WorldWideScience

Sample records for biological fluorescence imaging

  1. Ion beam induced fluorescence imaging in biological systems

    Science.gov (United States)

    Bettiol, Andrew A.; Mi, Zhaohong; Vanga, Sudheer Kumar; Chen, Ce-belle; Tao, Ye; Watt, Frank

    2015-04-01

    Imaging fluorescence generated by MeV ions in biological systems such as cells and tissue sections requires a high resolution beam (system and a fluorescent probe that has a high quantum efficiency and low bleaching rate. For cutting edge applications in bioimaging, the fluorescence imaging technique needs to break the optical diffraction limit allowing for sub-cellular structure to be visualized, leading to a better understanding of cellular function. In a nuclear microprobe this resolution requirement can be readily achieved utilizing low beam current techniques such as Scanning Transmission Ion Microscopy (STIM). In recent times, we have been able to extend this capability to fluorescence imaging through the development of a new high efficiency fluorescence detection system, and through the use of new novel fluorescent probes that are resistant to ion beam damage (bleaching). In this paper we demonstrate ion beam induced fluorescence imaging in several biological samples, highlighting the advantages and challenges associated with using this technique.

  2. Ion beam induced fluorescence imaging in biological systems

    International Nuclear Information System (INIS)

    Imaging fluorescence generated by MeV ions in biological systems such as cells and tissue sections requires a high resolution beam (<100 nm), a sensitive detection system and a fluorescent probe that has a high quantum efficiency and low bleaching rate. For cutting edge applications in bioimaging, the fluorescence imaging technique needs to break the optical diffraction limit allowing for sub-cellular structure to be visualized, leading to a better understanding of cellular function. In a nuclear microprobe this resolution requirement can be readily achieved utilizing low beam current techniques such as Scanning Transmission Ion Microscopy (STIM). In recent times, we have been able to extend this capability to fluorescence imaging through the development of a new high efficiency fluorescence detection system, and through the use of new novel fluorescent probes that are resistant to ion beam damage (bleaching). In this paper we demonstrate ion beam induced fluorescence imaging in several biological samples, highlighting the advantages and challenges associated with using this technique

  3. Exploiting Molecular Biology by Time-Resolved Fluorescence Imaging

    Science.gov (United States)

    Müller, Francis; Fattinger, Christof

    Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal fluorophore. With fluorescence lifetimes in the microsecond range and fluorescence quantum yield of 0.4 some water soluble complexes of Ruthenium like modified Ru(sulfobathophenanthroline) complexes fulfill these properties. They are outstanding fluorescent labels for ultrasensitive assays as illustrated in two examples, in drug discovery and in point of care testing.We discuss the fundamentals and the state-of-the-art of the most sensitive time-gated fluorescence assays. We reflect on how the imaging devices currently employed for readout of these assays might evolve in the future. Many contemporary biological investigations rely on highly sensitive in vitro assays for the analysis of specific molecules in biological specimens, and the main part of these assays depends on high-sensitivity fluorescence detection techniques for the final readout. The analyzed molecules and molecular interactions in the specimen need to be detected in the presence of other highly abundant biomolecules, while the analyzed molecules themselves are only present at nano-, pico-, or even femtomolar concentration.A short scientific rationale of fluorescence is presented. It emphasizes the use of fluorescent labels for sensitive assays in life sciences and specifies the main properties of an ideal

  4. Biological applications of fluorescence lifetime imaging beyond microscopy

    Science.gov (United States)

    Akers, Walter J.; Berezin, Mikhail Y.; Lee, Hyeran; Guo, Kevin; Almutairi, Adah; Fréchet, Jean M. J.; Fischer, Georg M.; Daltrozzo, Ewald; Achilefu, Samuel

    2010-02-01

    Fluorescence lifetime is a relatively new contrast mechanism for optical imaging in living subjects that relies on intrinsic properties of fluorophores rather than concentration dependent intensity. Drawing upon the success of fluorescence lifetime imaging microscopy (FLIM) for investigation of protein-protein interactions and intracellular physiology, in vivo fluorescence lifetime imaging (FLI) promises to dramatically increase the utility of fluorescencebased imaging in preclinical and clinical applications. Intrinsic fluorescence lifetime measurements in living tissues can distinguish pathologies such as cancer from healthy tissue. Unfortunately, intrinsic FLT contrast is limited to superficial measurements. Conventional intensity-based agents have been reported for measuring these phenomena in vitro, but translation into living animals is difficult due to optical properties of tissues. For this reason, contrast agents that can be detected in the near infrared (NIR) wavelengths are being developed by our lab and others to enhance the capabilities of this modality. FLT is less affected by concentration and thus is better for detecting small changes in physiology, as long as sufficient fluorescence signal can be measured. FLT can also improve localization of signals for improved deep tissue imaging. Examples of the utility of exogenous contrast agents will be discussed, including applications in monitoring physiologic functions, controlled drug release and cancer biology. Instrumentation for FLI will also be discussed, including planar and diffuse optical imaging in time and frequency domains. Future applications will also be discussed that are being developed in this exciting field that complement other optical modalities.

  5. Compressive Fluorescence Microscopy for Biological and Hyperspectral Imaging

    CERN Document Server

    Studer, Vincent; Chahid, Makhlad; Moussavi, Hamed; Candes, Emmanuel; Dahan, Maxime

    2012-01-01

    The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices---especially in optics---have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher dimensional signals which typically exhibits extreme redund...

  6. Photon-counting H33D detector for biological fluorescence imaging

    Science.gov (United States)

    Michalet, X.; Siegmund, O. H. W.; Vallerga, J. V.; Jelinsky, P.; Millaud, J. E.; Weiss, S.

    2006-11-01

    We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross-delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions.

  7. Fluorescence imaging for bacterial cell biology: from localization to dynamics, from ensembles to single molecules.

    Science.gov (United States)

    Yao, Zhizhong; Carballido-López, Rut

    2014-01-01

    Fluorescent proteins and developments in superresolution (nanoscopy) and single-molecule techniques bring high sensitivity, speed, and one order of magnitude gain in spatial resolution to live-cell imaging. These technologies have only recently been applied to prokaryotic cell biology, revealing the exquisite subcellular organization of bacterial cells. Here, we review the parallel evolution of fluorescence microscopy methods and their application to bacteria, mainly drawing examples from visualizing actin-like MreB proteins in the model bacterium Bacillus subtilis. We describe the basic principles of nanoscopy and conventional techniques and their advantages and limitations to help microbiologists choose the most suitable technique for their biological question. Looking ahead, multidimensional live-cell nanoscopy combined with computational image analysis tools, systems biology approaches, and mathematical modeling will provide movie-like, mechanistic, and quantitative description of molecular events in bacterial cells. PMID:25002084

  8. Near infrared fluorescence quenching properties of copper (II) ions for potential applications in biological imaging

    Science.gov (United States)

    Maji, Dolonchampa; Zhou, Mingzhou; Sarder, Pinaki; Achilefu, Samuel

    2014-03-01

    Fluorescence quenching properties of copper(II) ions have been used for designing Cu(II) sensitive fluorescent molecular probes. In this paper, we demonstrate that static quenching plays a key role in free Cu(II)-mediated fluorescence quenching of a near infrared (NIR) fluorescent dye cypate. The Stern-Volmer quenching constant was calculated to be KSV = 970,000 M-1 in 25 mM MES buffer, pH 6.5 at room temperature. We synthesized LS835, a compound containing cypate attached covalently to chelated Cu(II) to study fluorescence quenching by chelated Cu(II). The fluorescence quenching mechanism of chelated Cu(II) is predominantly dynamic or collisional quenching. The quenching efficiency of chelated Cu(II) was calculated to be 58% ± 6% in dimethylsulfoxide at room temperature. Future work will involve further characterization of the mechanism of NIR fluorescence quenching by Cu(II) and testing its reversibility for potential applications in designing fluorophore-quencher based molecular probes for biological imaging.

  9. Synthesis and Characterization of Water-Soluble Conjugated Oligoelectrolytes for Near-Infrared Fluorescence Biological Imaging.

    Science.gov (United States)

    Woo, Shin-Jae; Park, Sungmin; Jeong, Ji-Eun; Hong, Yoochan; Ku, Minhee; Kim, Bo Yun; Jang, Il Ho; Heo, Soon Chul; Wang, Taejun; Kim, Ki Hean; Yang, Jaemoon; Kim, Jae Ho; Woo, Han Young

    2016-06-29

    Near-infrared (NIR) fluorophores attract increasing attention as a molecular marker (or probe) for in vivo and in vitro biological fluorescence imaging. Three types of new NIR fluorescent conjugated oligoelectrolytes (COEs: Q-FlTBTTFl, Q-FlBBTFl, and Q-FlTBBTTFl) are synthesized with quaternized ammonium ionic groups in their side-chains for water solubility. The emission wavelength is modulated in the range 600-1300 nm, by adjusting the intramolecular charge transfer in the molecular backbone based on the electron-rich fluorene (and/or thiophene) and electron-deficient benzo[2,1,3]thiadiazole (or benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole) moieties. The COEs show a remarkably larger Stokes shift (147-276 nm) compared to commercial rhodamine and cyanine dyes in water, avoiding self-quenching and interference from the excitation backscattered light. The photoluminescence (PL) quantum efficiency is improved substantially by up to 27.8% in water by fabricating a vesicular complex, COE/v, with a block ionomer, poly[(ethylene oxide)-block-(sodium 2-acrylamido-2-methyl-1-propanesulfonate)]. In vitro cellular uptake images with the COEs are obtained with good biocompatibility by confocal single-photon and two-photon microscopy. The ex vivo and in vivo images of a mouse xenograft model treated with the Q-FlBBTFl/v exhibit a substantially stronger fluorescence signal at the tumor site than at the other organs, highlighting the potential of the COE/v as an NIR fluorescent imaging agent for the diagnosis of cancer. PMID:27267787

  10. Development of Two Color Fluorescent Imager and Integrated Fluidic System for Nanosatellite Biology Applications

    Science.gov (United States)

    Wu, Diana Terri; Ricco, Antonio Joseph; Lera, Matthew P.; Timucin, Linda R.; Parra, Macarena P.

    2012-01-01

    Nanosatellites offer frequent, low-cost space access as secondary payloads on launches of larger conventional satellites. We summarize the payload science and technology of the Microsatellite in-situ Space Technologies (MisST) nanosatellite for conducting automated biological experiments. The payload (two fused 10-cm cubes) includes 1) an integrated fluidics system that maintains organism viability and supports growth and 2) a fixed-focus imager with fluorescence and scattered-light imaging capabilities. The payload monitors temperature, pressure and relative humidity, and actively controls temperature. C. elegans (nematode, 50 m diameter x 1 mm long) was selected as a model organism due to previous space science experience, its completely sequenced genome, size, hardiness, and the variety of strains available. Three strains were chosen: two green GFP-tagged strains and one red tdTomato-tagged strain that label intestinal, nerve, and pharyngeal cells, respectively. The integrated fluidics system includes bioanalytical and reservoir modules. The former consists of four 150 L culture wells and a 4x5 mm imaging zone the latter includes two 8 mL fluid reservoirs for reagent and waste storage. The fluidic system is fabricated using multilayer polymer rapid prototyping: laser cutting, precision machining, die cutting, and pressure-sensitive adhesives it also includes eight solenoid-operated valves and one mini peristaltic pump. Young larval-state (L2) nematodes are loaded in C. elegans Maintenance Media (CeMM) in the bioanalytical module during pre-launch assembly. By the time orbit is established, the worms have grown to sufficient density to be imaged and are fed fresh CeMM. The strains are pumped sequentially into the imaging area, imaged, then pumped into waste. Reagent storage utilizes polymer bags under slight pressure to prevent bubble formation in wells or channels. The optical system images green and red fluorescence bands by excitation with blue (473 nm peak

  11. A Practical Approach to Quantitative Processing and Analysis of Small Biological Structures by Fluorescent Imaging

    Science.gov (United States)

    Noller, Crystal M.; Boulina, Maria; McNamara, George; Szeto, Angela; McCabe, Philip M.

    2016-01-01

    Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 μm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines.

  12. Correlated imaging of living biological cells with a soft X-ray microscope and a fluorescence microscope

    International Nuclear Information System (INIS)

    Soft X-ray microscope is a very powerful tool to observe cellular organelles of living biological. However the inner structures are very complicated and it is difficult to identify the organelles obtained with the soft X-ray microscopes. We have proposed a correlated imaging with a soft X-ray microscope and a fluorescence microscope that is to observe the same biological cells with the both microscopes at the same time. (author)

  13. Spectrally resolved multidepth fluorescence imaging

    Science.gov (United States)

    Luo, Yuan; Zervantonakis, Ioannis K.; Oh, Se Baek; Kamm, Roger D.; Barbastathis, George

    2011-09-01

    We present a multicolor fluorescence imaging modality to visualize in real-time tissue structures emitting multispectral fluorescent light from different focal depths. Each designated spectrum of fluorescent emission from a specific depth within a volumetric tissue is probed by a depth-spectrum selective holographic grating. The grating for each fluorescent color are multiplexed within a volume hologram, which enables simultaneously obtaining multicolored fluorescent information at different depths within a biological tissue sample. We demonstrate the imaging modality's ability to obtain laser-induced multicolored fluorescence images of a biological sample from different depths without scanning. We also experimentally demonstrate that the imaging modality can be simultaneously operated at both fluorescent and bright field modes to provide complementary information of volumetric tissue structures at different depths in real-time.

  14. Trends in fluorescence imaging and related techniques to unravel biological information

    OpenAIRE

    Haustein, Elke; Schwille, Petra

    2007-01-01

    Optical microscopy is among the most powerful tools that the physical sciences have ever provided biology. It is indispensable for basic lab work, as well as for cutting edge research, as the visual monitoring of life processes still belongs to the most compelling evidences for a multitude of biomedical applications. Along with the rapid development of new probes and methods for the analysis of laser induced fluorescence, optical microscopy over past years experienced a vast increase of both ...

  15. Combined application of dynamic light scattering imaging and fluorescence intravital microscopy in vascular biology

    International Nuclear Information System (INIS)

    The dynamic light scattering imaging (DLSI) system combined with the conventional fluorescence intravital microscope (FIM) has been applied for the examination of blood and lymph vessels in the mouse ear in vivo. While the CCD camera can be shared by both techniques the combined application of DLSI and FIM allows rapid switching between the modalities. In current study temporal speckles fluctuations are used for rendering blood vessels structure and monitoring blood perfusion with the higher spatial resolution, whereas FIM provides the images of lymphatic vessels. The results clearly demonstrate that combined application of DLSI and FIM approaches provides synchronic in vivo images of blood and lymph vessels with higher contrast and specificity. The use of this new dual-modal diagnostic system is particularly important and has a great potential to significantly expand the capabilities of vascular diagnostics providing synchronic in vivo images of blood and lymph vessels

  16. Use of a novel rover-mounted fluorescence imager and fluorescent probes to detect biological material in the Atacama Desert in daylight

    OpenAIRE

    Weinstein, S; Pane, D.; Warren-Rhodes, K.; Cockell, C; Ernst, L. A.; Minkley, E.; Fisher, G.; Emani, S.; Wettergreen, D. S.; Wagner, M.; Cabrol, J. N.; Waggoner, A S

    2005-01-01

    We deployed our fluorescence imaging system which detects fluorescence signals from sparse microorganisms and biofilms on Carnegie Mellon University’s autonomous rover Zoë. The results of the 2004 Atacama Desert field season, in Chile, are discussed.

  17. Use of a Novel Rover-mounted Fluorescence Imager and Fluorescent Probes to Detect Biological Material in the Atacama Desert in Daylight

    Science.gov (United States)

    Weinstein, S.; Pane, D.; Warren-Rhodes, K.; Cockell, C.; Ernst, L. A.; Minkley, E.; Fisher, G.; Emani, S.; Wettergreen, D. S.; Wagner, M.

    2005-01-01

    We have developed an imaging system, the Fluorescence Imager (FI), for detecting fluorescence signals from sparse microorganisms and biofilms during autonomous rover exploration. The fluorescence signals arise both from naturally occurring chromophores, such as chlorophyll of cyanobacteria and lichens, and from fluorescent probes applied to soil and rocks. Daylight imaging has been accomplished by a novel use of a high-powered flashlamp synchronized to a CCD camera. The fluorescent probes are cell permanent stains that have extremely low intrinsic fluorescence (quantum yields less than 0.01) and a large fluorescence enhancement (quantum yields greater than 0.4) when bound to the target. Each probe specifically targets either carbohydrates, proteins, nucleic acids or membrane lipids, the four classes of macromolecules found in terrestrial life. The intent of the probes is to interrogate the environment for surface and endolithic life forms.

  18. Fluorescent image tracking velocimeter

    Science.gov (United States)

    Shaffer, Franklin D.

    1994-01-01

    A multiple-exposure fluorescent image tracking velocimeter (FITV) detects and measures the motion (trajectory, direction and velocity) of small particles close to light scattering surfaces. The small particles may follow the motion of a carrier medium such as a liquid, gas or multi-phase mixture, allowing the motion of the carrier medium to be observed, measured and recorded. The main components of the FITV include: (1) fluorescent particles; (2) a pulsed fluorescent excitation laser source; (3) an imaging camera; and (4) an image analyzer. FITV uses fluorescing particles excited by visible laser light to enhance particle image detectability near light scattering surfaces. The excitation laser light is filtered out before reaching the imaging camera allowing the fluoresced wavelengths emitted by the particles to be detected and recorded by the camera. FITV employs multiple exposures of a single camera image by pulsing the excitation laser light for producing a series of images of each particle along its trajectory. The time-lapsed image may be used to determine trajectory and velocity and the exposures may be coded to derive directional information.

  19. 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.

  20. Multi Spectral Fluorescence Imager (MSFI)

    Science.gov (United States)

    Caron, Allison

    2016-01-01

    Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans). The multispectral fluorescence imager (MSFI) will have the capability to accommodate 10 cm x 10 cm Petri plates, various sized multi-well culture plates, and other custom culture containers. Features will include programmable temperature and light cycles, ethylene scrubbing (less than 25 ppb), CO2 control (between 400 ppm and ISS-ambient levels in units of 100 ppm) and sufficient airflow to prevent condensation that would interfere with imaging.

  1. Fluorescent Cell Imaging in Regenerative Medicine

    OpenAIRE

    Etai Sapoznik; Guoguang Niu; Yu Zhou; Murphy, Sean V.; Shay Soker

    2016-01-01

    Fluorescent protein imaging, a promising tool in biological research, incorporates numerous applications that can be of specific use in the field of regenerative medicine. To enhance tissue regeneration efforts, scientists have been developing new ways to monitor tissue development and maturation in vitro and in vivo. To that end, new imaging tools and novel fluorescent proteins have been developed for the purpose of performing deep-tissue high-resolution imaging. These new methods, such as i...

  2. Oligothiophenes as Fluorescent Markers for Biological Applications

    Directory of Open Access Journals (Sweden)

    Antonio Manetto

    2012-01-01

    Full Text Available This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (biomolecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described.

  3. Fluorescent Probes and Fluorescence (Microscopy Techniques — Illuminating Biological and Biomedical Research

    Directory of Open Access Journals (Sweden)

    Gregor P. C. Drummen

    2012-11-01

    Full Text Available Fluorescence, the absorption and re-emission of photons with longer wavelengths, is one of those amazing phenomena of Nature. Its discovery and utilization had, and still has, a major impact on biological and biomedical research, since it enables researchers not just to visualize normal physiological processes with high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo, to replace radioactive assays when possible, but also to shed light on many pathobiological processes underpinning disease states, which would otherwise not be possible. Compounds that exhibit fluorescence are commonly called fluorochromes or fluorophores and one of these fluorescent molecules in particular has significantly enabled life science research to gain new insights in virtually all its sub-disciplines: Green Fluorescent Protein. Because fluorescent proteins are synthesized in vivo, integration of fluorescent detection methods into the biological system via genetic techniques now became feasible. Currently fluorescent proteins are available that virtually span the whole electromagnetic spectrum. Concomitantly, fluorescence imaging techniques were developed, and often progress in one field fueled innovation in the other. Impressively, the properties of fluorescence were utilized to develop new assays and imaging modalities, ranging from energy transfer to image molecular interactions to imaging beyond the diffraction limit with super-resolution microscopy. Here, an overview is provided of recent developments in both fluorescence imaging and fluorochrome engineering, which together constitute the “fluorescence toolbox” in life science research.

  4. Fluorescence microscopy of single autofluorescent proteins for cellular biology

    OpenAIRE

    Cognet, Laurent; Coussen, Françoise; Choquet, Daniel; Lounis, Brahim

    2002-01-01

    International audience In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent prote...

  5. Far-field fluorescence microscopy beyond the diffraction limit: Fluorescence imaging with ultrahigh resolution

    OpenAIRE

    Rice, James H.

    2007-01-01

    Fluorescence microscopy is an important and extensively utilised tool for imaging biological systems. However, the image resolution that can be obtained has a limit as defined through the laws of diffraction. Demand for improved resolution has stimulated research into developing methods to image beyond the diffraction limit based on far-field fluorescence microscopy techniques. Rapid progress is being made in this area of science with methods emerging that enable fluorescence imaging in the f...

  6. 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…

  7. Phase-sensitive fluorescent imaging with coherent reconstruction

    CERN Document Server

    Field, Jeffrey J; Bartels, Randy A

    2015-01-01

    Optical imaging plays a critical role in advancing our understanding of three dimensional dynamics of biological systems. Coherent imaging (CI) methods exploit spatial phase information, encoded through propagation of coherent signal light emerging from a specimen, to extract a three-dimensional representation of the object from a single high-speed measurement. Until now, CI methods could not be applied to incoherent light, severely limiting their ability to image the most powerful biological probes available - fluorescent molecules - with sufficient speed and volume to observe important processes, such as neural processing in live specimens. We introduce a new imaging technique that transfers the spatial propagation phase of coherent illumination light to incoherent fluorescent light emission. The transfer of propagation phase allows CI techniques to be applied to fluorescent light imaging, and leads to large increases in imaging speed and depth of field. With this advance, biological imaging of fluorescent ...

