polymers shows intrinsic photoluminescence as polymers by themselves carrying integral fluorescent chemical structures in or pendent to their polymer backbone, such as Green Fluorescent protein (GFP), and the recently developed biodegradable photoluminescent polymer (BPLP). Thus there is no need to conjugate or encapsulate additional fluorescent materials for the latter. In the present review, we will review the fluorescent biodegradable polymers with emphases on material fluorescence mechanism, design criteria for fluorescence, and their cutting-edge applications in biomedical engineering. We expect that this review will provide insightful discussion on the fluorescent biomaterial design and lead to innovations for the development of the next generation of fluorescent biomaterials and fluorescence-based biomedical technology.

  15. Design Strategies for Fluorescent Biodegradable Polymeric Biomaterials

    OpenAIRE

    Zhang, Yi; Yang, Jian

    2012-01-01

    The marriage of biodegradable polymer and fluorescent imaging has resulted in an important area of polymeric biomaterials: biodegradable fluorescent polymers. Researchers have put significant efforts on developing versatile fluorescent biomaterials due to their promising in biological/biomedical labeling, tracking, monitoring, imaging, and diagnostic applications, especially in drug delivery, tissue engineering, and cancer imaging applications. Biodegradable fluorescent polymers can function ...

  16. Design Strategies for Fluorescent Biodegradable Polymeric Biomaterials

    Science.gov (United States)

    Zhang, Yi; Yang, Jian

    2013-01-01

    The marriage of biodegradable polymer and fluorescent imaging has resulted in an important area of polymeric biomaterials: biodegradable fluorescent polymers. Researchers have put significant efforts on developing versatile fluorescent biomaterials due to their promising in biological/biomedical labeling, tracking, monitoring, imaging, and diagnostic applications, especially in drug delivery, tissue engineering, and cancer imaging applications. Biodegradable fluorescent polymers can function not only as implant biomaterials but also as imaging probes. Currently, there are two major classes of biodegradable polymers used as fluorescent materials. The first class is the combination of non-fluorescent biodegradable polymers and fluorescent agents such as organic dyes and quantum dots. Another class of polymers shows intrinsic photoluminescence as polymers by themselves carrying integral fluorescent chemical structures in or pendent to their polymer backbone, such as Green Fluorescent protein (GFP), and the recently developed biodegradable photoluminescent polymer (BPLP). Thus there is no need to conjugate or encapsulate additional fluorescent materials for the latter. In the present review, we will review the fluorescent biodegradable polymers with emphases on material fluorescence mechanism, design criteria for fluorescence, and their cutting-edge applications in biomedical engineering. We expect that this review will provide insightful discussion on the fluorescent biomaterial design and lead to innovations for the development of the next generation of fluorescent biomaterials and fluorescence-based biomedical technology. PMID:23710326

  17. Fluorescent retroreflective signing of work zones : abstract

    NARCIS (Netherlands)

    Vos, A.P. de; Horst, A.R.A. van der; Alferdinck, J.W.A.M.; Kooi, F.L.

    1999-01-01

    Fluorescent retroreflective materials increase the brightness of traffic signs. In construction work zones a benefit is expected from the increased conspicuity of fluorescent retroreflective signs. Fluorescent material can be used instead of non-fluorescent materials both for the advance warning

  18. 21 CFR 892.1220 - Fluorescent scanner.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fluorescent scanner. 892.1220 Section 892.1220...) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1220 Fluorescent scanner. (a) Identification. A fluorescent scanner is a device intended to measure the induced fluorescent radiation in the body by exposing...