  8. Spectrally resolved fluorescent lifetime imaging

    OpenAIRE

    Hanley, Quentin S.

    2008-01-01

    Placing an imaging spectrograph or related components capable of generating a spectrum between a microscope and the image intensifier of a conventional fluorescence lifetime imaging (FLIM) system creates a spectrally resolved FLIM (SFLIM). This arrangement provides a number of opportunities not readily available to conventional systems using bandpass filters. The examples include: simultaneous viewing of multiple fluorophores; tracking of both the donor and acceptor; and observation of a rang...

  9. Biological applications of confocal fluorescence polarization microscopy

    Science.gov (United States)

    Bigelow, Chad E.

    Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine

  10. Nanoprobes for super-resolution fluorescence imaging at the nanoscale

    Institute of Scientific and Technical Information of China (English)

    HOU ShangGuo; LIANG Le; DENG SuHui; CHEN JianFang; HUANG Qing; CHENG Ya; FAN ChunHai

    2014-01-01

    Compared with other imaging techniques,fluorescence microscopy has become an essential tool to study cell biology due to its high compatibility with living cells.Owing to the resolution limit set by the diffraction of light,fluorescence microscopy could not resolve the nanostructures in the range of〈200 nm.Recently,many techniques have been emerged to overcome the diffraction barrier,providing nanometer spatial resolution.In the course of development,the progress in fluorescent probes has helped to promote the development of the high-resolution fluorescence nanoscopy.Here,we describe the contributions of the fluorescent probes to far-field super resolution imaging,focusing on concepts of the existing super-resolution nanoscopy based on the photophysics of fluorescent nanoprobes,like photoswitching,bleaching and blinking.Fluorescent probe technology is crucial in the design and implementation of super-resolution imaging methods.

  11. 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.

  12. Hyperspectral fluorescence lifetime imaging for optical biopsy.

    Science.gov (United States)

    Nie, Zhaojun; An, Ran; Hayward, Joseph E; Farrell, Thomas J; Fang, Qiyin

    2013-09-01

    A hyperspectral fluorescence lifetime imaging (FLIM) instrument is developed to study endogenous fluorophores in biological tissue as an optical biopsy tool. This instrument is able to spectrally, temporally, and spatially resolve fluorescence signal, thus providing multidimensional information to assist clinical tissue diagnosis. An acousto-optic tunable filter (AOTF) is used to realize rapid wavelength switch, and a photomultiplier tube and a high-speed digitizer are used to collect the time-resolved fluorescence decay at each wavelength in real time. The performance of this instrument has been characterized and validated on fluorescence tissue phantoms and fresh porcine skin specimens. This dual-arm AOTF design achieves high spectral throughput while allowing microsecond nonsequential, random wavelength switching, which is highly desirable for time-critical applications. In the results reported here, a motorized scanning stage is used to realize spatial scanning for two-dimensional images, while a rapid beam steering technique is feasible and being developed in an ongoing project. PMID:24002188

  13. Near-Infrared Fluorescent Materials for Sensing of Biological Targets

    Directory of Open Access Journals (Sweden)

    Julia Xiaojun Zhao

    2008-05-01

    Full Text Available Near-infrared fluorescent (NIRF materials are promising labeling reagents for sensitive determination and imaging of biological targets. In the near-infrared region biological samples have low background fluorescence signals, providing high signal to noise ratio. Meanwhile, near-infrared radiation can penetrate into sample matrices deeply due to low light scattering. Thus, in vivo and in vitro imaging of biological samples can be achieved by employing the NIRF probes. To take full advantage of NIRF materials in the biological and biomedical field, one of the key issues is to develop intense and biocompatible NIRF probes. In this review, a number of NIRF materials are discussed including traditional NIRF dye molecules, newly developed NIRF quantum dots and single-walled carbon nanotubes, as well as rare earth metal compounds. The use of some NIRF materials in various nanostructures is illustrated. The enhancement of NIRF using metal nanostructures is covered as well. The fluorescence mechanism and bioapplications of each type of the NIRF materials are discussed in details.

  14. Entangled-photon coincidence fluorescence imaging

    OpenAIRE

    Scarcelli, Giuliano; Yun, Seok H.

    2008-01-01

    We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as “detectors” breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations.

  15. Fluorescence microscopy of single autofluorescent proteins for cellular biology

    CERN Document Server

    Cognet, Laurent; Choquet, Daniel; Lounis, Brahim

    2002-01-01

    In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells.

  16. Fast x-ray fluorescence microtomography of hydrated biological samples.

    Directory of Open Access Journals (Sweden)

    Enzo Lombi

    Full Text Available Metals and metalloids play a key role in plant and other biological systems as some of them are essential to living organisms and all can be toxic at high concentrations. It is therefore important to understand how they are accumulated, complexed and transported within plants. In situ imaging of metal distribution at physiological relevant concentrations in highly hydrated biological systems is technically challenging. In the case of roots, this is mainly due to the possibility of artifacts arising during sample preparation such as cross sectioning. Synchrotron x-ray fluorescence microtomography has been used to obtain virtual cross sections of elemental distributions. However, traditionally this technique requires long data acquisition times. This has prohibited its application to highly hydrated biological samples which suffer both radiation damage and dehydration during extended analysis. However, recent advances in fast detectors coupled with powerful data acquisition approaches and suitable sample preparation methods can circumvent this problem. We demonstrate the heightened potential of this technique by imaging the distribution of nickel and zinc in hydrated plant roots. Although 3D tomography was still impeded by radiation damage, we successfully collected 2D tomograms of hydrated plant roots exposed to environmentally relevant metal concentrations for short periods of time. To our knowledge, this is the first published example of the possibilities offered by a new generation of fast fluorescence detectors to investigate metal and metalloid distribution in radiation-sensitive, biological samples.

  17. Optical imaging of biological tissues

    Science.gov (United States)

    Bouza Dominguez, Jorge

    In this thesis, a new time-dependent model for describing light propagation in biological media is proposed. The model is based on the simplified spherical harmonics approximation and is represented by a set of coupled parabolic partial differential equations (TD-pSPN equations). In addition, the model is extended for modeling the time-dependent response of fluorescent agents in biological tissues and the ensuing time-domain propagation of light therein. In a comparison with Monte Carlo simulations, it is shown that the TD-pSPN equations present unique features in its derivation that makes it a more accurate alternative to the diffusion equation (DE). The TD-pSPN model (for orders N > 1) outperforms the DE in the description of the propagation of light in near-nondiffusive media and in all the physical situations where DE fails. Often, only small orders of the SP N approximation are needed to obtain accurate results. A diffuse optical tomography (DOT) algorithm is also implemented based on the TD-pSPN equations as the forward model using constrained optimization methods. The algorithm uses time-dependent (TD) data directly. Such an approach is benefited from both the accuracy of the SPN models and the richness of TD data. In the calculation of the gradient of the objective function, a time-dependent adjoint differentiation method is introduced that reduces computation time. Several numerical experiments are performed for small geometry media with embedded inclusions that mimic small animal imaging. In these experiments, the values of the optical coefficients are varied within realistic bounds that are representative of those found in the range of the near-infrared spectrum, including high absorption values. Single and multi-parameter reconstructions (absorption and diffusion coefficients) are performed. The reconstructed images based on the TD-pSPN equations (N > 1) give better estimates of the optical properties of the media than the DE. On the other hand

  18. In vivo cellular imaging using fluorescent proteins - Methods and Protocols

    Directory of Open Access Journals (Sweden)

    M. Monti

    2012-12-01

    Full Text Available The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible to the cell often can be made visible with the use of fluorescent proteins. With this words, Robert M. Hoffman introduces In vivo Cellular Imaging Using Fluorescent proteins, the eighteen chapters book dedicated to the description of how fluorescence proteins have changed the way to analyze cellular processes in vivo. Modern researches aim to study new and less invasive methods able to follow the behavior of different cell types in different biological contexts: for example, how cancer cells migrate or how they respond to different therapies. Also, in vivo systems can help researchers to better understand animal embryonic development so as how fluorescence proteins may be used to monitor different processes in living organisms at the molecular and cellular level.

  19. Development of an oxidative dehydrogenation-based fluorescent probe for Cu{sup 2+} and its biological imaging in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan Jiangli, E-mail: fanjl@dlut.edu.cn [State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, High-tech District, Dalian 116024 (China); Liu Xiaojian; Hu Mingming; Zhu Hao; Song Fengling [State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, High-tech District, Dalian 116024 (China); Peng Xiaojun, E-mail: pengxj@dlut.edu.cn [State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, High-tech District, Dalian 116024 (China)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer The fluorescent probe contains an N, O and S tridentate ligand. Black-Right-Pointing-Pointer The probe is simple but highly sensitive and selective toward Cu{sup 2+}. Black-Right-Pointing-Pointer The mechanism is based on the Cu{sup 2+}-promoted dehydrogenation of amine in different organic and aqueous solutions. Black-Right-Pointing-Pointer It was successfully applied to visualize Cu{sup 2+} in living cells. - Abstract: Based on a boron dipyrromethene (BODIPY) derivative containing an N, O and S tridentate ligand, a Cu{sup 2+} fluorescent probe BTCu was developed. The detection mechanism was verified as Cu{sup 2+}-promoted oxidative dehydrogenation of an amine moiety, leading to a formation of a fluorescent Cu{sup +}-Schiff base complex. Free BTCu exhibited a maximum absorption wavelength at 496 nm, and a very weak maximum emission at 511 nm. Upon addition of various metals ions, it showed large fluorescence enhancement toward Cu{sup 2+} (417-fold in MeCN and 103-fold in MeCN/HEPES solution, respectively) with high selectivity. The detection limits are as low as 1.74 Multiplication-Sign 10{sup -8} M and 4.96 Multiplication-Sign 10{sup -8} M in the two different solutions, respectively. And BTCu could work in a wide pH range with an extraordinary low pK{sub a} of 1.21 {+-} 0.06. Using fluorescence microscopy, the probe was shown to be capable of penetrating into living cells and imaging intracellular Cu{sup 2+} changes.

  20. Intrinsic fluorescence of selenium nanoparticles for cellular imaging applications

    Science.gov (United States)

    Khalid, A.; Tran, Phong A.; Norello, Romina; Simpson, David A.; O'Connor, Andrea J.; Tomljenovic-Hanic, Snjezana

    2016-02-01

    Nanoparticles hold great potential in contributing to high-resolution bioimaging as well as for biomedical applications. Although, selenium (Se) nanoparticles (NPs) have been investigated owing to their potential roles in therapeutics, the imaging capability of these NPs has never been explored. This manuscript identifies the intrinsic fluorescence of Se NPs, which is highly beneficial for nanoscale imaging of biological structures. The emission of individual NPs and its evolution with time is explored. The photoluminescence spectra has revealed visible to near infrared emission for Se NPs. The work finally reflects on the role of this intrinsic fluorescence for in vitro imaging and tracking in fibroblast cells, without the need of any additional tags. This technique would overcome the limitations of the conventionally used methods of imaging with tagged fluorescent proteins and dyes, preventing possible adverse cellular effects or phototoxicity caused by the added fluorescent moieties.Nanoparticles hold great potential in contributing to high-resolution bioimaging as well as for biomedical applications. Although, selenium (Se) nanoparticles (NPs) have been investigated owing to their potential roles in therapeutics, the imaging capability of these NPs has never been explored. This manuscript identifies the intrinsic fluorescence of Se NPs, which is highly beneficial for nanoscale imaging of biological structures. The emission of individual NPs and its evolution with time is explored. The photoluminescence spectra has revealed visible to near infrared emission for Se NPs. The work finally reflects on the role of this intrinsic fluorescence for in vitro imaging and tracking in fibroblast cells, without the need of any additional tags. This technique would overcome the limitations of the conventionally used methods of imaging with tagged fluorescent proteins and dyes, preventing possible adverse cellular effects or phototoxicity caused by the added fluorescent

  1. Hadamard-transform fluorescence-lifetime imaging.

    Science.gov (United States)

    Mizuno, Takahiko; Iwata, Tetsuo

    2016-04-18

    We discuss a Hadamard-transform-based fluorescence-lifetime-imaging (HT-FLI) technique for fluorescence-lifetime-imaging microscopy (FLIM). The HT-FLI uses a Fourier-transform phase-modulation fluorometer (FT-PMF) for fluorescence-lifetime measurements, where the modulation frequency of the excitation light is swept linearly in frequency from zero to a specific maximum during a fixed duration of time. Thereafter, fluorescence lifetimes are derived through Fourier transforms for the fluorescence and reference waveforms. The FT-PMF enables the analysis of multi-component samples simultaneously. HT imaging uses electronic exchange of HT illumination mask patterns, and a high-speed, high-sensitivity photomultiplier, to eliminate frame-rate issues that accompany two-dimensional image detectors. PMID:27137259

  2. In Vivo Metal Ion Imaging Using Fluorescent Sensors.

    Science.gov (United States)

    Van de Bittner, Genevieve C; Hirayama, Tasuku

    2016-01-01

    In vivo imaging in living animals provides the ability to monitor alterations of signaling molecules, ions, and other biological components during various life stages and in disease. The data gained from in vivo imaging can be used for biological discovery or to determine elements of disease progression and can inform the development and translation of therapeutics. Herein, we present theories behind small-molecule, fluorescent, metal ion sensors as well as the methods for their successful application to in vivo metal ion imaging, including ex vivo validation. PMID:27283424

  3. Imaging an atomic beam using fluorescence

    Institute of Scientific and Technical Information of China (English)

    Ming He(何明); Jin Wang(王谨); Mingsheng Zhan(詹明生)

    2003-01-01

    A fluorescence detection scheme is applied to image an atomic beam. Using two laser diodes as the sources of detection light and pumping light respectively, the fluorescence image of the atomic beam is then observed by a commercial CCD-camera, which is corresponding to the atomic state and velocity distribution. The detection scheme has a great utilization in the experiments of cold atoms and atomic optics.

  4. Reflectance and fluorescence hyperspectral elastic image registration

    Science.gov (United States)

    Lange, Holger; Baker, Ross; Hakansson, Johan; Gustafsson, Ulf P.

    2004-05-01

    Science and Technology International (STI) presents a novel multi-modal elastic image registration approach for a new hyperspectral medical imaging modality. STI's HyperSpectral Diagnostic Imaging (HSDI) cervical instrument is used for the early detection of uterine cervical cancer. A Computer-Aided-Diagnostic (CAD) system is being developed to aid the physician with the diagnosis of pre-cancerous and cancerous tissue regions. The CAD system uses the fusion of multiple data sources to optimize its performance. The key enabling technology for the data fusion is image registration. The difficulty lies in the image registration of fluorescence and reflectance hyperspectral data due to the occurrence of soft tissue movement and the limited resemblance of these types of imagery. The presented approach is based on embedding a reflectance image in the fluorescence hyperspectral imagery. Having a reflectance image in both data sets resolves the resemblance problem and thereby enables the use of elastic image registration algorithms required to compensate for soft tissue movements. Several methods of embedding the reflectance image in the fluorescence hyperspectral imagery are described. Initial experiments with human subject data are presented where a reflectance image is embedded in the fluorescence hyperspectral imagery.

  5. Robust image registration of biological microscopic images

    OpenAIRE

    Ching-Wei Wang; Shuk-Man Ka; Ann Chen

    2014-01-01

    Image registration of biological data is challenging as complex deformation problems are common. Possible deformation effects can be caused in individual data preparation processes, involving morphological deformations, stain variations, stain artifacts, rotation, translation, and missing tissues. The combining deformation effects tend to make existing automatic registration methods perform poor. In our experiments on serial histopathological images, the six state of the art image registratio...

  6. Imaging and quantification of trace metals in thin biological specimens using microprobe techniques: synchrotron induced X-ray fluorescence microprobe and nuclear microprobe

    International Nuclear Information System (INIS)

    The combination of Synchrotron X-Ray Fluorescence microprobe (SXRF) with other micro-analytical techniques based on accelerated particle beams, such as Rutherford Backscattering Spectrometry (RBS), and Particle Induced X-ray Emission (PIXE) enable to quantify SXRF results of thin biological specimens. The same specimens were analysed by both techniques. The average thickness calculated by RBS of discrete tissue regions in equivalent sample sections was used to normalize SXRF data. Analysing the same sample region with PIXE technique, simultaneously to RBS, can validate the methodology once it will enable to compare concentrations obtained for elements that are detected by both SXRF and PIXE. Major departures were found for the quantification of Ca and Ti. For Cr and Fe a good agreement was found between both micro-analytical techniques. Differences below 20% were obtained for the majority of the elements and samples analysed. The procedure can be applied to produce quantitative elemental maps and it will be advantageous to quantify toxic elements present at low concentrations in tissues, such as Cr, Ni and Ph, which can only be efficiently assessed by SXRF. (authors)

  7. Hyperspectral Fluorescence and Reflectance Imaging Instrument

    Science.gov (United States)

    Ryan, Robert E.; O'Neal, S. Duane; Lanoue, Mark; Russell, Jeffrey

    2008-01-01

    The system is a single hyperspectral imaging instrument that has the unique capability to acquire both fluorescence and reflectance high-spatial-resolution data that is inherently spatially and spectrally registered. Potential uses of this instrument include plant stress monitoring, counterfeit document detection, biomedical imaging, forensic imaging, and general materials identification. Until now, reflectance and fluorescence spectral imaging have been performed by separate instruments. Neither a reflectance spectral image nor a fluorescence spectral image alone yields as much information about a target surface as does a combination of the two modalities. Before this system was developed, to benefit from this combination, analysts needed to perform time-consuming post-processing efforts to co-register the reflective and fluorescence information. With this instrument, the inherent spatial and spectral registration of the reflectance and fluorescence images minimizes the need for this post-processing step. The main challenge for this technology is to detect the fluorescence signal in the presence of a much stronger reflectance signal. To meet this challenge, the instrument modulates artificial light sources from ultraviolet through the visible to the near-infrared part of the spectrum; in this way, both the reflective and fluorescence signals can be measured through differencing processes to optimize fluorescence and reflectance spectra as needed. The main functional components of the instrument are a hyperspectral imager, an illumination system, and an image-plane scanner. The hyperspectral imager is a one-dimensional (line) imaging spectrometer that includes a spectrally dispersive element and a two-dimensional focal plane detector array. The spectral range of the current imaging spectrometer is between 400 to 1,000 nm, and the wavelength resolution is approximately 3 nm. The illumination system consists of narrowband blue, ultraviolet, and other discrete

  8. Nonlinear spectral imaging of biological tissues

    Science.gov (United States)

    Palero, J. A.

    2007-07-01

    The work presented in this thesis demonstrates live high resolution 3D imaging of tissue in its native state and environment. The nonlinear interaction between focussed femtosecond light pulses and the biological tissue results in the emission of natural autofluorescence and second-harmonic signal. Because biological intrinsic emission is generally very weak and extends from the ultraviolet to the visible spectral range, a broad-spectral range and high sensitivity 3D spectral imaging system is developed. Imaging the spectral characteristics of the biological intrinsic emission reveals the structure and biochemistry of the cells and extra-cellular components. By using different methods in visualizing the spectral images, discrimination between different tissue structures is achieved without the use of any stain or fluorescent label. For instance, RGB real color spectral images of the intrinsic emission of mouse skin tissues show blue cells, green hair follicles, and purple collagen fibers. The color signature of each tissue component is directly related to its characteristic emission spectrum. The results of this study show that skin tissue nonlinear intrinsic emission is mainly due to the autofluorescence of reduced nicotinamide adenine dinucleotide (phosphate), flavins, keratin, melanin, phospholipids, elastin and collagen and nonlinear Raman scattering and second-harmonic generation in Type I collagen. In vivo time-lapse spectral imaging is implemented to study metabolic changes in epidermal cells in tissues. Optical scattering in tissues, a key factor in determining the maximum achievable imaging depth, is also investigated in this work.

  9. Supercontinuum Stimulated Emission Depletion Fluorescence Lifetime Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Lesoine, Michael; Bose, Sayantan; Petrich, Jacob; Smith, Emily

    2012-06-13

    Supercontinuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated by using time-correlated single-photon counting (TCSPC) detection. The spatial resolution of the developed STED instrument was measured by imaging monodispersed 40-nm fluorescent beads and then determining their fwhm, and was 36 ± 9 and 40 ± 10 nm in the X and Y coordinates, respectively. The same beads measured by confocal microscopy were 450 ± 50 and 430 ± 30 nm, which is larger than the diffraction limit of light due to underfilling the microscope objective. Underfilling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 ± 0.1 ns) was measured for the fluorescent beads by using confocal or STED lifetime imaging. The instrument has been applied to study Alexa Fluor 594-phalloidin labeled F-actin-rich projections with dimensions smaller than the diffraction limit of light in cultured cells. Fluorescence lifetimes of the actin-rich projections range from 2.2 to 2.9 ns as measured by STED lifetime imaging.

  10. Two-photon excited fluorescence from biological aerosol particles

    International Nuclear Information System (INIS)

    We used a 40 MHz mode-locked 524 nm laser source to evaluate the utility of sub-picosecond excitation of fluorescence from 2-photon absorption in biological aerosols. Individual particles of biological composition, as well as other calibration particles, suspended in an inlet air flow were illuminated and measured as they passed through an optical chamber. To our knowledge, this was the first demonstration of 2-photon excited fluorescence from micron-sized particles composed of micro-organisms. We also observed a high fluorescence signal at visible wavelengths, which was not present with single-photon excitation.

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

    NARCIS (Netherlands)

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

    2014-01-01

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

  12. 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.

  13. Fluorescence optical imaging in anticancer drug delivery

    Czech Academy of Sciences Publication Activity Database

    Etrych, Tomáš; Lucas, H.; Janoušková, Olga; Chytil, Petr; Mueller, T.; Mäder, K.