  19. RAPID3? Aptly named!

    Science.gov (United States)

    Berthelot, J-M

    2014-01-01

    The RAPID3 score is the sum of three 0-10 patient self-report scores: pain, functional impairment on MDHAQ, and patient global estimate. It requires 5 seconds for scoring and can be used in all rheumatologic conditions, although it has mostly been used in rheumatoid arthritis where cutoffs for low disease activity (12/30) have been set. A RAPID3 score of ≤ 3/30 with 1 or 0 swollen joints (RAPID3 ≤ 3 + ≤ SJ1) provides remission criteria comparable to Boolean, SDAI, CDAI, and DAS28 remission criteria, in far less time than a formal joint count. RAPID3 performs as well as the DAS28 in separating active drugs from placebos in clinical trials. RAPID3 also predicts subsequent structural disease progression. RAPID3 can be determined at short intervals at home, allowing the determination of the area under the curve of disease activity between two visits and flare detection. However, RAPID3 should not be seen as a substitute for DAS28 and face to face visits in routine care. Monitoring patient status with only self-report information without a rheumatologist's advice (including joints and physical examination, and consideration of imaging and laboratory tests) may indeed be as undesirable for most patients than joint examination without a patient questionnaire. Conversely, combining the RAPID3 and the DAS28 may consist in faster or more sensitive confirmation that a medication is effective. Similarly, better enquiring of most important concerns of patients (pain, functional status and overall opinion on their disorder) should reinforces patients' confidence in their rheumatologist and treatments.

  20. Method to minimize ozone effect on Cy5 fluorescent intensity in DNA microarrays.

    Science.gov (United States)

    Kim, Youngjun; Seo, Hyun Hee; Jeong, Mi Seon; Lee, Ki Heon; Lee, In Ho; So, Kyeong A; Kim, Mi Kyung; Lee, Yoo-Kyung; Kim, Seon Ah; Kim, Tae Jin

    2017-12-01

    Cyanine 5 (Cy5) is an established fluorescent dye in microarray analysis. It is degraded rapidly when exposed to atmospheric ozone during post-hybridization washes, which leads to loss of fluorescent intensity. To minimize this undesirable effect, we coated microarray slides with sodium dodecyl sulfate (SDS) solution at post-hybridization washes. The fluorescent intensities on coated slides were more stable than those on uncoated slide. We also performed the microarrays with SDS solution for a year to check the solution's effectiveness along with seasonal changes of atmospheric ozone level. Consistent results in microarray analysis were obtained using Cy5 dye under atmospheric ozone. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Optofluidic fluorescent imaging cytometry on a cell phone.

    Science.gov (United States)

    Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

    2011-09-01

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in

  2. Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled Protein A Caused by Affinity Binding with Immunoglobulin G in Bovine Plasma

    Directory of Open Access Journals (Sweden)

    Kiyotaka Sakai

    2009-10-01

    Full Text Available Fluorescence enhancement of fluorescein isothiocyanate-labeled protein A (FITC-protein A caused by the binding with immunoglobulin G (IgG in bovine plasma was studied. FITC-protein A was immobilized onto a glass surface by covalent bonds. An increase in fluorescence intensity was dependent on IgG concentration ranging from 20 to 78 μg/mL in both phosphate buffer saline and bovine plasma. This method requires no separation procedure, and the reaction time is less than 15 min. A fluorescence enhancement assay by the affinity binding of fluorescence-labeled reagent is thus available for the rapid determination of biomolecules in plasma.

  3. Fluorescence detection in (sub-)nanoliter microarrays

    Science.gov (United States)

    van den Doel, L. Richard; Vellekoop, Michael J.; Sarro, Pasqualina M.; Picioreanu, S.; Moerman, R.; Frank, J.; van Dedem, G. W. K.; Hjelt, Kari H.; van Vliet, Lucas J.; Young, Ian T.

    1999-06-01

    The goal of our TU Delft interfaculty research program is to develop intelligent molecular diagnostic systems (IMDS) that can analyze liquid samples that contain a variety of biochemical compounds such as those associated with fermentation processes. One specific project within the IMDS program focuses on photon sensors. In order to analyze the liquid samples we use dedicated microarrays. At this stage, these are basically miniaturized micro titre plates. Typical dimensions of a vial are 200 X 200 X 20 micrometer3. These dimensions may be varied and the shape of the vials can be modified with a result that the volume of the vials varies from 0.5 to 1.6 nl. For all experiments, we have used vials with the shape of a truncated pyramid. These vials are fabricated in silicon by a wet etching process. For testing purposes the vials are filled with rhodamine solutions of various concentrations. To avoid evaporation glycerol-water (1:1, v/v) with a viscosity of 8.3 times the viscosity of water is used as solvent. We aim at wide field-of-view imaging at the expense of absolute sensitivity: the field-of-view increases quadratically with decreasing magnification. Small magnification, however, implies low Numerical Aperture (NA). The ability of a microscope objective to collect photons is proportional to the square of the NA. To image the entire microarray we have used an epi-illumination fluorescence microscope equipped with a low magnification (2.5 X/0.075) objective and a scientific CCD camera to integrate the photons emitted from the fluorescing particles in the solutions in the vials. From these experiments we found that for this setup the detection limit is on the order of micromolar concentrations of fluorescing particles. This translates to 108 molecules per vial.

  4. Implementation of rapid diagnostics with antimicrobial stewardship.

    Science.gov (United States)

    Minejima, Emi; Wong-Beringer, Annie

    2016-11-01

    Antimicrobial stewardship (ASP) is an intervention-based program to improve patient outcomes to infection while limiting spread of resistance and unintended consequences. Many rapid diagnostic tools are now FDA cleared for clinical use, with three evaluated across multiple settings: Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry, Verigene, and FilmArray. Areas covered: This review will focus on studies published that evaluated ASP intervention with rapid diagnostic implementation on outcomes of infection. A description of the key ASP personnel, rapid diagnostic notification methods, hours of notification, and scope of ASP intervention is summarized. Expert commentary: It is critical that ASPs continually re-evaluate and evolve with technological advances. Rapid diagnostic tools are powerful in their ability to identify organisms quickly. A trained clinician is needed to evaluate the results and interact with the providers to educate them on result interpretation and optimal antimicrobial selection to maximize treatment success.

  5. Fluorescence Quenching of a Conjugated Polymer by Synergistic Amine-Carboxylic Acid and π-π Interactions for Selective Detection of Aromatic Amines in Aqueous Solution.

    Science.gov (United States)

    Zhao, Yi-Jia; Miao, Kesong; Zhu, Zhengtao; Fan, Li-Juan

    2017-06-23

    Fluorescence sensing of amine in aqueous solution is challenging. The various basicity and chemical structures of amines may lead to poor selectivity in aqueous solution, and selective fluorescence detection of primary aromatic amine is rarely reported. This paper presents design and synthesis of a fluorescent conjugated polymer for rapid and selective sensing of aromatic amines in aqueous solution. The fluorescent conjugated polymer, poly[fluorenyl-alt-p-phenyleneethynylene] with pendant carboxylic acid groups and long alky chains, is synthesized via palladium-catalyzed Sonogashira coupling reaction. The fluorescence of the polymer is selectively quenched by the aromatic amines in aqueous solution, whereas the aliphatic amines enhance the fluorescence of the polymer. The high selectivity to the aromatic amines, particularly to the environmentally important p-phenylenediamine, likely originates from the amplified π-π fluorescence quenching synergized by amine and carboxylic acid interaction. Our results demonstrate an effective material design strategy that may be extended to fluorescence sensing of other aromatic compounds.

  6. Multipoint fluorescence correlation spectroscopy with total internal reflection fluorescence microscope.

    Science.gov (United States)

    Ohsugi, Yu; Kinjo, Masataka

    2009-01-01

    We report simultaneous determination of diffusion coefficients at different points of a cell membrane using a multipoint fluorescence correlation spectroscopy (FCS) system. A system carrying seven detection areas in the evanescent field is achieved by using seven optical fibers on the image plane in the detection port of an objective-type total internal reflection FCS (TIR-FCS) system. Fluctuation of fluorescence intensity is monitored and evaluated using seven photomultiplier tubes (PMTs) and a newly constructed multichannel correlator. We demonstrate simultaneous-multipoint FCS, with a 3-mus time resolution, to investigate heterogeneous structures such as cell membranes and membrane-binding molecular dynamics near glass surfaces in live cells.