    2016-01-01

    Roč. 226, 28 March (2016), s. 168-181. ISSN 0168-3659 R&D Projects: GA ČR(CZ) GA15-02986S; GA MŠk(CZ) LO1507 Institutional support: RVO:61389013 Keywords : fluorescence imaging * drug delivery * theranostics Subject RIV: CD - Macromolecular Chemistry Impact factor: 7.705, year: 2014

  14. Fluorescence lifetime imaging in biosciences: technologies and applications

    Institute of Scientific and Technical Information of China (English)

    Raluca NIESNER; Karl-Heinz GERICKE

    2008-01-01

    The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging tech-niques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analy-sis of the total fluorescence signal originating from the sam-ple, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macro-scopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved tech-niques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosci-ences, especially for fast intravital studies are discussed in this work.

  15. Distinguishability of Biological Material Using Ultraviolet Multi-Spectral Fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P.C.; Heinen, R.J.; Rigdon, L.D.; Rosenthal, S.E.; Shokair, I.R.; Siragusa, G.R.; Tisone, G.C.; Wagner, J.S.

    1998-10-14

    Recent interest in the detection and analysis of biological samples by spectroscopic methods has led to questions concerning the degree of distinguishability and biological variability of the ultraviolet (W) fluorescent spectra from such complex samples. We show that the degree of distinguishability of such spectra is readily determined numerically.

  16. Fluorescent nanoparticle probes for imaging of cancer.

    Science.gov (United States)

    Santra, Swadeshmukul; Malhotra, Astha

    2011-01-01

    Fluorescent nanoparticles (FNPs) have received immense popularity in cancer imaging in recent years because of their attractive optical properties. In comparison to traditional organic-based fluorescent dyes and fluorescent proteins, FNPs offer much improved sensitivity and photostability. FNPs in certain size range have a strong tendency to enter and retain in solid tumor tissue with abnormal (leaky) vasculature--a phenomenon known as Enhanced Permeation and Retention (EPR) effect, advancing their use for in vivo tumor imaging. Furthermore, large surface area of FNPs and their usual core-shell structure offer a platform for designing and fabricating multimodal/multifunctional nanoparticles (MMNPs). For effective cancer imaging, often the optical imaging modality is integrated with other nonoptical-based imaging modalities such as MRI, X-ray, and PET, thus creating multimodal nanoparticle (NP)-based imaging probes. Such multimodal NP probes can be further integrated with therapeutic drug as well as cancer targeting agent leading to multifunctional NPs. Biocompatibility of FNPs is an important criterion that must be seriously considered during FNP design. NP composition, size, and surface chemistry must be carefully selected to minimize potential toxicological consequences both in vitro and in vivo. In this article, we will mainly focus on three different types of FNPs: dye-loaded NPs, quantum dots (Qdots), and phosphores; briefly highlighting their potential use in translational research. PMID:21480546

  17. Biological imaging in radiation oncology

    Energy Technology Data Exchange (ETDEWEB)

    Grosu, A.L.; Wiedenmann, N.; Molls, M. [Klinik und Poliklinik fuer Strahlentherapie und Radiologische Onkologie Klinikum rechts der Isar, Technical Univ. of Munich (Germany)

    2005-07-01

    The goal of this study was to discuss the value of integrating biological imaging (PET, SPECT, MRS etc.) in radiation treatment planning and monitoring. Studies in patients with brain tumors have shown that, compared to CT and MRI alone, the image fusion of CT/MRI and amino acid SPECT or PET allows a more correct delineation of gross tumor volume (GTV) and planning target volume (PTV). For FDG-PET, comparable results with different techniques are reported in the literature also for bronchial carcinoma, ear-nose-and-throat tumors, and cervical carcinoma, or, in the case of MRS, for prostate cancer. Imaging of hypoxia, cell proliferation, apoptosis, tumor angiogenesis, and gene expression leads to the identification of differently aggressive areas of a biologically inhomogeneous tumor mass that can be individually and more appropriately targeted using innovative IMRT. Thus, a biological, inhomogeneous dose distribution can be generated, the so-called dose painting. In addition, the biological imaging can play a significant role in the evaluation of the therapy response after radiochemotherapy. Clinical studies in ear-nose-and-throat tumors, bronchial carcinoma, esophagus carcinoma, and cervical carcinoma suggest that the sensitivity and specificity of FDG-PET for the therapy response are higher compared to anatomical imaging (CT and MRI). Clinical and experimental studies are required to define the real impact of these investigations in radiation treatment planning, and especially in the evaluation of therapy response. (orig.)

  18. Registering plant dysfunction in artificial biosystems through fluorescence imaging technique

    Science.gov (United States)

    Nikolova, Alexandra; Krumov, Alexandar; Vassilev, Vesselin

    Humanity ambitions in space exploration and long-term men-operated space missions evoke an increasing interest to artificial ecosystem researches. Advanced studies of plant biosystems provoke development of new innovative technologies for plant cultivation in man-made environment. Closed ecosystems of different types and structure are now used for space horticulture, cultivation of genetically modified species, bio-products for pharmacies and industry etc. New technologies are required to monitor and control basic parameters of future bioregenerative life support system, especially of plants photosynthetic activity as the most fundamental biological process. Authors propose a conception for a non-invasive control of plant physiological status in closed biosystem through spatial registration of chlorophyll fluorescence. This approach allows an early detection of stress impact on plants, reveal the dynamic and direction of the negative influence and the level of plant stress. Technical requirements for obtaining plant fluorescence images are examined in close relation with plant illumination conditions. Problems related with optimised plant illumination are discussed. Examples of fluorescence images of healthy and stressed plants demonstrate the sensibility and rapidity of signal changes caused by plant dysfunction. Proposed conception could be used for developing new technical solutions in autocontrolled bio-support systems, based on real time analysis of fluorescence images.

  19. Fluorescence labeled microbubbles for multimodal imaging.

    Science.gov (United States)

    Barrefelt, Åsa; Zhao, Ying; Larsson, Malin K; Egri, Gabriella; Kuiper, Raoul V; Hamm, Jörg; Saghafian, Maryam; Caidahl, Kenneth; Brismar, Torkel B; Aspelin, Peter; Heuchel, Rainer; Muhammed, Mamoun; Dähne, Lars; Hassan, Moustapha

    2015-08-28

    Air-filled polyvinyl alcohol microbubbles (PVA-MBs) were recently introduced as a contrast agent for ultrasound imaging. In the present study, we explore the possibility of extending their application in multimodal imaging by labeling them with a near infrared (NIR) fluorophore, VivoTag-680. PVA-MBs were injected intravenously into FVB/N female mice and their dynamic biodistribution over 24 h was determined by 3D-fluorescence imaging co-registered with 3D-μCT imaging, to verify the anatomic location. To further confirm the biodistribution results from in vivo imaging, organs were removed and examined histologically using bright field and fluorescence microscopy. Fluorescence imaging detected PVA-MB accumulation in the lungs within the first 30 min post-injection. Redistribution to a low extent was observed in liver and kidneys at 4 h, and to a high extent mainly in the liver and spleen at 24 h. Histology confirmed PVA-MB localization in lung capillaries and macrophages. In the liver, they were associated with Kupffer cells; in the spleen, they were located mostly within the marginal-zone. Occasional MBs were observed in the kidney glomeruli and interstitium. The potential application of PVA-MBs as a contrast agent was also studied using ultrasound (US) imaging in subcutaneous and orthotopic pancreatic cancer mouse models, to visualize blood flow within the tumor mass. In conclusion, this study showed that PVA-MBs are useful as a contrast agent for multimodal imaging. PMID:26187672

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

    OpenAIRE

    2014-01-01

    Background In vivo bioluminescence, fluorescence, and single-photon emission computed tomography (SPECT) imaging provide complementary information about biological processes. However, to date these signatures are evaluated separately on individual preclinical systems. In this paper, we introduce a fully integrated bioluminescence-fluorescence-SPECT platform. Next to an optimization in logistics and image fusion, this integration can help improve understanding of the optical imaging (OI) resul...

  1. Biological oscillations: Fluorescence monitoring by confocal microscopy

    Science.gov (United States)

    Chattoraj, Shyamtanu; Bhattacharyya, Kankan

    2016-09-01

    Fluctuations play a vital role in biological systems. Single molecule spectroscopy has recently revealed many new kinds of fluctuations in biological molecules. In this account, we focus on structural fluctuations of an antigen-antibody complex, conformational dynamics of a DNA quadruplex, effects of taxol on dynamics of microtubules, intermittent red-ox oscillations at different organelles in a live cell (mitochondria, lipid droplets, endoplasmic reticulum and cell membrane) and stochastic resonance in gene silencing. We show that there are major differences in these dynamics between a cancer cell and the corresponding non-cancer cell.

  2. Radiometric calibration to consider in quantitative clinical fluorescence imaging measurements

    Science.gov (United States)

    Litorja, M.; Urbas, A.; Zong, Y.

    2015-03-01

    The fluorescent light detected by a clinical imager is assumed to be proportional only to the amount of fluorescent substance present in the sample and the level of excitation. Unfortunately, there are many factors that can add or subtract to the light signal directly attributable to the desired fluorescence emission, especially with fluorescence from inside the body imaged remotely. The quantification of fluorescence emission is feasible by calibrating the imager using international system of units (SI)-traceable physical and material calibration artifacts such that the detector's digital numbers (DN) can be converted to radiometric units. Here we discuss three calibration methods for quantitative clinical fluorescence imaging systems.

  3. Computed tomography based spectral imaging for fluorescence microscopy

    Science.gov (United States)

    Ford, Bridget Kathleen

    Multispectral imaging has been used for decades in remote sensing to enhance the classification, discrimination and characterization of materials. Only recently has this same technology been similarly applied to fixed biological samples in cytogenetics, pathology and medicine. A further extension to in vivo studies is often limited by the low levels of associated fluorescence as well as the increased temporal resolution required to analyze physiological changes. In addition, the cellular response to a specific agonist is often heterogeneous across the cellular field requiring a combination of sufficient spatial and temporal resolutions. A computed tomography imaging spectrometer (CTIS) has been developed which overcomes these limitations by simultaneously collecting extended range spectral information (470--740 nm, 5 nm sampling) across a 2-D field of view (200 mum x 200 mum, 0.96 mum sampling). The CTIS uses a computer generated hologram to produce a 5 x 5 array of images with differing amounts and directions of dispersion. This set of images allows the 3-D signal (x, y, lambda) from a fluorescent sample to be mapped onto a 2-D detector array. In this way, the full spectral and spatial information is acquired for a 2-D cellular field during a single integration time (presently 2 sec for biological specimens). The CTIS's design, calibration, and underlying theory are described in detail. In addition, the capability of the CTIS to simultaneously collect the fluorescence emission of multiple fluorophores across a 2-D cellular field is demonstrated. Specifically, the combined spectral variations of seminapthorhodafluor-I and enhanced green fluorescent protein were followed in rat insulinoma cells in order to extend the linear range of intracellular pH detection.

  4. Two photon fluorescence imaging of lipid membrane domains and potentials using advanced fluorescent probes

    Science.gov (United States)

    Kilin, Vasyl; Darwich, Zeinab; Richert, Ludovic; Didier, Pascal; Klymchenko, Andrey; Mély, Yves

    2013-02-01

    Biomembranes are ordered and dynamic nanoscale structures critical for cell functions. The biological functions of the membranes strongly depend on their physicochemical properties, such as electrostatics, phase state, viscosity, polarity and hydration. These properties are essential for the membrane structure and the proper folding and function of membrane proteins. To monitor these properties, fluorescence techniques and notably, two-photon microscopy appear highly suited due to their exquisite sensitivity and their capability to operate in complex biological systems, such as living cells and tissues. In this context, we have developed multiparametric environment-sensitive fluorescent probes tailored for precise location in the membrane bilayer. We notably developed probes of the 3-hydroxychromone family, characterized by an excited state intramolecular proton transfer reaction, which generates two tautomeric emissive species with well-separated emission bands. As a consequence, the response of these probes to changes in their environment could be monitored through changes in the ratios of the two bands, as well as through changes in the fluorescence lifetimes. Using two-photon ratiometric imaging and FLIM, these probes were used to monitor the surface membrane potential, and were applied to detect apoptotic cells and image membrane domains.

  5. LED light source for hyperspectral fluorescence imaging

    OpenAIRE

    Tendenes, Nils Ove

    2012-01-01

    This report deals with the possibility of creating a LED light source, to be used withhyperspectral fluorescence imaging. There are commercially available light sources thatcould be used, but they are expensive, they do not necessarily emit the right wavelength, the uniformity of the field is questionable and they are difficult to modify.First a batch of Light emitting diodes were acquired, these were subjected to a seriesof tests to classify their limitations and determine which diodes were ...

  6. Porphyrin-magnetite nanoconjugates for biological imaging

    LENUS (Irish Health Repository)

    Nowostawska, Malgorzata

    2011-04-08

    Abstract Background The use of silica coated magnetic nanoparticles as contrast agents has resulted in the production of highly stable, non-toxic solutions that can be manipulated via an external magnetic field. As a result, the interaction of these nanocomposites with cells is of vital importance in understanding their behaviour and biocompatibility. Here we report the preparation, characterisation and potential application of new "two-in-one" magnetic fluorescent nanocomposites composed of silica-coated magnetite nanoparticles covalently linked to a porphyrin moiety. Method The experiments were performed by administering porphyrin functionalised silica-coated magnetite nanoparticles to THP-1 cells, a human acute monocytic leukaemia cell line. Cells were cultured in RPMI 1640 medium with 25 mM HEPES supplemented with heat-inactivated foetal bovine serum (FBS). Results We have synthesised, characterised and analysed in vitro, a new multimodal (magnetic and fluorescent) porphyrin magnetic nanoparticle composite (PMNC). Initial co-incubation experiments performed with THP-1 macrophage cells were promising; however the PMNC photobleached under confocal microscopy study. β-mercaptoethanol (β-ME) was employed to counteract this problem and resulted not only in enhanced fluorescence emission, but also allowed for elongated imaging and increased exposure times of the PMNC in a cellular environment. Conclusion Our experiments have demonstrated that β-ME visibly enhances the emission intensity. No deleterious effects to the cells were witnessed upon co-incubation with β-ME alone and no increases in background fluorescence were recorded. These results should present an interest for further development of in vitro biological imaging techniques.

  7. Total internal reflection fluorescence microscopy imaging-guided confocal single-molecule fluorescence spectroscopy

    OpenAIRE

    Zheng, Desheng; Kaldaras, Leonora; Lu, H. Peter

    2012-01-01

    We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscop...

  8. Selective Detection of Neurotransmitters by Fluorescence and Chemiluminescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ziqiang Wang; Edward S. Yeung

    2001-08-06

    In recent years, luminescence imaging has been widely employed in neurochemical analysis. It has a number of advantages for the study of neuronal and other biological cells: (1) a particular molecular species or cellular constituent can be selectively visualized in the presence of a large excess of other species in a heterogeneous environment; (2) low concentration detection limits can be achieved because of the inherent sensitivity associated with fluorescence and chemiluminescence; (3) low excitation intensities can be used so that long-term observation can be realized while the viability of the specimen is preserved; and (4) excellent spatial resolution can be obtained with the light microscope so subcellular compartments can be identified. With good sensitivity, temporal and spatial resolution, the flux of ions and molecules and the distribution and dynamics of intracellular species can be measured in real time with specific luminescence probes, substrates, or with native fluorescence. A noninvasive detection scheme based on glutamate dehydrogenase (GDH) enzymatic assay combined with microscopy was developed to measure the glutamate release in cultured cells from the central nervous system (CNS). The enzyme reaction is very specific and sensitive. The detection limit with CCD imaging is down to {micro}M levels of glutamate with reasonable response time. They also found that chemiluminescence associated with the ATP-dependent reaction between luciferase and luciferin can be used to image ATP at levels down to 10 nM in the millisecond time scale. Similar imaging experiments should be feasible in a broad spectrum of biological systems.

  9. Selective Detection of Neurotransmitters by Fluorescence and Chemiluminescence Imaging

    International Nuclear Information System (INIS)

    In recent years, luminescence imaging has been widely employed in neurochemical analysis. It has a number of advantages for the study of neuronal and other biological cells: (1) a particular molecular species or cellular constituent can be selectively visualized in the presence of a large excess of other species in a heterogeneous environment; (2) low concentration detection limits can be achieved because of the inherent sensitivity associated with fluorescence and chemiluminescence; (3) low excitation intensities can be used so that long-term observation can be realized while the viability of the specimen is preserved; and (4) excellent spatial resolution can be obtained with the light microscope so subcellular compartments can be identified. With good sensitivity, temporal and spatial resolution, the flux of ions and molecules and the distribution and dynamics of intracellular species can be measured in real time with specific luminescence probes, substrates, or with native fluorescence. A noninvasive detection scheme based on glutamate dehydrogenase (GDH) enzymatic assay combined with microscopy was developed to measure the glutamate release in cultured cells from the central nervous system (CNS). The enzyme reaction is very specific and sensitive. The detection limit with CCD imaging is down to(micro)M levels of glutamate with reasonable response time. They also found that chemiluminescence associated with the ATP-dependent reaction between luciferase and luciferin can be used to image ATP at levels down to 10 nM in the millisecond time scale. Similar imaging experiments should be feasible in a broad spectrum of biological systems

  10. Nanoparticles and nanocomposites for fluorescence sensing and imaging

    International Nuclear Information System (INIS)

    The assortment of fluorescence reporters is changing dramatically. Traditionally explored intrinsic fluorescence of biological macromolecules and cellular pigments and of externally introduced organic dyes are presently in strong competition with new nanomaterials. Among them are conjugated polymers, semiconductor nanocrystals (quantum dots), up-converting nanocrystals, magic-size clusters of silver and gold, nanodiamonds and carbon dots. They demonstrate diverse photophysical behavior and allow one to obtain diverse information when used in analytical tools or when they form images in biological systems. Based on them, functional nanocomposites displaying a variety of useful features, thus extending dramatically the information content of output data, can be constructed. We describe their properties and compare them with those of small-molecular emitters, such as organic dyes. With their aid, one can modulate over a wide range the wavelengths of excitation and emission, the lifetimes and anisotropies and design the systems with ‘superenhancement’ and ‘superquenching’. Such unlimited possibilities are offered by combining different types of luminophores based on electronic conjugation, plasmonic effects or excited-state resonance energy transfer. This tutorial review provides a comparative analysis of the properties of new nanoscale materials and of their hybrid nanocomposites for applications in fluorescence sensing and imaging. (topical review)

  11. Fluorenyl benzothiadiazole and benzoselenadiazole near-IR fluorescent probes for two-photon fluorescence imaging (Conference Presentation)

    Science.gov (United States)

    Belfield, Kevin D.; Yao, Sheng; Kim, Bosung; Yue, Xiling

    2016-03-01

    Imaging biological samples with two-photon fluorescence (2PF) microscopy has the unique advantage of resulting high contrast 3D resolution subcellular image that can reach up to several millimeters depth. 2PF probes that absorb and emit at near IR region need to be developed. Two-photon excitation (2PE) wavelengths are less concerned as 2PE uses wavelengths doubles the absorption wavelength of the probe, which means 2PE wavelengths for probes even with absorption at visible wavelength will fall into NIR region. Therefore, probes that fluoresce at near IR region with high quantum yields are needed. A series of dyes based on 5-thienyl-2, 1, 3-benzothiadiazole and 5-thienyl-2, 1, 3-benzoselenadiazole core were synthesized as near infrared two-photon fluorophores. Fluorescence maxima wavelengths as long as 714 nm and fluorescence quantum yields as high as 0.67 were achieved. The fluorescence quantum yields of the dyes were nearly constant, regardless of solvents polarity. These diazoles exhibited large Stokes shift (two-photon absorption cross sections (up to 2,800 GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.

  12. Design Strategies of Fluorescent Biosensors Based on Biological Macromolecular Receptors

    Directory of Open Access Journals (Sweden)

    Takashi Morii

    2010-02-01

    Full Text Available Fluorescent biosensors to detect the bona fide events of biologically important molecules in living cells are increasingly demanded in the field of molecular cell biology. Recent advances in the development of fluorescent biosensors have made an outstanding contribution to elucidating not only the roles of individual biomolecules, but also the dynamic intracellular relationships between these molecules. However, rational design strategies of fluorescent biosensors are not as mature as they look. An insatiable request for the establishment of a more universal and versatile strategy continues to provide an attractive alternative, so-called modular strategy, which permits facile preparation of biosensors with tailored characteristics by a simple combination of a receptor and a signal transducer. This review describes an overview of the progress in design strategies of fluorescent biosensors, such as auto-fluorescent protein-based biosensors, protein-based biosensors covalently modified with synthetic fluorophores, and signaling aptamers, and highlights the insight into how a given receptor is converted to a fluorescent biosensor. Furthermore, we will demonstrate a significance of the modular strategy for the sensor design.

  13. In Vivo Fluorescence Reflectance Imaging with Subcutaneous Mouse Tumor Models.

    Science.gov (United States)

    Cao, Jie; Zhou, Mingzhou

    2016-01-01

    Optical imaging is undoubtedly one of the most versatile and widely used imaging techniques in both research and clinical practice. Among optical imaging technologies, fluorescence imaging is the most popularly used and has become an essential tool in biomedical science. A key component of fluorescence imaging is fluorescence-producing reporters, including fluorescent dyes and conjugates, as well as fluorescent proteins. For in vivo imaging applications, fluorophores with long emission at the near-infrared (NIR) region are generally preferred to overcome the photon attenuation in living tissue. Here, we describe the in vivo fluorescence imaging of an integrin αυβ3 targeted NIR fluorescent probe (cRGD-ICG-Der-02) using subcutaneous mouse tumor models. PMID:27283414

  14. Fluorescence lifetime imaging of oxygen in living cells

    NARCIS (Netherlands)

    Gerritsen, H.C.; Sanders, R.; Draaijer, A.; Ince, C.; Levine, Y.K.

    1997-01-01

    The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence life-time imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can

  15. Biological Imaging Capability in the ABRS Facility on ISS

    Science.gov (United States)

    Cox, David R.; Murdoch, T.; Regan, M. F.; Meshlberger, R. J.; Mortenson, T. E.; Albino, S. A.; Paul, A. L.; Ferl, R. J.