  7. Ultrasensitive fluorescence detection of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mathies, R.A.; Glazer, A.N.

    1992-01-01

    We have shown that a number of polycationic highly fluorescent dyes form complexes with double-stranded DNA (dsDNA) which are stable to electrophoresis and have characterized in detail such dsDNA complexes with TOTO (1,1[prime]-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole). TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with dsDNA, up to a maximum dye to DNA bp ratio of 1:4, with >1000-fold fluorescence enhancement. We have developed an assay using YOYO for the quantitation of single-stranded and dsDNA in solution applicable over a range of DNA concentrations from 0.5 to 100 ng per ml. The fluorescent dsDNA-dye complexes allow detection of dsDNA on agarose and acrylamide gels with picogram sensitivity. We have applied these complexes in multiplex mapping experiments for accurate sizing and quantitation of restriction fragments. We have shown that in gel shift experiments the stable dsDNA-dye complexes can be used to detect heteroduplex-Muts complexes with a sensitivity comparable to radioisotopic detection.

  8. Plasmonics Enhanced Smartphone Fluorescence Microscopy

    KAUST Repository

    Wei, Qingshan

    2017-05-12

    Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

  9. [Reading by fluorescent lamp light].

    Science.gov (United States)

    Höfling, G

    1979-08-01

    In dioptrics courses it has hitherto been taught that eyes of near-sighted patients who work in fluorescent light should be slightly under-corrected, since the near point is approximated in bluish light as a result of the chromatic aberration of the eye, in contrast to its location in white light or even the reddish light of incandescent lamps. The theory further states that this recommendation does not apply if close-range refraction occurs under fluorescent light. However, this is seldom the case. In our own investigations this approximation of the near point, under uniform illumination by fluorescent strip lights running across the ceiling, did not occur, at least not in "white" light (No. 33). On the contrary, under an incandescent lamp which was only half as powerful the near point was regularly closer to the eye than under white fluorescent light. The recommendation for under-correction of pulpit spectacles computed in incandescent light can therefore no longer be upheld.--While many patients complain of difficulties in diffuse incandescent light, these may be due to several other illumination factors which have not yet been fully investigated.--Some fundamental differences between diffuse fluorescent lighting and incandescent lamp lighting where there is a fall-off in luminosity are described.

  10. Fluorescence and phosphorescence of rutin

    Energy Technology Data Exchange (ETDEWEB)

    Bondarev, Stanislav L., E-mail: bondarev@imaph.bas-net.by [Minsk State Higher Radioengineering College, 220005 Minsk (Belarus); Knyukshto, Valeri N. [B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, 220072 Minsk (Belarus)

    2013-10-15

    Rutin is one of the most promising flavonoid from a pharmacological and biochemical point of view. Here we have explored its spectroscopic and photophysical properties at room temperature and 77 K using steady-state absorption-luminescence methods and pulse spectroscopy equipment. By excitation into the absorption band 1 of rutin in methanol at room temperature the normal Stokes' shifted fluorescence with a maximum at 415 nm and quantum yield of 2×10{sup −4} was revealed. However, by excitation into the bands 2 and 3 any emission wasn’t observed. At 77 K in ethanol glass we have observed fluorescence at 410 nm and phosphorescence at 540 nm for the first time. As a result the adequate energetic scheme including the lowest electronic excited singlet at 26000 cm{sup −1} and triplet at 19600 cm{sup −1} states was proposed. -- Highlights: • Rutin fluorescence and phosphorescence at 77 K were revealed for the first time. • Room temperature fluorescence is determined by maximum at 415 nm and yield of 2×10{sup −4}. • Violation of Vavilov–Kasha rule by excitation into the absorption bands 2 and 3. • Fluorescence and phosphorescence in rutin are caused by the allowed π, π{sup (⁎)} transitions.