    2010-01-01

    This slide presentation reviews the Advanced Biological Research System (ABRS) on the International Space Station (ISS) and its biological imaging capability. The ABRS is an environmental control chamber. It has two indpendently controlled Experiment Research Chambers (ERCs) with temperature, relative humidity and carbon dioxide controls. ABRS is a third generation plant growth system. Several experiments are reviewed, with particular interest in the use of Green Fluorescent Protein (GFP) a non-destructive plant stress reporting mechanism, naturally found in jellyfish.

  16. Gradient-Based Algorithm for Determining Tumor Volumes in Small Animals Using Planar Fluorescence Imaging Platform

    OpenAIRE

    Miller, Jessica P.; Egbulefu, Christopher; Prior, Julie L.; Zhou, Mingzhou; Achilefu, Samuel

    2016-01-01

    Planar fluorescence imaging is widely used in biological research because of its simplicity, use of non-ionizing radiation, and high-throughput data acquisition. In cancer research, where small animal models are used to study the in vivo effects of cancer therapeutics, the output of interest is often the tumor volume. Unfortunately, inaccuracies in determining tumor volume from surface-weighted projection fluorescence images undermine the data, and alternative physical or conventional tomogra...

  17. Biologic fluorescence decay characteristics: determination by Laguerre expansion technique

    Science.gov (United States)

    Snyder, Wendy J.; Maarek, Jean-Michel I.; Papaioannou, Thanassis; Marmarelis, Vasilis Z.; Grundfest, Warren S.

    1996-04-01

    Fluorescence decay characteristics are used to identify biologic fluorophores and to characterize interactions with the fluorophore environment. In many studies, fluorescence lifetimes are assessed by iterative reconvolution techniques. We investigated the use of a new approach: the Laguerre expansion of kernels technique (Marmarelis, V.Z., Ann. Biomed., Eng. 1993; 21, 573-589) which yields the fluorescence impulse response function by least- squares fitting of a discrete-time Laguerre functions expansion. Nitrogen (4 ns FWHM) and excimer (120 ns FWHM) laser pulses were used to excite the fluorescence of an anthracene and of type II collagen powder. After filtering (monochromator) and detection (MCP-PMT), the fluorescence response was digitized (digital storage oscilloscope) and transferred to a personal computer. Input and output data were deconvolved by the Laguerre expansion technique to compute the impulse response function which was then fitted to a multiexponential function for determination of the decay constants. A single exponential (time constant: 4.24 ns) best approximated the fluorescence decay of anthracene, whereas the Type II collagen response was best approximated by a double exponential (time constants: 2.24 and 9.92 ns) in agreement with previously reported data. The results of the Laguerre expansion technique were compared to the least-squares iterative reconvolution technique. The Laguerre expansion technique appeared computationally efficient and robust to experimental noise in the data. Furthermore, the proposed method does not impose a set multiexponential form to the decay.

  18. Quantitative fluorescence microscopy of macromolecules in gel and biological tissue

    Energy Technology Data Exchange (ETDEWEB)

    Tatarkova, Svetlana A [Department of Physics, University of Durham, Durham DH1 3LE (United Kingdom); School of Pharmacy, University of Manchester, Manchester M13 9PL (United Kingdom); Verma, Anita Kamra [School of Pharmacy, University of Manchester, Manchester M13 9PL (United Kingdom); Department of Zoology, K M College, University of Delhi, Delhi-110 007 (India); Berk, David A [School of Pharmacy, University of Manchester, Manchester M13 9PL (United Kingdom); Lloyd, Christopher J [School of Pharmacy, University of Manchester, Manchester M13 9PL (United Kingdom)

    2005-12-07

    Quantitative fluorescence microscopy provides valuable insight into drug delivery and pharmacokinetics. The technique is based on analysis of statistical fluctuations in fluorescence that arises as fluorophores pass through a small volume illuminated by a focused laser beam, and has been applied to measure particle motion and binding interactions in solutions, on surfaces and inside the cells. We examined the use of fluorescence correlation spectroscopy combined with a microscope (FCSM) to assess the transport of fluorescent beads and macromolecules in aqueous solutions, gels and living biological tissue. Obstructed diffusion of fluorescent beads in gels of various densities was tested to get a sensible estimate of diffusion in the interstitial tissue matrix consistent with previous reports. Fluorescently labelled liposomes as an artificial drug or gene carrying vehicles were used for pharmacokinetic tests of drug delivery in living tissue. The results indicate that FCS is an accurate and valuable tool for measuring the physical properties of gene vectors in vitro and for characterizing interactions with tissue in vivo.

  19. Smartphone microendoscopy for high resolution fluorescence imaging

    CERN Document Server

    Hong, Xiangqian; Mugler, Dale H; Yu, Bing

    2016-01-01

    High resolution optical endoscopes are increasingly used in diagnosis of various medical conditions of internal organs, such as the gastrointestinal tracts, but they are too expensive for use in resource-poor settings. On the other hand, smartphones with high resolution cameras and Internet access have become more affordable, enabling them to diffuse into most rural areas and developing countries in the past decade. In this letter we describe a smartphone microendoscope that can take fluorescence images with a spatial resolution of 3.1 {\\mu}m. Images collected from ex vivo, in vitro and in vivo samples using the device are also presented. The compact and cost-effective smartphone microendoscope may be envisaged as a powerful tool for detecting pre-cancerous lesions of internal organs in low and middle income countries.

  20. Semiconductor Nanocrystals for Biological Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Aihua; Gu, Weiwei; Larabell, Carolyn; Alivisatos, A. Paul

    2005-06-28

    Conventional organic fluorophores suffer from poor photo stability, narrow absorption spectra and broad emission feature. Semiconductor nanocrystals, on the other hand, are highly photo-stable with broad absorption spectra and narrow size-tunable emission spectra. Recent advances in the synthesis of these materials have resulted in bright, sensitive, extremely photo-stable and biocompatible semiconductor fluorophores. Commercial availability facilitates their application in a variety of unprecedented biological experiments, including multiplexed cellular imaging, long-term in vitro and in vivo labeling, deep tissue structure mapping and single particle investigation of dynamic cellular processes. Semiconductor nanocrystals are one of the first examples of nanotechnology enabling a new class of biomedical applications.

  1. Tunable fluorescence from patterned silver nano-island arrays for sensitive sub-cell imaging

    International Nuclear Information System (INIS)

    Surface-enhanced fluorescence, a burgeoning technique in biological detection, provides largely enhanced fluorescence signal by exciting localized surfaces plasmons resonance with fluorescent dyes. Nanostructure and surroundings brings great impact on the emission signal, however, insufficient physics about the process limits further improvement on the nanostructure design. In this study, optical properties of Rhodamin-6G molecules on patterned silver nano-island arrays are tailored by precisely controlling the distance between the dyes and silver arrays. The fluorescence signal depends on the distance and the largest enhancement of 10 folds is achieved when the distance is 10 nm. The results are theoretically corroborated by finite difference time domain simulation and applied to cytoskeleton fluorescence imaging using phalloidin–fluorescein isothiocyanate. Our study provides insights into the physical mechanisms associated with the fluorescence enhancement and quenching, and our experiments suggest potential applications to high-sensitivity sub-cell imaging. (paper)

  2. Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy

    OpenAIRE

    Lin, Po-Yen; Lyu, Hong-Chou; Hsu, Chin-Ying Stephen; Chang, Chia-Seng; Kao, Fu-Jen

    2010-01-01

    In this study, multiphoton excitation was utilized to image normal and carious dental tissues noninvasively. Unique structures in dental tissues were identified using the available multimodality (second harmonic, autofluorescence, and fluorescence lifetime analysis) without labeling. The collagen in dentin exhibits a strong second harmonic response. Both dentin and enamel emit strong autofluorescence that reveals in detail morphological features (such as dentinal tubules and enamel rods) and,...

  3. Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

    OpenAIRE

    Niino, Yusuke; Hotta, Kohji; Oka, Kotaro

    2010-01-01

    Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca2+. 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 indica...

  4. Bacterial imaging with photostable upconversion fluorescent nanoparticles.

    Science.gov (United States)

    Ong, Li Ching; Ang, Lei Yin; Alonso, Sylvie; Zhang, Yong

    2014-03-01

    Autofluorescence, photodamage and photobleaching are often encountered when using downconverting fluorophores and fluorescent proteins for bacteria labeling. These caveats represent a serious limitation when trying to map bacteria dissemination for prolonged periods. Upconversion nanoparticles (UCNs), which are able to convert low energy near-infrared (NIR) excitation light into higher energy visible or NIR light, can address these limitations. These particles' unique optical properties translate into attractive advantages of minimal autofluorescence, reduced photodamage, deeper tissue penetration and prolonged photostability. Here, we report a UCN-based bacteria labeling strategy using Escherichia coli as prototypic bacteria. A comparative analysis highlighted the superior photostability of UCN-labeled bacteria over green fluorescent protein-expressing bacteria. Infection study of UCN-labeled bacteria in dendritic cells indicated co-localization of the UCN signal with bacterial position for up to 6 h post-infection. Furthermore, long-term monitoring of the same infected cells demonstrated the potential to utilize photostable UCN-based imaging for bacterial trafficking purposes. PMID:24412082

  5. Combination of fluorescence imaging and local spectrophotometry in fluorescence diagnostics of early cancer of larynx and bronchi

    International Nuclear Information System (INIS)

    The results of comparative studies of autofluorescence and 5-ALA-induced fluorescence of protoporphyrin IX, used in the diagnostics of early cancer of larynx and bronchi, are presented. The autofluorescence and 5-ALA-induced fluorescence images of larynx and bronchial tissues are analysed during the endoscopic study. The method of local spectrophotometry is used to verify findings obtained from fluorescence images. It is shown that such a combined approach can be efficiently used to improve the diagnostics of precancer and early cancer, to detect a primary multiple tumours, as well as for the diagnostics of a residual tumour or an early recurrence after the endoscopic, surgery or X-ray treatment. The developed approach allows one to minimise the number of false-positive results and to reduce the number of biopsies, which are commonly used in the white-light bronchoscopy search for occult cancerous loci. (laser biology and medicine)

  6. Semiconductor Quantum Rods as Single Molecule FluorescentBiological Labels

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Aihua; Gu, Weiwei; Boussert, Benjamine; Koski, Kristie; Gerion, Daniele; Manna, Liberato; Le Gros, Mark; Larabell, Carolyn; Alivisatos, A. Paul

    2006-05-29

    In recent years, semiconductor quantum dots have beenapplied with great advantage in a wide range of biological imagingapplications. The continuing developments in the synthesis of nanoscalematerials and specifically in the area of colloidal semiconductornanocrystals have created an opportunity to generate a next generation ofbiological labels with complementary or in some cases enhanced propertiescompared to colloidal quantum dots. In this paper, we report thedevelopment of rod shaped semiconductor nanocrystals (quantum rods) asnew fluorescent biological labels. We have engineered biocompatiblequantum rods by surface silanization and have applied them fornon-specific cell tracking as well as specific cellular targeting. Theproperties of quantum rods as demonstrated here are enhanced sensitivityand greater resistance for degradation as compared to quantum dots.Quantum rods have many potential applications as biological labels insituations where their properties offer advantages over quantumdots.

  7. Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging.

    Science.gov (United States)

    Nishimura, Hirohito; Ritchie, Ken; Kasai, Rinshi S; Goto, Miki; Morone, Nobuhiro; Sugimura, Hiroyuki; Tanaka, Koichiro; Sase, Ichiro; Yoshimura, Akihiko; Nakano, Yoshitaro; Fujiwara, Takahiro K; Kusumi, Akihiro

    2013-09-16

    Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1-2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed. PMID:24043702

  8. Another treatment of fluorescence polarization microspectroscopy and imaging.

    Science.gov (United States)

    Fisz, Jacek J

    2009-04-16

    -angular excitation and/or detection apertures are displayed and discussed in a systematic way. A few words of comment are added on the application of the symmetry adapted calibration (SAC) method to TRFPM experiments. A very important aim of this article is to provide a correct and more complete description of fluorescence polarization microspectroscopy and imaging of macroscopically isotropic media (i.e., solutions, solutions of labeled macromolecules, membrane suspensions, or biological cells), that can be immediately applied in the experimental practice in the life and medical sciences and also in different areas of nano(bio)technology. PMID:19309131

  9. Fabrication of SERS-fluorescence dual modal nanoprobes and application to multiplex cancer cell imaging

    Science.gov (United States)

    Lee, Sangyeop; Chon, Hyangah; Yoon, Soo-Young; Lee, Eun Kyu; Chang, Soo-Ik; Lim, Dong Woo; Choo, Jaebum

    2011-12-01

    We report a highly sensitive optical imaging technology using surface-enhanced Raman scattering (SERS)-fluorescence dual modal nanoprobes (DMNPs). Fluorescence microscopy is a well-known imaging technique that shows specific protein distributions within cells. However, most currently available fluorescent organic dyes have relatively weak emission intensities and are rapidly photo-bleached. Thus more sensitive and stable probes are needed. In this work we develop DMNPs, which can be used for both SERS and fluorescence detection. SERS detection is a powerful technique that allows ultrasensitive chemical or biochemical analysis through unlimited multiplexing and single molecule sensitivity. Combining advantages of fluorescence and SERS allows these dual modal nanostructures to be used as powerful probes for novel biomedical imaging. In this work, the fabrication and characterization of the SERS-fluorescence DMNPs and application to biological imaging were investigated using markers CD24 and CD44, which are co-expressed in MDA-MB-231 breast cancer cells, as a model system. SERS imaging with DMNPs was found to be a powerful tool to determine the co-localization of CD24 and CD44 in the cell.We report a highly sensitive optical imaging technology using surface-enhanced Raman scattering (SERS)-fluorescence dual modal nanoprobes (DMNPs). Fluorescence microscopy is a well-known imaging technique that shows specific protein distributions within cells. However, most currently available fluorescent organic dyes have relatively weak emission intensities and are rapidly photo-bleached. Thus more sensitive and stable probes are needed. In this work we develop DMNPs, which can be used for both SERS and fluorescence detection. SERS detection is a powerful technique that allows ultrasensitive chemical or biochemical analysis through unlimited multiplexing and single molecule sensitivity. Combining advantages of fluorescence and SERS allows these dual modal nanostructures to be used

  10. Biological detection and tagging using tailorable, reactive, highly fluorescent chemosensors.

    Energy Technology Data Exchange (ETDEWEB)

    Shepodd, Timothy J.; Zifer, Thomas; McElhanon, James Ross; Rahn, Larry A.

    2006-11-01

    This program was focused on the development of a fluorogenic chemosensor family that could tuned for reaction with electrophilic (e.g. chemical species, toxins) and nucleophilic (e.g. proteins and other biological molecules) species. Our chemosensor approach utilized the fluorescent properties of well-known berberine-type alkaloids. In situ chemosensor reaction with a target species transformed two out-of-plane, weakly conjugated, short-wavelength chromophores into one rigid, planar, conjugated, chromophore with strong long wavelength fluorescence (530-560 nm,) and large Stokes shift (100-180 nm). The chemosensor was activated with an isourea group which allowed for reaction with carboxylic acid moieties found in amino acids.

  11. Fluorescence Imaging Study of Impinging Underexpanded Jets

    Science.gov (United States)

    Inman, Jennifer A.; Danehy, Paul M.; Nowak, Robert J.; Alderfer, David W.

    2008-01-01

    An experiment was designed to create a simplified simulation of the flow through a hole in the surface of a hypersonic aerospace vehicle and the subsequent impingement of the flow on internal structures. In addition to planar laser-induced fluorescence (PLIF) flow visualization, pressure measurements were recorded on the surface of an impingement target. The PLIF images themselves provide quantitative spatial information about structure of the impinging jets. The images also help in the interpretation of impingement surface pressure profiles by highlighting the flow structures corresponding to distinctive features of these pressure profiles. The shape of the pressure distribution along the impingement surface was found to be double-peaked in cases with a sufficiently high jet-exit-to-ambient pressure ratio so as to have a Mach disk, as well as in cases where a flow feature called a recirculation bubble formed at the impingement surface. The formation of a recirculation bubble was in turn found to depend very sensitively upon the jet-exit-to-ambient pressure ratio. The pressure measured at the surface was typically less than half the nozzle plenum pressure at low jet pressure ratios and decreased with increasing jet pressure ratios. Angled impingement cases showed that impingement at a 60deg angle resulted in up to a factor of three increase in maximum pressure at the plate compared to normal incidence.

  12. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    OpenAIRE

    Morrell-Falvey, J. L.; Qi, H.; Doktycz, M. J.; Venkatraman, S.

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features ...

  13. Imaging cell biology in live animals: Ready for prime time

    OpenAIRE

    Weigert, Roberto; Porat-Shliom, Natalie; Amornphimoltham, Panomwat

    2013-01-01

    Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobi...

  14. Reflectance and Fluorescence Spectral Recovery via Actively Lit RGB Images.

    Science.gov (United States)

    Fu, Ying; Lam, Antony; Sato, Imari; Okabe, Takahiro; Sato, Yoichi

    2016-07-01

    In recent years, fluorescence analysis of scenes has received attention in computer vision. Fluorescence can provide additional information about scenes, and has been used in applications such as camera spectral sensitivity estimation, 3D reconstruction, and color relighting. In particular, hyperspectral images of reflective-fluorescent scenes provide a rich amount of data. However, due to the complex nature of fluorescence, hyperspectral imaging methods rely on specialized equipment such as hyperspectral cameras and specialized illuminants. In this paper, we propose a more practical approach to hyperspectral imaging of reflective-fluorescent scenes using only a conventional RGB camera and varied colored illuminants. The key idea of our approach is to exploit a unique property of fluorescence: the chromaticity of fluorescent emissions are invariant under different illuminants. This allows us to robustly estimate spectral reflectance and fluorescent emission chromaticity. We then show that given the spectral reflectance and fluorescent chromaticity, the fluorescence absorption and emission spectra can also be estimated. We demonstrate in results that all scene spectra can be accurately estimated from RGB images. Finally, we show that our method can be used to accurately relight scenes under novel lighting. PMID:27295456

  15. Functionalization of hydroxyl terminated polybutadiene with biologically active fluorescent molecule

    Indian Academy of Sciences (India)

    R Murali Sankar; Subhadeep Saha; K Seeni Meera; Tushar Jana

    2009-10-01

    A biologically active molecule, 2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (CBDT), has been covalently attached at the terminal carbon atoms of the hydroxyl terminated polybutadiene (HTPB) backbone. The modification of HTPB backbone by CBDT molecule does not affect the unique physico-chemical properties such as fluidity, hydroxyl value and microstructure of the parent HTPB. The formation of hydrogen bonding between the terminal hydroxyl groups and the nitrogen atoms of triazine moiety is the driving force for the terminal attachment chemistry. The functionalized HTPB (HTPB–CBDT) shows a strong fluorescence emission at 385 nm.

  16. Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies

    International Nuclear Information System (INIS)

    Optical visualization of nanoscale morphological changes taking place in living biological cells during such important processes as endo- and exocytosis is challenging due to the low refractive index of lipid membranes. In this paper we summarize and discuss advances in the powerful combination of two complementary live imaging techniques, ion conductance and fluorescence confocal microscopy, that allows cell membrane topography to be related with molecular-specific fluorescence at high spatial and temporal resolution. We demonstrate the feasibility of the use of ion conductance microscopy to image apical plasma membrane of mouse embryo trophoblast outgrowth cells at a resolution sufficient to depict single endocytic pits. This opens the possibility to study individual endocytic events in embryo trophoblast outgrowth cells where endocytosis plays a crucial role during early stages of embryo development. (special issue article)

  17. Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies

    Science.gov (United States)

    Shevchuk, A. I.; Novak, P.; Velazquez, M. A.; Fleming, T. P.; Korchev, Y. E.

    2013-09-01

    Optical visualization of nanoscale morphological changes taking place in living biological cells during such important processes as endo- and exocytosis is challenging due to the low refractive index of lipid membranes. In this paper we summarize and discuss advances in the powerful combination of two complementary live imaging techniques, ion conductance and fluorescence confocal microscopy, that allows cell membrane topography to be related with molecular-specific fluorescence at high spatial and temporal resolution. We demonstrate the feasibility of the use of ion conductance microscopy to image apical plasma membrane of mouse embryo trophoblast outgrowth cells at a resolution sufficient to depict single endocytic pits. This opens the possibility to study individual endocytic events in embryo trophoblast outgrowth cells where endocytosis plays a crucial role during early stages of embryo development.