  11. Plasmonics Enhanced Smartphone Fluorescence Microscopy.

    Science.gov (United States)

    Wei, Qingshan; Acuna, Guillermo; Kim, Seungkyeum; Vietz, Carolin; Tseng, Derek; Chae, Jongjae; Shir, Daniel; Luo, Wei; Tinnefeld, Philip; Ozcan, Aydogan

    2017-05-18

    Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

  12. Stress wave focusing transducers

    Energy Technology Data Exchange (ETDEWEB)

    Visuri, S.R., LLNL

    1998-05-15

    Conversion of laser radiation to mechanical energy is the fundamental process behind many medical laser procedures, particularly those involving tissue destruction and removal. Stress waves can be generated with laser radiation in several ways: creation of a plasma and subsequent launch of a shock wave, thermoelastic expansion of the target tissue, vapor bubble collapse, and ablation recoil. Thermoelastic generation of stress waves generally requires short laser pulse durations and high energy density. Thermoelastic stress waves can be formed when the laser pulse duration is shorter than the acoustic transit time of the material: {tau}{sub c} = d/c{sub s} where d = absorption depth or spot diameter, whichever is smaller, and c{sub s} = sound speed in the material. The stress wave due to thermoelastic expansion travels at the sound speed (approximately 1500 m/s in tissue) and leaves the site of irradiation well before subsequent thermal events can be initiated. These stress waves, often evolving into shock waves, can be used to disrupt tissue. Shock waves are used in ophthalmology to perform intraocular microsurgery and photodisruptive procedures as well as in lithotripsy to fragment stones. We have explored a variety of transducers that can efficiently convert optical to mechanical energy. One such class of transducers allows a shock wave to be focused within a material such that the stress magnitude can be greatly increased compared to conventional geometries. Some transducer tips could be made to operate regardless of the absorption properties of the ambient media. The size and nature of the devices enable easy delivery, potentially minimally-invasive procedures, and precise tissue- targeting while limiting thermal loading. The transducer tips may have applications in lithotripsy, ophthalmology, drug delivery, and cardiology.

  13. Fluorescent diagnostics of organic pollution in natural waters: A neural network approach

    Energy Technology Data Exchange (ETDEWEB)

    Orlov, Y.V.; Persiantsev, I.G.; Rebrik, S.P. [Nuclear Physics Institute, Moscow (Russian Federation)] [and others

    1995-12-31

    Rapid diagnosis of pollution is one of the key tasks in the field of ecological monitoring of natural and technogeneous environment. One of the promising methods of fluorescent diagnosis of organic pollution of water environment is the registration and analysis of two-dimensional Spectral Fluorescent Signatures (SFS). The neural networks - based system suggested in this paper is intended for solving the problem of detection, identification, and concentration measurement of water environmental pollution. The suggested system uses SFS as input pattern and allows one to build a rapid diagnosis system for ecological monitoring.

  14. Risks and Benefits of Rapid Clozapine Titration.

    Science.gov (United States)

    Lochhead, Jeannie D; Nelson, Michele A; Schneider, Alan L

    2016-05-18

    Clozapine is often considered the gold standard for the treatment of schizophrenia. Clinical guidelines suggest a gradual titration over 2 weeks to reduce the risks of adverse events such as seizures, hypotension, agranulocytosis, and myocarditis. The slow titration often delays time to therapeutic response. This raises the question of whether, in some patients, it may be safe to use a more rapid clozapine titration. The following case illustrates the potential risks associated with the use of multiple antipsychotics and rapid clozapine titration. We present the case of a young man with schizophrenia who developed life threatening neuroleptic malignant syndrome (NMS) during rapid clozapine titration and treatment with multiple antipsychotics. We were unable to find another case in the literature of NMS associated with rapid clozapine titration. This case is meant to urge clinicians to carefully evaluate the risks and benefits of rapid clozapine titration, and to encourage researchers to further evaluate the safety of rapid clozapine titration. Rapid clozapine titration has implications for decreasing health care costs associated with prolonged hospitalizations, and decreasing the emotional suffering associated with uncontrolled symptoms of psychosis. Clozapine is considered the most effective antipsychotic available thus efforts should focus on developing strategies that would allow for safest and most efficient use of clozapine to encourage its utilization for treatment resistance schizophrenia.

  15. Risks and benefits of rapid clozapine titration

    Directory of Open Access Journals (Sweden)

    Jeannie D. Lochhead

    2016-05-01

    Full Text Available Clozapine is often considered the gold standard for the treatment of schizophrenia. Clinical guidelines suggest a gradual titration over 2 weeks to reduce the risks of adverse events such as seizures, hypotension, agranulocytosis, and myocarditis. The slow titration often delays time to therapeutic response. This raises the question of whether, in some patients, it may be safe to use a more rapid clozapine titration. The following case illustrates the potential risks associated with the use of multiple antipsychotics and rapid clozapine titration. We present the case of a young man with schizophrenia who developed life threatening neuroleptic malignant syndrome (NMS during rapid clozapine titration and treatment with multiple antipsychotics. We were unable to find another case in the literature of NMS associated with rapid clozapine titration. This case is meant to urge clinicians to carefully evaluate the risks and benefits of rapid clozapine titration, and to encourage researchers to further evaluate the safety of rapid clozapine titration. Rapid clozapine titration has implications for decreasing health care costs associated with prolonged hospitalizations, and decreasing the emotional suffering associated with uncontrolled symptoms of psychosis. Clozapine is considered the most effective antipsychotic available thus efforts should focus on developing strategies that would allow for safest and most efficient use of clozapine to encourage its utilization for treatment resistance schizophrenia.

  16. Attentional Focus Effects in Standing Long Jump Performance: Influence of a Broad and Narrow Internal Focus.

    Science.gov (United States)

    Becker, Kevin A; Smith, Peter J K

    2015-07-01

    The content of instructions that strength coaches give can have a significant impact on how an athlete or client performs. Research on motor learning has shown an advantage of instructions focusing on the effects of movements (external focus) over those focusing on the movements themselves (internal focus) in the performance of motor skills. Internally focused cues are abundant in coaching, therefore the purpose of this study was to test whether some internally focused cues might be more helpful than others. Participants (68) were randomly assigned to either an external focus (EX), broad internal focus (B-IN), narrow internal focus (N-IN), or a control group (CON), and performed 5 standing long jumps. All groups were instructed that the goal was to jump as far as possible. In addition, the EX group was told to "jump as far past the start line as possible." The B-IN group was told to "use your legs." The N-IN group was told to "extend your knees as rapidly as possible," and the CON group received no additional instruction. An analysis of covariance showed that the EX group (198.09 ± 31.89 cm) jumped significantly farther than both the B-IN group (173.74 ± 35.36 cm), p = 0.010 and the N-IN group (178.53 ± 31.17 cm), p = 0.049, with no group different from the CON group. The results suggest that a broad internal focus is no more effective than a narrow internal focus, and that an external focus leads to the greatest jump distance. Strength and conditioning professionals should carefully word their instructions to induce an external focus of attention whenever possible.

  17. Blue LEDs feasibility for tissue fluorescence analysis

    Science.gov (United States)

    Dets, Sergiy M.; Denisov, Nikolay A.