  18. Intraoperative fluorescent imaging of intracranial tumors: a review.

    Science.gov (United States)

    Behbahaninia, Milad; Martirosyan, Nikolay L; Georges, Joseph; Udovich, Joshua A; Kalani, M Yashar S; Feuerstein, Burt G; Nakaji, Peter; Spetzler, Robert F; Preul, Mark C

    2013-05-01

    A review of fluorescent imaging for intracranial neoplasms is presented. Complete resection of brain cancer is seldom possible because of the goal to preserve brain tissue and the inability to visualize individual infiltrative tumor cells. Verification of histology and identification of tumor invasion in macroscopically normal-appearing brain tissue determine prognosis after resection of malignant gliomas. Therefore, imaging modalities aim to facilitate intraoperative decision-making. Intraoperative fluorescent imaging techniques have the potential to enable precise histopathologic diagnosis and to detect tumor remnants in the operative field. Macroscopic fluorescence imaging is effective for gross tumor detection. Microscopic imaging techniques enhance the sensitivity of the macroscopic observations and provide real-time histological information. Further development of clinical grade fluorescent agents specifically targeting tumor cells could improve the diagnostic and prognostic yield of intraoperative imaging. PMID:23523009

  19. High resolution fluorescent bio-imaging with electron beam excitation.

    Science.gov (United States)

    Kawata, Yoshimasa; Nawa, Yasunori; Inami, Wataru

    2014-11-01

    We have developed electron beam excitation assisted (EXA) optical microscope[1-3], and demonstrated its resolution higher than 50 nm. In the microscope, a light source in a few nanometers size is excited by focused electron beam in a luminescent film. The microscope makes it possible to observe dynamic behavior of living biological specimens in various surroundings, such as air or liquids. Scan speed of the nanometric light source is faster than that in conventional near-field scanning optical microscopes. The microscope enables to observe optical constants such as absorption, refractive index, polarization, and their dynamic behavior on a nanometric scale. The microscope opens new microscopy applications in nano-technology and nano-science.Figure 1(a) shows schematic diagram of the proposed EXA microscope. An electron beam is focused on a luminescent film. A specimen is put on the luminescent film directly. The inset in Fig. 1(a) shows magnified image of the luminescent film and the specimen. Nanometric light source is excited in the luminescent film by the focused electron beam. The nanometric light source illuminates the specimen, and the scattered or transmitted radiation is detected with a photomultiplier tube (PMT). The light source is scanned by scanning of the focused electron beam in order to construct on image. Figure 1(b) shows a luminescence image of the cells acquired with the EXA microscope, and Fig. 1(c) shows a phase contrast microscope image. Cells were observed in culture solution without any treatments, such as fixation and drying. The shape of each cell was clearly recognized and some bright spots were observed in cells. We believe that the bright spots indicated with arrows were auto-fluorescence of intracellular granules and light- grey regions were auto-fluorescence of cell membranes. It is clearly demonstrated that the EXA microscope is useful tool for observation of living biological cells in physiological conditions.jmicro;63/suppl_1/i

  20. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

    Science.gov (United States)

    Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

  1. Video-rate two-photon excited fluorescence lifetime imaging system with interleaved digitization.

    Science.gov (United States)

    Dow, Ximeng Y; Sullivan, Shane Z; Muir, Ryan D; Simpson, Garth J

    2015-07-15

    A fast (up to video rate) two-photon excited fluorescence lifetime imaging system based on interleaved digitization is demonstrated. The system is compatible with existing beam-scanning microscopes with minor electronics and software modification. Proof-of-concept demonstrations were performed using laser dyes and biological tissue. PMID:26176453

  2. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development.

    Science.gov (United States)

    Icha, Jaroslav; Schmied, Christopher; Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments. PMID:27167079

  3. Tip-enhanced fluorescence imaging of quantum dots

    OpenAIRE

    Huang, F.M.; Festy, F; Richards, D. R.

    2005-01-01

    We have imaged the fluorescence from a single quantum dot cluster using an apertureless scanning near-field optical microscope. When a sharp gold tip is brought within a few nanometers from the sample surface, the resulting enhancement in quantum dot fluorescence in the vicinity of the tip leads to a resolution of about 60 nm. We determine this enhancement of the fluorescence to be about fourfold in magnitude, which is consistent with the value expected as a result of competition between fluo...

  4. Shaping Small Bioactive Molecules to Untangle Their Biological Function: A Focus on Fluorescent Plant Hormones.

    Science.gov (United States)

    Lace, Beatrice; Prandi, Cristina

    2016-08-01

    Modern biology overlaps with chemistry in explaining the structure and function of all cellular processes at the molecular level. Plant hormone research is perfectly located at the interface between these two disciplines, taking advantage of synthetic and computational chemistry as a tool to decipher the complex biological mechanisms regulating the action of plant hormones. These small signaling molecules regulate a wide range of developmental processes, adapting plant growth to ever changing environmental conditions. The synthesis of small bioactive molecules mimicking the activity of endogenous hormones allows us to unveil many molecular features of their functioning, giving rise to a new field, plant chemical biology. In this framework, fluorescence labeling of plant hormones is emerging as a successful strategy to track the fate of these challenging molecules inside living organisms. Thanks to the increasing availability of new fluorescent probes as well as advanced and innovative imaging technologies, we are now in a position to investigate many of the dynamic mechanisms through which plant hormones exert their action. Such a deep and detailed comprehension is mandatory for the development of new green technologies for practical applications. In this review, we summarize the results obtained so far concerning the fluorescent labeling of plant hormones, highlighting the basic steps leading to the design and synthesis of these compelling molecular tools and their applications. PMID:27378726

  5. Quantification of tumor fluorescence during intraoperative optical cancer imaging.

    Science.gov (United States)

    Judy, Ryan P; Keating, Jane J; DeJesus, Elizabeth M; Jiang, Jack X; Okusanya, Olugbenga T; Nie, Shuming; Holt, David E; Arlauckas, Sean P; Low, Phillip S; Delikatny, E James; Singhal, Sunil

    2015-01-01

    Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small changes in fluorescence, sensitivity of signal detection at increasing depths and practicality of use. In vitro, spectroscopy was superior in detecting incremental differences in fluorescence than luminescence and digital imaging (Ln[SBR] = 6.8 ± 0.6, 2.4 ± 0.3, 2.6 ± 0.1, p = 0.0001). In fluorescent tumor cells, digital imaging measured higher SBRs than luminescence (6.1 ± 0.2 vs. 4.3 ± 0.4, p = 0.001). Spectroscopy was more sensitive than luminometry and digital imaging in identifying murine tumor fluorescence (SBR = 41.7 ± 11.5, 5.1 ± 1.8, 4.1 ± 0.9, p = 0.0001), and more sensitive than digital imaging at detecting fluorescence at increasing depths (SBR = 7.0 ± 3.4 vs. 2.4 ± 0.5, p = 0.03). Lastly, digital imaging was the most practical and least time-consuming. All methods detected incremental differences in fluorescence. Spectroscopy was the most sensitive for small changes in fluorescence. Digital imaging was the most practical considering its wide field of view, background noise filtering capability, and sensitivity to increasing depth. PMID:26563091

  6. Analysis of human aorta using fluorescence lifetime imaging microscopy (FLIM)

    Science.gov (United States)

    Vieira-Damiani, Gislaine; Adur, J.; Ferro, D. P.; Adam, R. L.; Pelegati, V.; Thomáz, A.; Cesar, C. L.; Metze, K.

    2012-03-01

    The use of photonics has improved our understanding of biologic phenomena. For the study of the normal and pathologic architecture of the aorta the use of Two-Photon Excited Fluorescence (TPEF) and Second Harmonic Generation showed interesting details of morphologic changes of the elastin-collagen architecture during aging or development of hypertension in previous studies. In this investigation we tried to apply fluorescence lifetime imaging (FLIM) for the morphologic analysis of human aortas. The aim of our study was to use FLIM in non-stained formalin-fixed and paraffin-embedded samples of the aorta ascendants in hypertensive and normotensive patients of various ages, examining two different topographical regions. The FLIM-spectra of collagen and elastic fibers were clearly distinguishable, thus permitting an exact analysis of unstained material on the microscopic level. Moreover the FLIM spectrum of elastic fibers revealed variations between individual cases, which indicate modifications on a molecular level and might be related to FLIM age or diseases states and reflect modifications on a molecular level.

  7. Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes

    OpenAIRE

    Guo, Hengchang; Aleyasin, Hossein; Howard, Scott S.; Dickinson, Bryan C; Lin, Vivian S.; Haskew-Layton, Renee E.; Xu, Chris; Chen, Yu; Ratan, Rajiv R.

    2013-01-01

    Abstract. We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H2O2) production in brain cells. For selective imaging of H2O2 over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H2O2 and mitochondria peroxy yellow 1 detects mitochondrial H2O2. Two-photon absorption cross sections for th...

  8. Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes

    OpenAIRE

    Guo, Hengchang; Aleyasin, Hossein; Howard, Scott S.; Dickinson, Bryan C; Lin, Vivian S.; Haskew-Layton, Renee E.; Xu, Chris; Chen, Yu; Ratan, Rajiv R.

    2013-01-01

    Abstract. We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide ( H 2 O 2 ) production in brain cells. For selective imaging of H 2 O 2 over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H 2 O 2 and mitochondria peroxy yellow 1 detects mitochondrial H 2 O 2 . Two-photon absorption cross ...

  9. Detection of rheumatoid arthritis in humans by fluorescence imaging

    Science.gov (United States)

    Ebert, Bernd; Dziekan, Thomas; Weissbach, Carmen; Mahler, Marianne; Schirner, Michael; Berliner, Birgitt; Bauer, Daniel; Voigt, Jan; Berliner, Michael; Bahner, Malte L.; Macdonald, Rainer

    2010-02-01

    The blood pool agent indo-cyanine green (ICG) has been investigated in a prospective clinical study for detection of rheumatoid arthritis using fluorescence imaging. Temporal behavior as well as spatial distribution of fluorescence intensity are suited to differentiate healthy and inflamed finger joints after i.v. injection of an ICG bolus.

  10. Easily Accessible and Highly Selective "Turn-on" Fluorescent Sensor for Imaging Cadmium in Living Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; ZHANG Ying-mu; LI Yao-xian; ZHAO Qing

    2013-01-01

    A new schiff base of phenothiazine derivative was designed for ratiometric sensing of Cd2+ selectively.Upon the addition of Cd2+ to the solution of phenothiazine derivative,the fluorescence intensity of it was enhanced in a linear fashion and the maximum fluorescence peak exhibited a blue shift from 575 nm to 525 nm.This ratiometric fluorescent sensor displays a very high sensitivity(detection limits were 0.34 and 1.0 μmol/L of Cd2+ using the visual fluorescence changes and UV-Vis changes,respectively),a rapid response time(<10 s) and high selectivity for Cd2+ over other transition metal ions.Moreover,the living cells image experiments also demonstrate the value of the sensor in fluorescent visualization of Cd2+ in the environmental and biological systems.

  11. Patch-based anisotropic diffusion scheme for fluorescence diffuse optical tomography—part 2: image reconstruction

    Science.gov (United States)

    Correia, Teresa; Koch, Maximilian; Ale, Angelique; Ntziachristos, Vasilis; Arridge, Simon

    2016-02-01

    Fluorescence diffuse optical tomography (fDOT) provides 3D images of fluorescence distributions in biological tissue, which represent molecular and cellular processes. The image reconstruction problem is highly ill-posed and requires regularisation techniques to stabilise and find meaningful solutions. Quadratic regularisation tends to either oversmooth or generate very noisy reconstructions, depending on the regularisation strength. Edge preserving methods, such as anisotropic diffusion regularisation (AD), can preserve important features in the fluorescence image and smooth out noise. However, AD has limited ability to distinguish an edge from noise. We propose a patch-based anisotropic diffusion regularisation (PAD), where regularisation strength is determined by a weighted average according to the similarity between patches around voxels within a search window, instead of a simple local neighbourhood strategy. However, this method has higher computational complexity and, hence, we wavelet compress the patches (PAD-WT) to speed it up, while simultaneously taking advantage of the denoising properties of wavelet thresholding. Furthermore, structural information can be incorporated into the image reconstruction with PAD-WT to improve image quality and resolution. In this case, the weights used to average voxels in the image are calculated using the structural image, instead of the fluorescence image. The regularisation strength depends on both structural and fluorescence images, which guarantees that the method can preserve fluorescence information even when it is not structurally visible in the anatomical images. In part 1, we tested the method using a denoising problem. Here, we use simulated and in vivo mouse fDOT data to assess the algorithm performance. Our results show that the proposed PAD-WT method provides high quality and noise free images, superior to those obtained using AD.

  12. Cell imaging by transient fluorescence detected infrared microscopy

    Science.gov (United States)

    Ohmori, Tsutomu; Sakai, Makoto; Ishihara, Miya; Kikuchi, Makoto; Fujii, Masaaki

    2008-02-01

    Transient fluorescence detected infrared (TFD-IR) microscopy was developed to overcome the diffraction limit of infrared (IR) light without a near-field system. This microscopic technique is based on TFD-IR spectroscopy, which converts information on IR absorption to fluorescence intensity by further electronic excitation of vibrationally excited molecules by a probing UV/visible light. Roots of Arabidopsis thaliana and living A549 cells with fluorescent dyes were chosen as samples. In the measurements using the TFD-IR microscope, tunable IR picosecond laser pulses were used in the wavelength range from 2700 to 3700 nm, corresponding to CH, NH, and OH stretching modes. Fluorescence images of the root cells of A. thaliana by the TFD-IR scheme were obtained with super-resolution compared with the resolution of conventional IR microscopy. The resolution is estimated to be less than 2.6 μm by fitting of a gaussian function. However, the TFD-IR images were dominated mainly by the fluorescent dyes because they were almost the same as a conventional fluorescence image. To investigate other contributions hidden by that of fluorescent dyes, we plotted the fluorescence intensity in several 5 μm squares at various IR wavelengths, called a TFD-IR spectrum. For root cells of A. thaliana, the TFD-IR spectra show shapes similar to those of a conventional IR absorption spectrum of the fluorescent dye. Therefore, the TFD-IR images are not due to the cellular components. For an A549 cell, the TFD-IR spectra were different from a conventional IR absorption spectrum of fluorescent dyes in the wavelength region shorter than 3100 nm. We speculate that the spectral difference is due to the cellular components, possibly assigned to the combination band related to amino groups of cellular components bonded covalently to the fluorescent dyes.

  13. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    OpenAIRE

    Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

    2014-01-01

    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human...

  14. Quantification of tumor fluorescence during intraoperative optical cancer imaging

    OpenAIRE

    Judy, Ryan P.; Keating, Jane J.; Elizabeth M. DeJesus; Jack X. Jiang; Okusanya, Olugbenga T.; Shuming Nie; Holt, David E.; Arlauckas, Sean P.; Low, Phillip S.; E. James Delikatny; Sunil Singhal

    2015-01-01

    Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small ch...

  15. 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.

  16. Image processing for drift compensation in fluorescence microscopy

    DEFF Research Database (Denmark)

    Petersen, Steffen; Thiagarajan, Viruthachalam; Coutinho, Isabel;

    2013-01-01

    Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove....../reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame....... We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the “Shift and Add” approach leading to an image of identical size asthe individual image. We have also reconstructed the image using a 3 fold larger...

  17. FISHji: new ImageJ macros for the quantification of fluorescence in epifluorescence images

    OpenAIRE

    Fontenete, S.; Carvalho, D; Lourenço, Anália; N.Guimarães; Madureira, P.; Figueiredo, C.; Azevedo, N. F.

    2016-01-01

    Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing sof...

  18. Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging.

    Science.gov (United States)

    Stoltzfus, Caleb R; Rebane, Aleksander

    2016-05-01

    We study the optimal conditions for high throughput two-photon excited fluorescence (2PEF) and three-photon excited fluorescence (3PEF) imaging using femtosecond lasers. We derive relations that allow maximization of the rate of imaging depending on the average power, pulse repetition rate, and noise characteristics of the laser, as well as on the size and structure of the sample. We perform our analysis using ~100 MHz, ~1 MHz and 1 kHz pulse rates and using both a tightly-focused illumination beam with diffraction-limited image resolution, as well loosely focused illumination with a relatively low image resolution, where the latter utilizes separate illumination and fluorescence detection beam paths. Our theoretical estimates agree with the experiments, which makes our approach especially useful for optimizing high throughput imaging of large samples with a field-of-view up to 10x10 cm(2). PMID:27231620

  19. Imaging chemical extraction by polymer inclusion membranes using fluorescence microscopy

    International Nuclear Information System (INIS)

    Polymer inclusion membranes (PIMs) transport chemicals between bodies of liquid by simultaneously performing chemical extraction and back-extraction. The internal chemical and physical mechanisms by which this transport occurs are, however, poorly understood. Also, some PIMs, which are otherwise optimal for their task, age and lose function after only days, limiting their feasibility for industrial upscaling. Through the application of fluorescence imaging methods we are able for the first time to see where chemical extraction occurs in the membrane. Extraction of fluorescein from solution by PIMs demonstrates inhomogeneities that do not correlate to surface morphology. Fluorescence lifetime imaging demonstrates that regions of increased extraction have distinctly different fluorescence lifetimes to that of the surrounding PIM indicating localized chemical environments, and this is observed to change with membrane age. Fluorescence imaging is shown to allow probing and novel understanding of PIM internal chemical morphology. (paper)

  20. SHG nanoprobes: advancing harmonic imaging in biology.

    Science.gov (United States)

    Dempsey, William P; Fraser, Scott E; Pantazis, Periklis

    2012-05-01

    Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination - converting two photons into one photon of half the incident wavelength - and can be detected by conventional two-photon microscopy. Because the optical signal of SHG nanoprobes is based on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long-term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution. PMID:22392481

  1. Photoswitchable Spiropyran Dyads for Biological Imaging.

    Science.gov (United States)

    Xiong, Yaoyao; Rivera-Fuentes, Pablo; Sezgin, Erdinc; Vargas Jentzsch, Andreas; Eggeling, Christian; Anderson, Harry L

    2016-08-01

    The synthesis of a small-molecule dyad consisting of a far-red-emitting silicon rhodamine dye that is covalently linked to a photochromic spironaphthothiopyran unit, which serves as a photoswitchable quencher, is reported. This system can be switched reversibly between the fluorescent and nonfluorescent states using visible light at wavelengths of 405 and 630 nm, respectively, and it works effectively in aqueous solution. Live-cell imaging demonstrates that this dyad has several desirable features, including excellent membrane permeability, fast and reversible modulation of fluorescence by visible light, and good contrast between the bright and dark states. PMID:27456166

  2. Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing

    International Nuclear Information System (INIS)

    Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB

  3. A novel fluorescent label based on biological fluores-cent nanoparticles and its application in cell recognition

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Uniform-sized fluorescent nanoparticles have been prepared by employing silica as the shell and a highly luminescent dye complex of ruthenium ion and bipyridyl, tris(2,2′-bipyridyl) dichlororuthenium(Ⅱ) hexahydrate as the core of the nanoparticles. A novel fluorescent label method is proposed, which is based on the biological fluorescent nanoparticles on the foundation of nanotechnology, biotechnology and fluorescent label technology. In comparison with the conventional fluorophores as fluorescent labels such as fluorescein isothiocyanate (FITC) label, this new label shows more superiority in photochemical stability, detection sensitivity and application scope for the biomedicine research. SmIgG+ B lymphocytes isolated from the circulating blood of human beings can be easily recognized by using this new fluorescent label.

  4. Microholographic imaging of biological samples

    International Nuclear Information System (INIS)

    A camera system suitable for x-ray microholography has been constructed. Visible light Fourier transform microholograms of biological samples and other test targets have been recorded and reconstructed digitally using a glycerol microdrop as a reference wave source. Current results give a resolution of ∼4 - 10 λ with λ = 514.5 nm. 11 refs., 1 fig

  5. Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy.

    Directory of Open Access Journals (Sweden)

    Víctor M Campa

    Full Text Available The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.

  6. Hyperspectral fluorescence imaging with multi wavelength LED excitation

    Science.gov (United States)

    Luthman, A. Siri; Dumitru, Sebastian; Quirós-Gonzalez, Isabel; Bohndiek, Sarah E.

    2016-04-01

    Hyperspectral imaging (HSI) can combine morphological and molecular information, yielding potential for real-time and high throughput multiplexed fluorescent contrast agent imaging. Multiplexed readout from targets, such as cell surface receptors overexpressed in cancer cells, could improve both sensitivity and specificity of tumor identification. There remains, however, a need for compact and cost effective implementations of the technology. We have implemented a low-cost wide-field multiplexed fluorescence imaging system, which combines LED excitation at 590, 655 and 740 nm with a compact commercial solid state HSI system operating in the range 600 - 1000 nm. A key challenge for using reflectance-based HSI is the separation of contrast agent fluorescence from the reflectance of the excitation light. Here, we illustrate how it is possible to address this challenge in software, using two offline reflectance removal methods, prior to least-squares spectral unmixing. We made a quantitative comparison of the methods using data acquired from dilutions of contrast agents prepared in well-plates. We then established the capability of our HSI system for non-invasive in vivo fluorescence imaging in small animals using the optimal reflectance removal method. The HSI presented here enables quantitative unmixing of at least four fluorescent contrast agents (Alexa Fluor 610, 647, 700 and 750) simultaneously in living mice. A successful unmixing of the four fluorescent contrast agents was possible both using the pure contrast agents and with mixtures. The system could in principle also be applied to imaging of ex vivo tissue or intraoperative imaging in a clinical setting. These data suggest a promising approach for developing clinical applications of HSI based on multiplexed fluorescence contrast agent imaging.

  7. A dual oxygenation and fluorescence imaging platform for reconstructive surgery

    Science.gov (United States)

    Ashitate, Yoshitomo; Nguyen, John N.; Venugopal, Vivek; Stockdale, Alan; Neacsu, Florin; Kettenring, Frank; Lee, Bernard T.; Frangioni, John V.; Gioux, Sylvain

    2013-03-01

    There is a pressing clinical need to provide image guidance during surgery. Currently, assessment of tissue that needs to be resected or avoided is performed subjectively, leading to a large number of failures, patient morbidity, and increased healthcare costs. Because near-infrared (NIR) optical imaging is safe, noncontact, inexpensive, and can provide relatively deep information (several mm), it offers unparalleled capabilities for providing image guidance during surgery. These capabilities are well illustrated through the clinical translation of fluorescence imaging during oncologic surgery. In this work, we introduce a novel imaging platform that combines two complementary NIR optical modalities: oxygenation imaging and fluorescence imaging. We validated this platform during facial reconstructive surgery on large animals approaching the size of humans. We demonstrate that NIR fluorescence imaging provides identification of perforator arteries, assesses arterial perfusion, and can detect thrombosis, while oxygenation imaging permits the passive monitoring of tissue vital status, as well as the detection and origin of vascular compromise simultaneously. Together, the two methods provide a comprehensive approach to identifying problems and intervening in real time during surgery before irreparable damage occurs. Taken together, this novel platform provides fully integrated and clinically friendly endogenous and exogenous NIR optical imaging for improved image-guided intervention during surgery.

  8. Quantitative sensing of microviscosity in protocells and amyloid materials using fluorescence lifetime imaging of molecular rotors

    Science.gov (United States)

    Thompson, Alex J.; Tang, T.-Y. Dora; Herling, Therese W.; Che Hak, C. Rohaida; Mann, Stephen; Knowles, Tuomas P. J.; Kuimova, Marina K.