    2000-04-01

    We considered the limited number of light-induced fluorescence applications for marketed ultra-bright blue LEDs where they can compete with versatile laser sources. Satisfactory optical output and miniature size as well as low power consumption of blue LEDs emitting at 470 nm allow to consider them as a promising alternatives to metal vapor or gas lasers used in many expires LIF applications. Available to authors LEDs form Hewlett-Packard, Micro Electronics Corp., Nichia Chemical Industries Ltd. and Toyoda Gosei Co. were tested to comply with demands to a tissue excitation source for portable spectroscopes. The optical performance of LEDs has shown that selected group of InGaN LEDs could be successfully used for that. The miniature illuminator that includes LED, focusing condenser, filter set and distal fiberoptic light concentrator was designed and tested in conjunction with portable CCD- equipped spectroscope. Operating in dark condition the proposed LED illuminator provides the level of fluorescence signal sufficient to detect spectral abnormalities in human Caucasian skin and excised gastrointestinal samples. All tissue autofluorescence data taken under LED illumination were compared with readings under He-Cd laser excitation and showed a good match. A new diagnostic designs based on LEDs were considered for clinical use.

  18. Fluorescence confocal microscopy for pathologists.

    Science.gov (United States)

    Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

    2014-03-01

    Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on

  19. Rapid small lot manufacturing

    Energy Technology Data Exchange (ETDEWEB)

    Harrigan, R.W.

    1998-05-09

    The direct connection of information, captured in forms such as CAD databases, to the factory floor is enabling a revolution in manufacturing. Rapid response to very dynamic market conditions is becoming the norm rather than the exception. In order to provide economical rapid fabrication of small numbers of variable products, one must design with manufacturing constraints in mind. In addition, flexible manufacturing systems must be programmed automatically to reduce the time for product change over in the factory and eliminate human errors. Sensor based machine control is needed to adapt idealized, model based machine programs to uncontrolled variables such as the condition of raw materials and fabrication tolerances.

  20. Dual-modality, fluorescent, PLGA encapsulated bismuth nanoparticles for molecular and cellular fluorescence imaging and computed tomography.

    Science.gov (United States)

    Swy, Eric R; Schwartz-Duval, Aaron S; Shuboni, Dorela D; Latourette, Matthew T; Mallet, Christiane L; Parys, Maciej; Cormode, David P; Shapiro, Erik M

    2014-11-07

    Reports of molecular and cellular imaging using computed tomography (CT) are rapidly increasing. Many of these reports use gold nanoparticles. Bismuth has similar CT contrast properties to gold while being approximately 1000-fold less expensive. Herein we report the design, fabrication, characterization, and CT and fluorescence imaging properties of a novel, dual modality, fluorescent, polymer encapsulated bismuth nanoparticle construct for computed tomography and fluorescence imaging. We also report on cellular internalization and preliminary in vitro and in vivo toxicity effects of these constructs. 40 nm bismuth(0) nanocrystals were synthesized and encapsulated within 120 nm Poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles by oil-in-water emulsion methodologies. Coumarin-6 was co-encapsulated to impart fluorescence. High encapsulation efficiency was achieved ∼70% bismuth w/w. Particles were shown to internalize within cells following incubation in culture. Bismuth nanocrystals and PLGA encapsulated bismuth nanoparticles exhibited >90% and >70% degradation, respectively, within 24 hours in acidic, lysosomal environment mimicking media and both remained nearly 100% stable in cytosolic/extracellular fluid mimicking media. μCT and clinical CT imaging was performed at multiple X-ray tube voltages to measure concentration dependent attenuation rates as well as to establish the ability to detect the nanoparticles in an ex vivo biological sample. Dual fluorescence and CT imaging is demonstrated as well. In vivo toxicity studies in rats revealed neither clinically apparent side effects nor major alterations in serum chemistry and hematology parameters. Calculations on minimal detection requirements for in vivo targeted imaging using these nanoparticles are presented. Indeed, our results indicate that these nanoparticles may serve as a platform for sensitive and specific targeted molecular CT and fluorescence imaging.