    2014-03-01

    Molecular rotors are fluorophores that have a fluorescence quantum yield that depends upon intermolecular rotation. The fluorescence quantum yield, intensity and lifetime of molecular rotors all vary as functions of viscosity, as high viscosities inhibit intermolecular rotation and cause an increase in the non-radiative decay rate. As such, molecular rotors can be used to probe viscosity on microscopic scales. Here, we apply fluorescence lifetime imaging microscopy (FLIM) to measure the fluorescence lifetimes of three different molecular rotors, in order to determine the microscopic viscosity in two model systems with significant biological interest. First, the constituents of a novel protocell - a model of a prebiotic cell - were studied using the molecular rotors BODIPY C10 and kiton red. Second, amyloid formation was investigated using the molecular rotor Cy3.

  9. Small BODIPY Probes for Combined Dual (19) F MRI and Fluorescence Imaging.

    Science.gov (United States)

    Huynh, Anh Minh; Müller, Andreas; Kessler, Sonja M; Henrikus, Sarah; Hoffmann, Caroline; Kiemer, Alexandra K; Bücker, Arno; Jung, Gregor

    2016-07-19

    The combination of the two complementary imaging modalities (19) F magnetic resonance imaging (MRI) and fluorescence imaging (FLI) possesses high potential for biological and medical applications. Herein we report the first design, synthesis, dual detection validation, and cytotoxic testing of four promising BODIPY dyes for dual (19) F MRI-fluorescence detection. Using straightforward Steglich reactions, small fluorinated alcohols were easily covalently tethered to a BODIPY dye in high yields, leaving its fluorescence properties unaffected. The synthesized compounds were analyzed with various techniques to demonstrate their potential utility in dual imaging. As expected, the chemically and magnetically equivalent trifluoromethyl groups of the agents exhibited a single NMR signal. The determined longitudinal relaxation times T1 and the transverse relaxation times T2 , both in the lower second range, enabled the imaging of four compounds in vitro. The most auspicious dual (19) F MRI-fluorescence agent was also successfully imaged in a mouse post-mortem within a 9.4 T small-animal tomograph. Toxicological assays with human cells (primary HUVEC and HepG2 cell line) also indicated the possibility for animal testing. PMID:27347843

  10. Smartphone microendoscopy for high resolution fluorescence imaging

    OpenAIRE

    Hong, Xiangqian; Nagarajan, Vivek K.; Mugler, Dale H.; Yu, Bing

    2016-01-01

    High resolution optical endoscopes are increasingly used in diagnosis of various medical conditions of internal organs, such as the gastrointestinal tracts, but they are too expensive for use in resource-poor settings. On the other hand, smartphones with high resolution cameras and Internet access have become more affordable, enabling them to diffuse into most rural areas and developing countries in the past decade. In this letter we describe a smartphone microendoscope that can take fluoresc...

  11. Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging

    NARCIS (Netherlands)

    Zhao, Q.; Schelen, B.; Schouten, R., et al.

    2012-01-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device des

  12. Enhanced speed in fluorescence imaging using beat frequency multiplexing

    Science.gov (United States)

    Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke

    2016-03-01

    Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

  13. Single camera imaging system for color and near-infrared fluorescence image guided surgery

    OpenAIRE

    Chen, Zhenyue; Zhu, Nan; Pacheco, Shaun; Wang, Xia; Liang, Rongguang

    2014-01-01

    Near-infrared (NIR) fluorescence imaging systems have been developed for image guided surgery in recent years. However, current systems are typically bulky and work only when surgical light in the operating room (OR) is off. We propose a single camera imaging system that is capable of capturing NIR fluorescence and color images under normal surgical lighting illumination. Using a new RGB-NIR sensor and synchronized NIR excitation illumination, we have demonstrated that the system can acquire ...

  14. A portable fluorescence microscopic imaging system for cholecystectomy

    Science.gov (United States)

    Ye, Jian; Yang, Chaoyu; Gan, Qi; Ma, Rong; Zhang, Zeshu; Chang, Shufang; Shao, Pengfei; Zhang, Shiwu; Liu, Chenhai; Xu, Ronald

    2016-03-01

    In this paper we proposed a portable fluorescence microscopic imaging system to prevent iatrogenic biliary injuries from occurring during cholecystectomy due to misidentification of the cystic structures. The system consisted of a light source module, a CMOS camera, a Raspberry Pi computer and a 5 inch HDMI LCD. Specifically, the light source module was composed of 690 nm and 850 nm LEDs, allowing the CMOS camera to simultaneously acquire both fluorescence and background images. The system was controlled by Raspberry Pi using Python programming with the OpenCV library under Linux. We chose Indocyanine green(ICG) as a fluorescent contrast agent and then tested fluorescence intensities of the ICG aqueous solution at different concentration levels by our fluorescence microscopic system compared with the commercial Xenogen IVIS system. The spatial resolution of the proposed fluorescence microscopic imaging system was measured by a 1951 USAF resolution target and the dynamic response was evaluated quantitatively with an automatic displacement platform. Finally, we verified the technical feasibility of the proposed system in mouse models of bile duct, performing both correct and incorrect gallbladder resection. Our experiments showed that the proposed system can provide clear visualization of the confluence between the cystic duct and common bile duct or common hepatic duct, suggesting that this is a potential method for guiding cholecystectomy. The proposed portable system only cost a total of $300, potentially promoting its use in resource-limited settings.

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

    Directory of Open Access Journals (Sweden)

    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

  16. A Ratiometric Fluorescence Imaging System for Surgical Guidance

    Directory of Open Access Journals (Sweden)

    Brian C. Wilson

    2008-10-01

    Full Text Available A 3-chip CCD imaging system has been developed for quantitative in vivo fluorescence imaging. This incorporates a ratiometric algorithm to correct for the effects of tissue optical absorption and scattering, imaging “geometry” and tissue autofluorescence background. The performance was characterized, and the algorithm was validated in tissue-simulating optical phantoms for quantitative measurement of the fluorescent molecule protoporphyrin IX (PpIX. The technical feasibility to use this system for fluorescence-guided surgical resection of malignant brain tumor tissue was assessed in an animal model in which PpIX was induced exogenously in the tumor cells by systemic administration of aminolevulinic acid (ALA.

  17. Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish

    Science.gov (United States)

    Choi, Seon-Ae; Park, Chul Soon; Kwon, Oh Seok; Giong, Hoi-Khoanh; Lee, Jeong-Soo; Ha, Tai Hwan; Lee, Chang-Soo

    2016-05-01

    Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic ‘naphthalene’ moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems.

  18. Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish.

    Science.gov (United States)

    Choi, Seon-Ae; Park, Chul Soon; Kwon, Oh Seok; Giong, Hoi-Khoanh; Lee, Jeong-Soo; Ha, Tai Hwan; Lee, Chang-Soo

    2016-01-01

    Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic 'naphthalene' moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems. PMID:27188400

  19. Use of fluorescence and scanning electron microscopy as tools in teaching biology

    Science.gov (United States)

    Ghosh, Nabarun; Silva, Jessica; Vazquez, Aracely; Das, A. B.; Smith, Don W.

    2011-06-01

    Recent nationwide surveys reveal significant decline in students' interest in Math and Sciences. The objective of this project was to inspire young minds in using various techniques involved in Sciences including Scanning Electron Microscopy. We used Scanning Electron Microscope in demonstrating various types of Biological samples. An SEM Tabletop model in the past decade has revolutionized the use of Scanning Electron Microscopes. Using SEM Tabletop model TM 1000 we studied biological specimens of fungal spores, pollen grains, diatoms, plant fibers, dust mites, insect parts and leaf surfaces. We also used fluorescence microscopy to view, to record and analyze various specimens with an Olympus BX40 microscope equipped with FITC and TRITC fluorescent filters, a mercury lamp source, DP-70 digital camera with Image Pro 6.0 software. Micrographs were captured using bright field microscopy, the fluoresceinisothiocyanate (FITC) filter, and the tetramethylrhodamine (TRITC) filter settings at 40X. A high pressure mercury lamp or UV source was used to excite the storage molecules or proteins which exhibited autofluorescence. We used fluorescent microscopy to confirm the localization of sugar beet viruses in plant organs by viewing the vascular bundles in the thin sections of the leaves and other tissues. We worked with the REU summer students on sample preparation and observation on various samples utilizing the SEM. Critical Point Drying (CPD) and metal coating with the sputter coater was followed before observing some cultured specimen and the samples that were soft in textures with high water content. SEM Top allowed investigating the detailed morphological features that can be used for classroom teaching. Undergraduate and graduate researchers studied biological samples of Arthropods, pollen grains and teeth collected from four species of snakes using SEM. This project inspired the research students to pursue their career in higher studies in science and 45% of the

  20. Tumor-stem cells interactions by fluorescence imaging

    Science.gov (United States)

    Meleshina, Aleksandra V.; Cherkasova, Elena I.; Sergeeva, Ekaterina; Turchin, Ilya V.; Kiseleva, Ekaterina V.; Dashinimaev, Erdem B.; Shirmanova, Marina V.; Zagaynova, Elena V.

    2013-02-01

    Recently, great deal of interest is investigation the function of the stem cells (SC) in tumors. In this study, we studied «recipient-tumor- fluorescent stem cells » system using the methods of in vivo imaging and laser scanning microscopy (LSM). We used adipose-derived adult stem (ADAS) cells of human lentiviral transfected with the gene of fluorescent protein Turbo FP635. ADAS cells were administrated into nude mice with transplanted tumor HeLa Kyoto (human cervical carcinoma) at different stages of tumor growth (0-8 days) intravenously or into tumor. In vivo imaging was performed on the experimental setup for epi - luminescence bioimaging (IAP RAS, Nizhny Novgorod). The results of the imaging showed localization of fluorophore tagged stem cells in the spleen on day 5-9 after injection. The sensitivity of the technique may be improved by spectral separation autofluorescence and fluorescence of stem cells. We compared the results of in vivo imaging and confocal laser scanning microscopy (LSM 510 META, Carl Zeiss, Germany). Internal organs of the animals and tumor tissue were investigated. It was shown that with i.v. injection of ADAS, bright fluorescent structures with spectral characteristics corresponding to TurboFP635 protein are locally accumulated in the marrow, lungs and tumors of animals. These findings indicate that ADAS cells integrate in the animal body with transplanted tumor and can be identified by fluorescence bioimaging techniques in vivo and ex vivo.

  1. Snapshot imaging Fraunhofer line discriminator for detection of plant fluorescence

    Science.gov (United States)

    Gupta Roy, S.; Kudenov, M. W.

    2015-05-01

    Non-invasive quantification of plant health is traditionally accomplished using reflectance based metrics, such as the normalized difference vegetative index (NDVI). However, measuring plant fluorescence (both active and passive) to determine photochemistry of plants has gained importance. Due to better cost efficiency, lower power requirements, and simpler scanning synchronization, detecting passive fluorescence is preferred over active fluorescence. In this paper, we propose a high speed imaging approach for measuring passive plant fluorescence, within the hydrogen alpha Fraunhofer line at ~656 nm, using a Snapshot Imaging Fraunhofer Line Discriminator (SIFOLD). For the first time, the advantage of snapshot imaging for high throughput Fraunhofer Line Discrimination (FLD) is cultivated by our system, which is based on a multiple-image Fourier transform spectrometer and a spatial heterodyne interferometer (SHI). The SHI is a Sagnac interferometer, which is dispersion compensated using blazed diffraction gratings. We present data and techniques for calibrating the SIFOLD to any particular wavelength. This technique can be applied to quantify plant fluorescence at low cost and reduced complexity of data collection.

  2. Development of novel fluorescent probe 3-perylene diphenylphosphine for determination of lipid hydroperoxide with fluorescent image analysis

    International Nuclear Information System (INIS)

    A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, 1H NMR, and 13C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies. Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials

  3. Coded Aperture Imaging for Fluorescent X-rays-Biomedical Applications

    Energy Technology Data Exchange (ETDEWEB)

    Haboub, Abdel; MacDowell, Alastair; Marchesini, Stefano; Parkinson, Dilworth

    2013-06-01

    Employing a coded aperture pattern in front of a charge couple device pixilated detector (CCD) allows for imaging of fluorescent x-rays (6-25KeV) being emitted from samples irradiated with x-rays. Coded apertures encode the angular direction of x-rays and allow for a large Numerical Aperture x- ray imaging system. The algorithm to develop the self-supported coded aperture pattern of the Non Two Holes Touching (NTHT) pattern was developed. The algorithms to reconstruct the x-ray image from the encoded pattern recorded were developed by means of modeling and confirmed by experiments. Samples were irradiated by monochromatic synchrotron x-ray radiation, and fluorescent x-rays from several different test metal samples were imaged through the newly developed coded aperture imaging system. By choice of the exciting energy the different metals were speciated.

  4. A fast responsive two-photon fluorescent probe for imaging H₂O₂ in lysosomes with a large turn-on fluorescence signal.

    Science.gov (United States)

    Ren, Mingguang; Deng, Beibei; Wang, Jian-Yong; Kong, Xiuqi; Liu, Zhan-Rong; Zhou, Kai; He, Longwei; Lin, Weiying

    2016-05-15

    Hydrogen peroxide (H2O2) plays a crucial role in many biological processes in the human body. As our understanding of the complexity of physiological H2O2 in lysosome, investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed a new example of a fast responsive and lysosome-targeted two-photon H2O2 fluorescent probe (Lyso-HP) with a large turn-on fluorescence signal (80-fold fluorescence enhancement). The addition of H2O2 to Lyso-HP results a dramatic fluorescence enhancement around 550 nm. The probe could image exogenous and endogenous H2O2 in living cells and the probe was located in lysosomes with high colocalization coefficient (0.96) compared with LysoTracker. The large fluorescence enhancement of the two-photon probe Lyso-HP renders it attractive for imaging H2O2 in living tissues with deep tissue penetration. Significantly, the probe is feasible for fluorescently monitoring H2O2 level changes in lysosomes and suitable for fluorescence imaging of H2O2 in living tissues with deep penetration by using two-photon microscopy. PMID:26710341

  5. Implantable imaging device for brain functional imaging system using flavoprotein fluorescence

    Science.gov (United States)

    Sunaga, Yoshinori; Yamaura, Hiroshi; Haruta, Makito; Yamaguchi, Takahiro; Motoyama, Mayumi; Ohta, Yasumi; Takehara, Hiroaki; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Yoshimura, Yumiko; Ohta, Jun

    2016-03-01

    The autofluorescence of mitochondrial flavoprotein is very useful for functional brain imaging because the fluorescence intensity of flavoprotein changes as per neural activities. In this study, we developed an implantable imaging device for green fluorescence imaging and detected fluorescence changes of flavoprotein associated with visual stimulation using the device. We examined the device performance using anesthetized mice. We set the device on the visual cortex and measured fluorescence changes of flavoprotein in response to visual stimulation. A full-field sinusoidal grating with a vertical orientation was used for applying to activate the visual cortex. We successfully observed visually evoked fluorescence changes in the mouse visual cortex using our implantable device. This result suggests that we can observe the fluorescence changes of flavoprotein associated with visual stimulation in a freely moving mouse by using this technology.

  6. Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes.

    Science.gov (United States)

    Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

    2016-01-01

    Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application. PMID:26931282

  7. Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

    Energy Technology Data Exchange (ETDEWEB)

    Gartia, Manas Ranjan [Department of Nuclear, Plasma and Radiological Engineering, University of Illinois, Urbana, IL 61801 (United States); Hsiao, Austin; Logan Liu, G [Department of Bioengineering, University of Illinois, Urbana, IL 61801 (United States); Sivaguru, Mayandi [Institute for Genomic Biology, University of Illinois, Urbana, IL 61801 (United States); Chen Yi, E-mail: loganliu@illinois.edu [Department of Electrical and Computer Engineering, University of Illinois, Urbana, IL 61801 (United States)

    2011-09-07

    We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.

  8. Functionalized silica nanoparticles: a platform for fluorescence imaging at the cell and small animal levels.

    Science.gov (United States)

    Wang, Kemin; He, Xiaoxiao; Yang, XiaoHai; Shi, Hui

    2013-07-16

    Going in vivo, including living cells and the whole body, is very important for gaining a better understanding of the mystery of life and requires specialized imaging techniques. The diversity, composition, and temporal-spatial variation of life activities from cells to the whole body require the analysis techniques to be fast-response, noninvasive, highly sensitive, and stable, in situ and in real-time. Functionalized nanoparticle-based fluorescence imaging techniques have the potential to meet such needs through real-time and noninvasive visualization of biological events in vivo. Functionalized silica nanoparticles (SiNPs) doped with fluorescent dyes appear to be an ideal and flexible platform for developing fluorescence imaging techniques used in living cells and the whole body. We can select and incorporate different dyes inside the silica matrix either noncovalently or covalently. These form the functionalized hybrid SiNPs, which support multiplex labeling and ratiometric sensing in living systems. Since the silica matrix protects dyes from outside quenching and degrading factors, this enhances the photostability and biocompatibility of the SiNP-based probes. This makes them ideal for real-time and long-time tracking. One nanoparticle can encapsulate large numbers of dye molecules, which amplifies their optical signal and temporal-spatial resolution response. Integrating fluorescent dye-doped SiNPs with targeting ligands using various surface modification techniques can greatly improve selective recognition. Along with the endocytosis, functionalized SiNPs can be efficiently internalized into cells for noninvasive localization, assessment, and monitoring. These unique characteristics of functionalized SiNPs substantially support their applications in fluorescence imaging in vivo. In this Account, we summarize our efforts to develop functionalized dye-doped SiNPs for fluorescence imaging at the cell and small animal levels. We first discuss how to design and

  9. FluoSTIC: miniaturized fluorescence image-guided surgery system

    Science.gov (United States)

    Gioux, Sylvain; Coutard, Jean-Guillaume; Berger, Michel; Grateau, Henri; Josserand, Véronique; Keramidas, Michelle; Righini, Christian; Coll, Jean-Luc; Dinten, Jean-Marc

    2012-10-01

    Over the last few years, near-infrared (NIR) fluorescence imaging has witnessed rapid growth and is already used in clinical trials for various procedures. However, most clinically compatible imaging systems are optimized for large, open-surgery procedures. Such systems cannot be employed during head and neck oncologic surgeries because the system is not able to image inside deep cavities or allow the surgeon access to certain tumors due to the large footprint of the system. We describe a miniaturized, low-cost, NIR fluorescence system optimized for clinical use during oral oncologic surgeries. The system, termed FluoSTIC, employs a miniature, high-quality, consumer-grade lipstick camera for collecting fluorescence light and a novel custom circular optical fiber array for illumination that combines both white light and NIR excitation. FluoSTIC maintains fluorescence imaging quality similar to that of current large-size imaging systems and is 22 mm in diameter and 200 mm in height and weighs less than 200 g.

  10. Imaging cellular and molecular biological functions

    Energy Technology Data Exchange (ETDEWEB)

    Shorte, S.L. [Institut Pasteur, 75 - Paris (France). Plateforme d' Imagerie Dynamique PFID-Imagopole; Frischknecht, F. (eds.) [Heidelberg Univ. Medical School (Germany). Dept. of Parasitology

    2007-07-01

    'Imaging cellular and molecular biological function' provides a unique selection of essays by leading experts, aiming at scientist and student alike who are interested in all aspects of modern imaging, from its application and up-scaling to its development. Indeed the philosophy of this volume is to provide student, researcher, PI, professional or provost the means to enter this applications field with confidence, and to construct the means to answer their own specific questions. (orig.)

  11. microlith : Image Simulation for Biological Phase Microscopy

    CERN Document Server

    Mehta, Shalin B

    2013-01-01

    Accurate simulation of image formation remains under-exploited for biological phase microscopy methods that employ partially coherent illumination, despite being important for the design of imaging systems and the reconstruction algorithms. We present an open-source MATLAB toolbox, microlith (https://code.google.com/p/microlith), that provides accurate simulation of the 3D image of a thin specimen under any partially coherent imaging system, including coherent or incoherent systems. We demonstrate the accuracy of the microlith toolbox by comparing simulated images and experimental images of a phase-only Siemens star test target using dark field and differential interference contrast microscopes. The comparison leads to intriguing insights about the sensitivity of the dark-field microscope to sub-resolution features and effects of specimen birefringence on differential interference contrast.

  12. 2D/3D cryo x-ray fluorescence imaging at the bionanoprobe at the advanced photon source

    Energy Technology Data Exchange (ETDEWEB)

    Chen, S., E-mail: sichen@aps.anl.gov; Vine, D. J.; Lai, B. [Advanced Photon Source, Argonne National Laboratory, Lemont, IL 60439 (United States); Paunesku, T.; Yuan, Y.; Woloschak, G. E. [Department of Radiation Oncology, Northwester University, Chicago, IL 60611 (United States); Deng, J. [Applied Physics, Northwestern University, Evanston, IL 60208 (United States); Jin, Q.; Hong, Y. P. [Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208 (United States); Flachenecker, C.; Hornberger, B. [Carl Zeiss X-ray Microscopy, Pleasanton, CA 94588 (United States); Brister, K. [Synchrotron Research Center, Northwestern University, Argonne, IL 60439 (United States); Jacobsen, C. [Advanced Photon Source, Argonne National Laboratory, Lemont, IL 60439 (United States); Applied Physics, Northwestern University, Evanston, IL 60208 (United States); Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208 (United States); Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208 (United States); Vogt, S. [Advanced Photon Source, Argonne National Laboratory, Lemont, IL 60439 (United States); Department of Radiation Oncology, Northwester University, Chicago, IL 60611 (United States)

    2016-01-28

    Trace elements, particularly metals, play very important roles in biological systems. Synchrotron-based hard X-ray fluorescence microscopy offers the most suitable capabilities to quantitatively study trace metals in thick biological samples, such as whole cells and tissues. In this manuscript, we have demonstrated X-ray fluorescence imaging of frozen-hydrated whole cells using the recent developed Bionanoprobe (BNP). The BNP provides spatial resolution down to 30 nm and cryogenic capabilities. Frozen-hydrated biological cells have been directly examined on a sub-cellular level at liquid nitrogen temperatures with minimal sample preparation.

  13. Chlorophyll fluorescence analysis and imaging in plant stress and disease

    Energy Technology Data Exchange (ETDEWEB)

    Daley, P.F.

    1994-12-01

    Quantitative analysis of chlorophyll fluorescence transients and quenching has evolved rapidly in the last decade. Instrumentation capable of fluorescence detection in bright actinic light has been used in conjunction with gas exchange analysis to build an empirical foundation relating quenching parameters to photosynthetic electron transport, the state of the photoapparatus, and carbon fixation. We have developed several instruments that collect video images of chlorophyll fluorescence. Digitized versions of these images can be manipulated as numerical data arrays, supporting generation of quenching maps that represent the spatial distribution of photosynthetic activity in leaves. We have applied this technology to analysis of fluorescence quenching during application of stress hormones, herbicides, physical stresses including drought and sudden changes in humidity of the atmosphere surrounding leaves, and during stomatal oscillations in high CO{sub 2}. We describe a recently completed portable fluorescence imaging system utilizing LED illumination and a consumer-grade camcorder, that will be used in long-term, non-destructive field studies of plant virus infections.

  14. The Cyan Fluorescent Protein (CFP Transgenic Mouse as a Model for Imaging Pancreatic Exocrine Cells

    Directory of Open Access Journals (Sweden)

    Hop S Tran Cao

    2009-03-01

    Full Text Available The use of fluorescent proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various fluorescent proteins. Objective To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. Design Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan. Fluorescence imaging was performed under the cyan fluorescent protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA. Intervention All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and H&E staining. Fluorescence microscopy was performed with an Olympus IMT-2 inverted fluorescence microscope. Main outcome measure Determination of fluorescence intensity of different organs. Results Surprisingly, we found that there is differential enhancement of fluorescence among organs; most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the cyan fluorescent protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. Conclusions The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer.

  15. In vivo dual-modality imaging of lymphatic systems using indocyanine green in rats: three-dimensional photoacoustic imaging and planar fluorescence imaging

    Science.gov (United States)

    Kim, Chulhong; Song, Kwang Hyun; Wang, Lihong V.

    2010-02-01

    The purpose of this study is to map non-invasively sentinel lymph nodes (SLNs) and lymphatic vessels of rats in vivo using FDA-approved indocyanine green (ICG) and two non-ionizing imaging modalities: volumetric spectroscopic photoacoustic (PA) imaging, which measures optical absorption, and planar fluorescence imaging, which measures fluorescent emission. SLNs and lymphatic vessels were clearly visible after a 0.2 ml-intradermal-injection of 1 mM ICG in both imaging systems. We also imaged deeply positioned lymph nodes in vivo by layering biological tissues on top of rats. These two modalities, when used together with ICG, have the potential to map SLNs in axillary staging and to study tumor metastasis in breast cancer patients.

  16. A novel colorimetric and off–on fluorescent sensor for Hg2+ and its application in live cell imaging

    International Nuclear Information System (INIS)

    A novel rhodamine-based fluorescent probe L2 with two “S” groups was synthesized and characterized. As expected, L2 exhibited high selectivity and sensitivity for Hg2+ over other commonly coexistent metal ions. Its selectivity is excellent, and the detection limit was measured to be 1 ppm. The significant changes in the fluorescence color could be used for naked-eye detection. Furthermore, fluorescence imaging experiments of Hg2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems. -- Highlights: • A new rhodamine-based chemodosimeter (L2) was synthesized and characterized. • L2 exhibited high selectivity and excellent sensitivity detection of Hg2+. • The significant changes in color could be used for naked-eye detection. • The fluorescence imaging experiments of Hg2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems

  17. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Emilio J Gualda

    2014-08-01

    Full Text Available The development of three dimensional cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex three dimensional matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy is becoming an excellent tool for fast imaging of such three-dimensional biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

  18. Fluorescence lifetime imaging of human skin and hair

    Science.gov (United States)

    Ehlers, A.; Riemann, I.; Anhut, T.; Kaatz, M.; Elsner, P.; König, K.

    2006-02-01

    Multiphoton imaging has developed into an important technique for in-vivo research in life sciences. With the laser System DermaInspect (JenLab, Germany) laser radiation from a Ti:Sapphire laser is used to generate multiphotonabsorption deep in the human skin in vivo. The resulting autofluorescence radiation arises from endogenous fluorophores such as NAD(P)H, flavines, collagen, elastin, porphyrins und melanin. Second harmonic generation (SHG) was used to detect collagen structures in the dermal layer. Femtosecond laser multiphoton imaging offers the possibility of high resolution optical tomography of human skin as well as fluorescence lifetime imaging (FLIM) with picosecond time resolution. In this work a photon detector with ultrashort rise time of less than 30ps was applied to FLIM measurements of human skin and hair with different pigmentation. Fluorescence lifetime images of different human hair types will be discussed.

  19. An individually coated near-infrared fluorescent protein as a safe and robust nanoprobe for in vivo imaging

    Science.gov (United States)

    Yang, Yu; Xiang, Kun; Yang, Yi-Xin; Wang, Yan-Wen; Zhang, Xin; Cui, Yangdong; Wang, Haifang; Zhu, Qing-Qing; Fan, Liqiang; Liu, Yuanfang; Cao, Aoneng

    2013-10-01

    A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging.A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging. Electronic supplementary information (ESI

  20. Fluorescent Tobacco mosaic virus-Derived Bio-Nanoparticles for Intravital Two-Photon Imaging

    Science.gov (United States)

    Niehl, Annette; Appaix, Florence; Boscá, Sonia; van der Sanden, Boudewijn; Nicoud, Jean-François; Bolze, Frédéric; Heinlein, Manfred

    2016-01-01

    Multi-photon intravital imaging has become a powerful tool to investigate the healthy and diseased brain vasculature in living animals. Although agents for multi-photon fluorescence microscopy of the microvasculature are available, issues related to stability, bioavailability, toxicity, cost or chemical adaptability remain to be solved. In particular, there is a need for highly fluorescent dyes linked to particles that do not cross the blood brain barrier (BBB) in brain diseases like tumor or stroke to estimate the functional blood supply. Plant virus particles possess a number of distinct advantages over other particles, the most important being the multi-valency of chemically addressable sites on the particle surface. This multi-valency, together with biological compatibility and inert nature, makes plant viruses ideal carriers for in vivo imaging agents. Here, we show that the well-known Tobacco mosaic virus is a suitable nanocarrier for two-photon dyes and for intravital imaging of the mouse brain vasculature. PMID:26793221

  1. Nonlinear spectral imaging of biological tissues

    NARCIS (Netherlands)

    Palero, J.A.

    2007-01-01

    The work presented in this thesis demonstrates live high resolution 3D imaging of tissue in its native state and environment. The nonlinear interaction between focussed femtosecond light pulses and the biological tissue results in the emission of natural autofluorescence and second-harmonic signal.

  2. Lymphatic Imaging in Humans with Near-Infrared Fluorescence

    OpenAIRE

    Rasmussen, John C.; Tan, I-Chih; Marshall, Milton V.; Fife, Caroline E.; Sevick-Muraca, Eva M

    2009-01-01

    While the lymphatic system is increasingly associated with diseases of prevalence, study of these diseases is difficult owing to the paucity of imaging techniques with the sensitivity and temporal resolution to discriminate lymphatic function. Herein, we review the known, pertinent features of the human lymphatic system in health and disease and set the context for a number of emerging studies that use near-infrared fluorescence imaging to non-invasively assess tumor draining lymphatic basins...

  3. Plant response to destruxins visualized by imaging of chlorophyll fluorescence

    Czech Academy of Sciences Publication Activity Database

    Soukupová, Julie; Smatanová, Sylvie; Nedbal, Ladislav; Jegorov, A.

    2003-01-01

    Roč. 118, č. 118 (2003), s. 399-405. ISSN 0031-9317 Institutional research plan: CEZ:MSM 123100001; CEZ:AV0Z6087904 Keywords : fungal infection, destruxins * fluorescence imaging Subject RIV: GF - Plant Pathology, Vermin, Weed, Plant Protection Impact factor: 1.767, year: 2003

  4. Preparation of Fluorescent Graphene Quantum Dots as Biological Imaging Marker for Cells%荧光石墨烯量子点制备及其在细胞成像中的应用

    Institute of Scientific and Technical Information of China (English)

    谢文菁; 傅英懿; 马红; 张沫; 范楼珍

    2012-01-01

    Currently, graphene has attracted much attention in the fields of bioimaging, biolabeling and drug delivery. Theoretical and experimental studies have shown that the graphene quantum dots (GQDs) are expected to show good optical properties due to their quantum confinement and edge effect. In this report, using the electrochemical assay the fluorescent GQDs with a diameter between 5 and 10 nm could be obtained via electrolysing graphite in alkaline condition and with hydrazine hydrate as a reducing agent at room temperature. The structure of the GQDs was confimed by means of transmission electron microscope (TEM) and atomic force microscope (AFM). The finding showed that the GQDs have an uniform size, and most of them are separate graphene. The GQDs mainly consist of single layer with less than 1 nm. Their features and properties were characterized by fourier transform infrared spectroscopy (FTIR), photoluminescence spectra (PL), UV-visible spectroscopy (UV-vis) and X-ray diffraction (XRD). The results indicated that the GQDs have bright yellow luminescence with a 14 % quantum yield, which is higher than that of traditional carbon quantum dots reported previously. When they were excited by different excitation wavelengths, the intensity of photoluminescence increased to the maximum, and then decreased gradually. The fluorescent emission peak of the GQDs remained unshifted, suggesting a novel kind of quantum dots different from those of graphene oxide quantum dots depending excitation wavelengths. The luminescence of GQDs arises from the graphene modified with the phthalhydrazide-like groups and hydrazide groups at the edge. The highly fluorescent GQDs have high water solubility, good photostability and biocompatibility, indicating that the GQDs can easily enter the cells. By incorporating the GQDs with A549 (lung cancer) and MCF-7 (breast cancer) cells through MTT assay, the newly obtained GQDs exhibited low cytotoxicity with an advantage of strong photoluminescence

  5. Visible to Near-Infrared Fluorescence Enhanced Cellular Imaging on Plasmonic Gold Chips.

    Science.gov (United States)

    Koh, Byumseok; Li, Xiaoyang; Zhang, Bo; Yuan, Bing; Lin, Yi; Antaris, Alexander L; Wan, Hao; Gong, Ming; Yang, Jiang; Zhang, Xiaodong; Liang, Yongye; Dai, Hongjie

    2016-01-27

    Rapid and sensitive detections of a variety of surface and intracellular proteins, nucleic acids, and other cellular biomarkers are important to elucidating biological signaling pathways and to devising disease diagnostics and therapeutics. Here, sensitive imaging and detection of cellular proteins on fluorescence-enhancing, nanostructured plasmonic gold (pGold) chips is presented. Imaging of fluorescently labeled cellular biomarkers on pGold is enhanced by 2-30-fold in the visible to near infrared (NIR) range of ≈500-900 nm. The high fluorescence enhancement of >700 nm significantly improves the dynamic range and signal/background ratios of NIR imaging, allowing high-performance multicolor imaging in the visible-NIR range using high quantum yield (QY) visible dyes and lower QY NIR fluorophores. Further, multiple cellular proteins of single cells of various cell types can be detected through microarraying of cells, useful for potentially hundreds and thousands different types of cells assayed on a single chip down to small cell numbers. This work suggests a simple, high throughput, high sensitivity, and multiplexed single-cell analysis method on fluorescence enhancing plasmonic substrates in the entire visible to NIR window. PMID:26663862

  6. PARPi-FL - a Fluorescent PARP1 Inhibitor for Glioblastoma Imaging

    Directory of Open Access Journals (Sweden)

    Christopher P. Irwin

    2014-05-01

    Full Text Available New intravital optical imaging technologies have revolutionized our understanding of mammalian biology and continue to evolve rapidly. However, there are only a limited number of imaging probes available to date. In this study, we investigated in mouse models of glioblastoma whether a fluorescent small molecule inhibitor of the DNA repair enzyme PARP1, PARPi-FL, can be used as an imaging agent to detect glioblastomas in vivo. We demonstrated that PARPi-FL has appropriate biophysical properties, low toxicity at concentrations used for imaging, high stability in vivo, and accumulates selectively in glioblastomas due to high PARP1 expression. Importantly, subcutaneous and orthotopic glioblastoma xenografts were imaged with high contrast clearly defining tumor tissue from normal surrounding tissue. This research represents a step toward exploring and developing PARPi-FL as an optical intraoperative imaging agent for PARP1 in the clinic.

  7. Miniaturized side-viewing imaging probe for fluorescence lifetime imaging (FLIM): validation with fluorescence dyes, tissue structural proteins and tissue specimens

    Science.gov (United States)

    Elson, D. S.; Jo, J. A.; Marcu, L.

    2007-05-01

    We report a side viewing fibre-based endoscope that is compatible with intravascular imaging and fluorescence lifetime imaging microscopy (FLIM). The instrument has been validated through testing with fluorescent dyes and collagen and elastin powders using the Laguerre expansion deconvolution technique to calculate the fluorescence lifetimes. The instrument has also been tested on freshly excised unstained animal vascular tissues.

  8. Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

    Directory of Open Access Journals (Sweden)

    D. A. Healy

    2014-02-01

    particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAP observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.

  9. Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

    Science.gov (United States)

    Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.

    2014-08-01

    particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are relatively insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse-mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAPs observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.

  10. Fluorescence lifetime to image epidermal ionic concentrations

    Science.gov (United States)

    Behne, Martin J.; Barry, Nicholas P.; Moll, Ingrid; Gratton, Enrico; Mauro, Theodora M.

    2004-09-01

    Measurements of ionic concentrations in skin have traditionally been performed with an array of methods which either did not reveal detailed localization information, or only provided qualitative, not quantitative information. FLIM combines a number of advantages into a method ideally suited to visualize concentrations of ions such as H+ in intact, unperturbed epidermis and stratum corneum (SC). Fluorescence lifetime is dye concentration-independent, the method requires only low light intensities and is therefore not prone to photobleaching or phototoxic artifacts, and because multiphoton lasers of IR wavelength are used, light penetrates deep into intact tissue. The standard method to measure SC pH is the flat pH electrode, which provides reliable information only about surface pH changes, without further vertical or subcellular spatial resolution; i.e., specific microdomains such as the corneocyte interstices are not resolved, and the deeper SC is inaccessible without resorting to inherently disruptive stripping methods. Furthermore, the concept of a gradient of pH through the SC stems from such stripping experiments, but other confirmation for this concept is lacking. Our investigations into the SC pH distribution so far have revealed the crucial role of the Sodium/Hydrogen Antiporter NHE1 in generation of SC acidity, the colocalization of enzymatic lipid processing activity in the SC with acidic domains of the SC, and the timing and localization of emerging acidity in the SC of newborns. Together, these results have led to an improved understanding of the SC pH, its distribution, origin, and regulation. Future uses for this method include measurements of other ions important for epidermal processes, such as Ca2+, and a quantitative approach to topical drug penetration.

  11. Sulfonate-based fluorescent probes for imaging hydrogen peroxide in living cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Based on the mechanism of H2O2-mediated hydrolysis of sulfonates, two fluorescein disulfonates compounds (FS-1 and FS-2) were designed and synthesized as the highly selective and sensitive fluorescent probes for imaging H2O2 in living cells. The probes were detected with elemental analysis, IR, 1H NMR and 13C NMR. Upon reaction with H2O2, the probes exhibit strong fluorescence responses and high selectivity for H2O2 over other reactive oxygen species and some biological compounds. Furthermore, the sulfonate-based probes, as novel fluorescent reagents, are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells by using confocal microscopy.

  12. Hyperspectral Microscopy of Near-Infrared Fluorescence Enables 17-Chirality Carbon Nanotube Imaging.

    Science.gov (United States)

    Roxbury, Daniel; Jena, Prakrit V; Williams, Ryan M; Enyedi, Balázs; Niethammer, Philipp; Marcet, Stéphane; Verhaegen, Marc; Blais-Ouellette, Sébastien; Heller, Daniel A

    2015-01-01

    The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity, and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species' fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems. This approach resolved up to 17 distinct (n,m) species (chiralities) with single nanotube spatial resolution in live mammalian cells, murine tissues ex vivo, and zebrafish endothelium in vivo. We anticipate that this approach will facilitate multiplexed nanotube imaging in biomedical applications while enabling deep-tissue optical penetration, and single-molecule resolution in vivo. PMID:26387482

  13. Reviewing the relevance of fluorescence in biological systems.

    Science.gov (United States)

    Lagorio, M Gabriela; Cordon, Gabriela B; Iriel, Analia

    2015-09-26

    Fluorescence is emitted by diverse living organisms. The analysis and interpretation of these signals may give information about their physiological state, ways of communication among species and the presence of specific chemicals. In this manuscript we review the state of the art in the research on the fluorescence emitted by plant leaves, fruits, flowers, avians, butterflies, beetles, dragonflies, millipedes, cockroaches, bees, spiders, scorpions and sea organisms and discuss its relevance in nature. PMID:26103563

  14. In Vivo Fluorescence Imaging in the Second Near-Infrared Window Using Carbon Nanotubes.

    Science.gov (United States)

    Hong, Guosong; Dai, Hongjie

    2016-01-01

    In vivo fluorescence imaging in the second near-infrared window (NIR-II window, 1000-1700 nm) is a powerful imaging technique that emerged in recent years. This imaging tool allows for noninvasive, deep-tissue visualization and interrogation of anatomical features and functions with improved imaging resolution and contrast at greater tissue penetration depths than traditional fluorescence imaging. Here, we present the detailed protocol for conducting NIR-II fluorescence imaging in live animals, including the procedures for preparation of biocompatible and NIR-II fluorescent carbon nanotube solution, live animal administration and NIR-II fluorescence image acquisition. PMID:27283426

  15. Onychomycosis diagnosis using fluorescence and infrared imaging systems

    Science.gov (United States)

    da Silva, Ana Paula; Fortunato, Thereza Cury; Stringasci, Mirian D.; Kurachi, Cristina; Bagnato, Vanderlei S.; Inada, Natalia M.

    2015-06-01

    Onychomycosis is a common disease of the nail plate, constituting approximately half of all cases of nail infection. Onychomycosis diagnosis is challenging because it is hard to distinguish from other diseases of the nail lamina such as psoriasis, lichen ruber or eczematous nails. The existing methods of diagnostics so far consist of clinical and laboratory analysis, such as: Direct Mycological examination and culture, PCR and histopathology with PAS staining. However, they all share certain disadvantages in terms of sensitivity and specificity, time delay, or cost. This study aimed to evaluate the use of infrared and fluorescence imaging as new non-invasive diagnostic tools in patients with suspected onychomycosis, and compare them with established techniques. For fluorescence analysis, a Clinical Evince (MM Optics®) was used, which consists of an optical assembly with UV LED light source wavelength 400 nm +/- 10 nm and the maximum light intensity: 40 mW/cm2 +/- 20%. For infrared analysis, a Fluke® Camera FKL model Ti400 was used. Patients with onychomycosis and control group were analyzed for comparison. The fluorescence images were processed using MATLAB® routines, and infrared images were analyzed using the SmartView® 3.6 software analysis provided by the company Fluke®. The results demonstrated that both infrared and fluorescence could be complementary to diagnose different types of onychomycosis lesions. The simplicity of operation, quick response and non-invasive assessment of the nail patients in real time, are important factors to be consider for an implementation.

  16. Highly stable organic fluorescent nanorods for living- cell imaging

    Institute of Scientific and Technical Information of China (English)

    Minhuan Lan[1,3; Jinfeng Zhang[1,3; Xiaoyue Zhu[1; Pengfei Wang[2; Xianfeng Chen[1; Chun-Sing Lee[1; Wenjun Zhang[1

    2015-01-01

    Metal-free, organic-dye-based fluorescent nanorods were fabricated through a simple solvent-exchange procedure. The as-prepared nanorods exhibit low toxicity to living cells and excellent photostability. Furthermore, they are stable in solutions of various pHs and high ionic strength and in solutions with interfering metal ions. Compared with the free DPP-Br molecules in THF, these nanorods exhibit larger Stokes shift, broader absorption spectra, and greatly improved photostability. We successfully demonstrated the application of the nanorods, including their aforementioned beneficial characteristics, as a good fluorescence probe for bio-imaging.

  17. Fluorescent supramolecular micelles for imaging-guided cancer therapy

    Science.gov (United States)

    Sun, Mengmeng; Yin, Wenyan; Dong, Xinghua; Yang, Wantai; Zhao, Yuliang; Yin, Meizhen

    2016-02-01

    A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth-inhibitory studies reveal a better therapeutic effect of FSMs after CPT encapsulation when compared with the free CPT drug. The multifunctional FSM nanomedicine platform as a nanovehicle has great potential for fluorescence imaging-guided cancer therapy.A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth

  18. Nanoscale fluorescence lifetime imaging with a single diamond NV center

    CERN Document Server

    Beams, Ryan; Johnson, Timothy W; Oh, Sang-Hyun; Novotny, Lukas; Vamivakas, Nick

    2013-01-01

    Solid-state quantum emitters, such as artificially engineered quantum dots or naturally occurring defects in solids, are being investigated for applications ranging from quantum information science and optoelectronics to biomedical imaging. Recently, these same systems have also been studied from the perspective of nanoscale metrology. In this letter we study the near-field optical properties of a diamond nanocrystal hosting a single nitrogen vacancy center. We find that the nitrogen vacancy center is a sensitive probe of the surrounding electromagnetic mode structure. We exploit this sensitivity to demonstrate nanoscale fluorescence lifetime imaging microscopy (FLIM) with a single nitrogen vacancy center by imaging the local density of states of an optical antenna.

  19. Full Field Supercritical Angle Fluorescence Microscopy for live cell imaging

    CERN Document Server

    Barroca, Thomas; Delahaye, Julie; Lévêque-Fort, Sandrine; Fort, Emmanuel

    2013-01-01

    We introduce a full field fluorescence imaging technique with axial confinement of about 100 nm at the sample/substrate interface. Contrary to standard surface imaging techniques, this confinement is obtained through emission filtering. This technique is based on supercritical emission selectivity. It can be implemented on any epifluorescence microscope with a commercial high numerical aperture objective and offers a real time surface imaging capability. This technique is of particular interest for live cell membrane and adhesion studies. Using HEK cells, we show that one can observe simultaneously the surface and in-depth cell phenomena.

  20. Chlorophyll fluorescence imaging of cadmium-treated white cabbage plants

    Directory of Open Access Journals (Sweden)

    Borek M.

    2013-04-01

    Full Text Available The chlorophyll fluorescence imaging technique is a valuable tool to study the impact of heavy metal stress in plants. The aim of this paper was to investigate the influence of Cd on photosynthetic apparatus of white cabbage (Brassica oleracea subsp. capitata f. alba plants. Two cabbage cultivars ‘Ditmarska Najwcześniejsza’ (‘DN’; early and ‘Amager Polana’ (‘AP’; late were used. Cd was applied before planting seedlings (10 mg Cd kg−1 DM of soil.. Measurements were performed at the 3rd leaf after 2 weeks of planting. The level of Cd-induced stress to plants was estimated by chlorophyll (Chl content (photometrically and analyses of images and numeric values of the major fluorescence parameters of Chl (Chl fluorescence imaging system FluorCam. Cd negatively affected the chlorophyll content and Fv/Fm, Fv’/Fm’, Φ PSII and qP in leaves of early cultivar of white cabbage. However, in the case of late cv. we did not observe such distinct changes. It suggests that late cultivars. are more resistant to Cd than the early ones. Considering methodological aspect of the study, Chl fluorescence imaging can better reveal some alterations within the leaf, because numeric values of specific parameters, which are the averaged data collected from the whole leaf, cannot reflect the tissue specificity. Abbreviations: HM – heavy metal, Cd – cadmium, Chl – chlorophyll, Fv/Fm – photochemical efficiency of PSII in the dark-adapted state, F‘v’/F‘m’ – PSII maximum efficiency, Φ PSII – quantum efficiency of PSII electron transport, NPQ – nonphotochemical quenching of maximal Chl fluorescence, qP – photochemical quenching coefficient.

  1. Imaging Atherosclerotic Plaque Calcification: Translating Biology.

    Science.gov (United States)

    Bailey, Grant; Meadows, Judith; Morrison, Alan R

    2016-08-01

    Calcification of atherosclerotic lesions was long thought to be an age - related, passive process, but increasingly data has revealed that atherosclerotic calcification is a more active process, involving complex signaling pathways and bone-like genetic programs. Initially, imaging of atherosclerotic calcification was limited to gross assessment of calcium burden, which is associated with total atherosclerotic burden and risk of cardiovascular mortality and of all cause mortality. More recently, sophisticated molecular imaging studies of the various processes involved in calcification have begun to elucidate information about plaque calcium composition and consequent vulnerability to rupture, leading to hard cardiovascular events like myocardial infarction. As such, there has been renewed interest in imaging calcification to advance risk assessment accuracy in an evolving era of precision medicine. Here we summarize recent advances in our understanding of the biologic process of atherosclerotic calcification as well as some of the molecular imaging tools used to assess it. PMID:27339750

  2. Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects

    Science.gov (United States)

    Chen, Xucai; Wang, Jianjun; Versluis, Michel; de Jong, Nico; Villanueva, Flordeliza S.

    2013-06-01

    High speed imaging has application in a wide area of industry and scientific research. In medical research, high speed imaging has the potential to reveal insight into mechanisms of action of various therapeutic interventions. Examples include ultrasound assisted thrombolysis, drug delivery, and gene therapy. Visual observation of the ultrasound, microbubble, and biological cell interaction may help the understanding of the dynamic behavior of microbubbles and may eventually lead to better design of such delivery systems. We present the development of a high speed bright field and fluorescence imaging system that incorporates external mechanical waves such as ultrasound. Through collaborative design and contract manufacturing, a high speed imaging system has been successfully developed at the University of Pittsburgh Medical Center. We named the system "UPMC Cam," to refer to the integrated imaging system that includes the multi-frame camera and its unique software control, the customized modular microscope, the customized laser delivery system, its auxiliary ultrasound generator, and the combined ultrasound and optical imaging chamber for in vitro and in vivo observations. This system is capable of imaging microscopic bright field and fluorescence movies at 25 × 106 frames per second for 128 frames, with a frame size of 920 × 616 pixels. Example images of microbubble under ultrasound are shown to demonstrate the potential application of the system.

  3. Reconstruction of Elemental Distribution Images from Synchrotron Radiation X-Ray Fluorescence Spectra

    Science.gov (United States)

    Toque, Jay Arre; Ide-Ektessabi, Ari

    Synchrotron radiation X-ray fluorescence spectroscopy (SRXRF) is a powerful technique for studying trace elements in biological samples and other materials in general. Its features including capability to perform measurements in air and water, noncontact and nondestructive assay are superior to other elemental analysis techniques. In this study, a technique for reconstructing elemental distribution mapping of trace elements from spectral data was developed. The reconstruction was made possible by using the measured fluorescent signals to obtain local differences in elemental concentrations. The proposed technique features interpolation and background subtraction using matrix transformations of the spectral data to produce an enhanced distribution images. It is achieved by employing polychromatic or monochromatic color assignments proportional to the fluorescence intensities for displaying single-element or multiple-element distributions respectively. Some typical applications (i.e., macrophage and tissue surrounding an implant) were presented and the samples were imaged using the proposed method. The distribution images of the trace elements of the selected samples were used in conjunction with other analytical techniques to draw relevant observations, which cannot be achieved using conventional techniques such as metallic uptake and corresponding cellular response. The elemental distribution images produced from this study were found to have better quality compared to images produced using other analytical techniques (e.g., SIMS, PIXE, XPS, etc).

  4. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

    International Nuclear Information System (INIS)

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8–7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence

  5. Full-direct method for imaging pharmacokinetic parameters in dynamic fluorescence molecular tomography

    International Nuclear Information System (INIS)

    Images of pharmacokinetic parameters (also known as parametric images) in dynamic fluorescence molecular tomography (FMT) can provide three-dimensional metabolic information for biological studies and drug development. However, the ill-posed nature of FMT and the high temporal variation of fluorophore concentration together make it difficult to obtain accurate parametric images in small animals in vivo. In this letter, we present a method to directly reconstruct the parametric images from the boundary measurements based on hybrid FMT/X-ray computed tomography (XCT) system. This method can not only utilize structural priors obtained from the XCT system to mitigate the ill-posedness of FMT but also make full use of the temporal correlations of boundary measurements to model the high temporal variation of fluorophore concentration. The results of numerical simulation and mouse experiment demonstrate that the proposed method leads to significant improvements in the reconstruction quality of parametric images

  6. Full-direct method for imaging pharmacokinetic parameters in dynamic fluorescence molecular tomography

    Science.gov (United States)

    Zhang, Guanglei; Pu, Huangsheng; He, Wei; Liu, Fei; Luo, Jianwen; Bai, Jing

    2015-02-01

    Images of pharmacokinetic parameters (also known as parametric images) in dynamic fluorescence molecular tomography (FMT) can provide three-dimensional metabolic information for biological studies and drug development. However, the ill-posed nature of FMT and the high temporal variation of fluorophore concentration together make it difficult to obtain accurate parametric images in small animals in vivo. In this letter, we present a method to directly reconstruct the parametric images from the boundary measurements based on hybrid FMT/X-ray computed tomography (XCT) system. This method can not only utilize structural priors obtained from the XCT system to mitigate the ill-posedness of FMT but also make full use of the temporal correlations of boundary measurements to model the high temporal variation of fluorophore concentration. The results of numerical simulation and mouse experiment demonstrate that the proposed method leads to significant improvements in the reconstruction quality of parametric images.

  7. Full-direct method for imaging pharmacokinetic parameters in dynamic fluorescence molecular tomography

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Guanglei, E-mail: guangleizhang@bjtu.edu.cn [Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084 (China); Department of Biomedical Engineering, School of Computer and Information Technology, Beijing Jiaotong University, Beijing 100044 (China); Pu, Huangsheng; Liu, Fei; Bai, Jing [Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084 (China); He, Wei [China Institute of Sport Science, Beijing 100061 (China); Luo, Jianwen, E-mail: luo-jianwen@tsinghua.edu.cn [Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084 (China); Center for Biomedical Imaging Research, School of Medicine, Tsinghua University, Beijing 100084 (China)

    2015-02-23

    Images of pharmacokinetic parameters (also known as parametric images) in dynamic fluorescence molecular tomography (FMT) can provide three-dimensional metabolic information for biological studies and drug development. However, the ill-posed nature of FMT and the high temporal variation of fluorophore concentration together make it difficult to obtain accurate parametric images in small animals in vivo. In this letter, we present a method to directly reconstruct the parametric images from the boundary measurements based on hybrid FMT/X-ray computed tomography (XCT) system. This method can not only utilize structural priors obtained from the XCT system to mitigate the ill-posedness of FMT but also make full use of the temporal correlations of boundary measurements to model the high temporal variation of fluorophore concentration. The results of numerical simulation and mouse experiment demonstrate that the proposed method leads to significant improvements in the reconstruction quality of parametric images.

  8. Scintillating Optical Fiber Imagers for biology

    International Nuclear Information System (INIS)

    S.O.F.I (Scintillating Optical Fiber Imager) is a detector developed to replace the autoradiographic films used in molecular biology for the location of radiolabelled (32P) DNA molecules in blotting experiments. It analyses samples on a 25 x 25 cm2 square area still 25 times faster than autoradiographic films, with a 1.75 and 3 mm resolution for two orthogonal directions. This device performs numerised images with a dynamic upper than 100 which allows the direct quantitation of the analysed samples. First, this thesis describes the S.O.F.I. development (Scintillating Optical Fibers, coding of these fibers and specific electronic for the treatment of the Multi-Anode Photo-Multiplier signals) and experiments made in collaboration with molecular biology laboratories. In a second place, we prove the feasibility of an automatic DNA sequencer issued from S.O.F.I

  9. Combined use of hard X-ray phase contrast imaging and X-ray fluorescence microscopy for sub-cellular metal quantification.

    OpenAIRE

    Kosior, Ewelina,; Bohic, Sylvain; Suhonen, Heikki; Ortega, Richard; Devès, Guillaume; Carmona, Asuncion; Marchi, Florence; Guillet, Jean Francois; Cloetens, Peter

    2012-01-01

    Hard X-ray fluorescence microscopy and magnified phase contrast imaging are combined to obtain quantitative maps of the projected metal concentration in whole cells. The experiments were performed on freeze dried cells at the nano-imaging station ID22NI of the European Synchrotron Radiation Facility (ESRF). X-ray fluorescence analysis gives the areal mass of most major, minor and trace elements; it is validated using a biological standard of known composition. Quantitative phase contrast imag...

  10. Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins

    Science.gov (United States)

    Krumholz, Arie; Shcherbakova, Daria M.; Xia, Jun; Wang, Lihong V.; Verkhusha, Vladislav V.

    2014-02-01

    Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.

  11. Deep vascular imaging in wounds by two-photon fluorescence microscopy.

    Directory of Open Access Journals (Sweden)

    Ciceron O Yanez

    Full Text Available Deep imaging within tissue (over 300 μm at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM. The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.

  12. Formation of ternary complexes with MgATP: effects on the detection of Mg2+ in biological samples by bidentate fluorescent sensors.

    Science.gov (United States)

    Schwartz, Sarina C; Pinto-Pacheco, Brismar; Pitteloud, Jean-Philippe; Buccella, Daniela

    2014-03-17

    Fluorescent indicators based on β-keto-acid bidentate coordination motifs display superior metal selectivity profiles compared to current o-aminophenol-N,N,O-triacetic acid (APTRA) based chelators for the study of biological magnesium. These low denticity chelators, however, may allow for the formation of ternary complexes with Mg(2+) and common ligands present in the cellular milieu. In this work, absorption, fluorescence, and NMR spectroscopy were employed to study the interaction of turn-on and ratiometric fluorescent indicators based on 4-oxo-4H-quinolizine-3-carboxylic acid with Mg(2+) and ATP, the most abundant chelator of biological magnesium, thus revealing the formation of ternary complexes under conditions relevant to fluorescence imaging. The formation of ternary species elicits comparable or greater optical changes than those attributed to the formation of binary complexes alone. Dissociation of the fluorescent indicators from both ternary and binary species have apparent equilibrium constants in the low millimolar range at pH 7 and 25 °C. These results suggest that these bidentate sensors are incapable of distinguishing between free Mg(2+) and MgATP based on ratio or intensity-based steady-state fluorescence measurements, thus posing challenges in the interpretation of results from fluorescence imaging of magnesium in nucleotide-rich biological samples. PMID:24593871

  13. Combined phase and X-Ray fluorescence imaging at the sub-cellular level

    International Nuclear Information System (INIS)

    This work presents some recent developments in the field of hard X-ray imaging applied to biomedical research. As the discipline is evolving quickly, new questions appear and the list of needs becomes bigger. Some of them are dealt with in this manuscript. It has been shown that the ID22NI beamline of the ESRF can serve as a proper experimental setup to investigate diverse aspects of cellular research. Together with its high spatial resolution, high flux and high energy range the experimental setup provides bigger field of view, is less sensitive to radiation damages (while taking phase contrast images) and suits well chemical analysis with emphasis on endogenous metals (Zn, Fe, Mn) but also with a possibility for exogenous one's like these found in nanoparticles (Au, Pt, Ag) study. Two synchrotron-based imaging techniques, fluorescence and phase contrast imaging were used in this research project. They were correlated with each other on a number of biological cases, from bacteria E.coli to various cells (HEK 293, PC12, MRC5VA, red blood cells). The explorations made in the chapter 5 allowed preparation of more established and detailed analysis, described in the next chapter where both techniques, X-ray fluorescence and phase contrast imaging, were exploited in order to access absolute metal projected mass fraction in a whole cell. The final image presents for the first time true quantitative information at the sub-cellular level, not biased by the cell thickness. Thus for the first time a fluorescence map serves as a complete quantitative image of a cell without any risk of misinterpretation. Once both maps are divided by each other pixel by pixel (fluorescence map divided by the phase map) they present a complete and final result of the metal (Zn in this work) projected mass fraction in ppm of dry weight. For the purpose of this calculation the analysis was extended to calibration (non-biological) samples. Polystyrene spheres of a known diameter and known

  14. Integrating reflectance and fluorescence imaging for apple disorder classification

    Science.gov (United States)

    Ariana, Diwan P.; Guyer, Daniel E.; Shrestha, Bim P.

    2004-03-01

    Multispectral imaging in reflectance and fluorescence modes combined with neural network analysis was used to classify various types of apple disorder from three apple varieties (Honey Crisp, Red Cort, and Red Delicious). Eighteen images from a combination of filter sets and three different imaging modes (reflectance, visible light induced fluorescence, and UV induced fluorescence) were acquired for each apple sample as a base for pixel-level classification into normal or disorder tissue. Two classification schemes, a 2-class and a multiple class, were developed and tested in this study. In the 2-class scheme, pixels were categorized into normal or disorder tissue, whereas in the multiple class scheme, pixels were categorized into normal, bitter pit, black rot, decay, soft scald, and superficial scald tissues. Results indicate that single variety training under the 2-class scheme yielded highest accuracy with total accuracy of 95, 97, and 100 % for Honey Crisp, Red Cort, or Red Delicious respectively. In the multiple-class scheme, the classification accuracy of Honey Crisp apple for normal, bitter pit, black rot, decay, and soft scald tissue was 94, 93, 97, 97, and 94 % respectively. Through variable selection analysis, in the 2-class scheme, fluorescence models yielded higher total classification accuracy compared to reflection models. For Red Cort and Red Delicious, models with only FUV yield more than 95% classification accuracy, demonstrating a potential of fluorescence to detect superficial scald. Several important wavelengths, including 680, 740, 905 and 940 nm, were identified from the filter combination analysis. The results indicate the potential of this technique to accurately recognize different types of disorder on apple.

  15. Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

    Science.gov (United States)

    Yu, Dan; Dong, Zhiqiang; Gustafson, William Clay; Ruiz-González, Rubén; Signor, Luca; Marzocca, Fanny; Borel, Franck; Klassen, Matthew P; Makhijani, Kalpana; Royant, Antoine; Jan, Yuh-Nung; Weiss, William A; Guo, Su; Shu, Xiaokun

    2016-02-01

    Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice. PMID:26549191

  16. Image Processing for Automated Analysis of the Fluorescence In-Situ Hybridization (FISH) Microscopic Images

    Czech Academy of Sciences Publication Activity Database

    Schier, Jan; Kovář, Bohumil; Kočárek, E.; Kuneš, Michal

    Berlin Heidelberg: Springer-Verlag, 2011, s. 622-633. (Lecture Notes in Computer Science ). ISBN 978-3-642-24081-2. [5th International Conference, ICHIT 2011. Daejeon (KR), 22.09.2011-24.09.2011] R&D Projects: GA TA ČR TA01010931 Institutional research plan: CEZ:AV0Z10750506 Keywords : fluorescence in-situ hybridization * image processing * image segmentation Subject RIV: IN - Informatics, Computer Science http://library.utia.cas.cz/separaty/2011/ZS/shier-image processing for automated analysis of the fluorescence in-situ hybridization (fish) microscopic images.pdf

  17. Proton-induced x-ray fluorescence CT imaging

    International Nuclear Information System (INIS)

    Purpose: To demonstrate the feasibility of proton-induced x-ray fluorescence CT (pXFCT) imaging of gold in a small animal sized object by means of experiments and Monte Carlo (MC) simulations. Methods: First, proton-induced gold x-ray fluorescence (pXRF) was measured as a function of gold concentration. Vials of 2.2 cm in diameter filled with 0%–5% Au solutions were irradiated with a 220 MeV proton beam and x-ray fluorescence induced by the interaction of protons, and Au was detected with a 3 × 3 mm2 CdTe detector placed at 90° with respect to the incident proton beam at a distance of 45 cm from the vials. Second, a 7-cm diameter water phantom containing three 2.2-diameter vials with 3%–5% Au solutions was imaged with a 7-mm FWHM 220 MeV proton beam in a first generation CT scanning geometry. X-rays scattered perpendicular to the incident proton beam were acquired with the CdTe detector placed at 45 cm from the phantom positioned on a translation/rotation stage. Twenty one translational steps spaced by 3 mm at each of 36 projection angles spaced by 10° were acquired, and pXFCT images of the phantom were reconstructed with filtered back projection. A simplified geometry of the experimental data acquisition setup was modeled with the MC TOPAS code, and simulation results were compared to the experimental data. Results: A linear relationship between gold pXRF and gold concentration was observed in both experimental and MC simulation data (R2 > 0.99). All Au vials were apparent in the experimental and simulated pXFCT images. Specifically, the 3% Au vial was detectable in the experimental [contrast-to-noise ratio (CNR) = 5.8] and simulated (CNR = 11.5) pXFCT image. Due to fluorescence x-ray attenuation in the higher concentration vials, the 4% and 5% Au contrast were underestimated by 10% and 15%, respectively, in both the experimental and simulated pXFCT images. Conclusions: Proton-induced x-ray fluorescence CT imaging of 3%–5% gold solutions in a small animal

  18. Proton-induced x-ray fluorescence CT imaging

    Energy Technology Data Exchange (ETDEWEB)

    Bazalova-Carter, Magdalena, E-mail: bazalova@stanford.edu; Xing, Lei [Department of Radiation Oncology, Stanford University, Stanford, California 94305-5847 and Global Station for Quantum Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo 060-8648 (Japan); Ahmad, Moiz [Department of Radiation Oncology, Stanford University, Stanford, California 94305-5847 (United States); Matsuura, Taeko; Takao, Seishin; Shirato, Hiroki; Umegaki, Kikuo [Department of Medical Physics, Proton Beam Therapy Center, Hokkaido University Hospital, Sapporo 060-8648, Japan and Global Station for Quantum Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo 060-8648 (Japan); Matsuo, Yuto [Department of Medical Physics, Proton Beam Therapy Center, Hokkaido University Hospital, Sapporo 060-8648 (Japan); Fahrig, Rebecca [Department of Radiology, Stanford University, Stanford, California 94305 (United States)

    2015-02-15

    Purpose: To demonstrate the feasibility of proton-induced x-ray fluorescence CT (pXFCT) imaging of gold in a small animal sized object by means of experiments and Monte Carlo (MC) simulations. Methods: First, proton-induced gold x-ray fluorescence (pXRF) was measured as a function of gold concentration. Vials of 2.2 cm in diameter filled with 0%–5% Au solutions were irradiated with a 220 MeV proton beam and x-ray fluorescence induced by the interaction of protons, and Au was detected with a 3 × 3 mm{sup 2} CdTe detector placed at 90° with respect to the incident proton beam at a distance of 45 cm from the vials. Second, a 7-cm diameter water phantom containing three 2.2-diameter vials with 3%–5% Au solutions was imaged with a 7-mm FWHM 220 MeV proton beam in a first generation CT scanning geometry. X-rays scattered perpendicular to the incident proton beam were acquired with the CdTe detector placed at 45 cm from the phantom positioned on a translation/rotation stage. Twenty one translational steps spaced by 3 mm at each of 36 projection angles spaced by 10° were acquired, and pXFCT images of the phantom were reconstructed with filtered back projection. A simplified geometry of the experimental data acquisition setup was modeled with the MC TOPAS code, and simulation results were compared to the experimental data. Results: A linear relationship between gold pXRF and gold concentration was observed in both experimental and MC simulation data (R{sup 2} > 0.99). All Au vials were apparent in the experimental and simulated pXFCT images. Specifically, the 3% Au vial was detectable in the experimental [contrast-to-noise ratio (CNR) = 5.8] and simulated (CNR = 11.5) pXFCT image. Due to fluorescence x-ray attenuation in the higher concentration vials, the 4% and 5% Au contrast were underestimated by 10% and 15%, respectively, in both the experimental and simulated pXFCT images. Conclusions: Proton-induced x-ray fluorescence CT imaging of 3%–5% gold solutions in a

  19. A novel indocyanine green nanoparticle probe for non invasive fluorescence imaging in vivo

    Science.gov (United States)

    Navarro, Fabrice P.; Berger, Michel; Goutayer, Mathieu; Guillermet, Stéphanie; Josserand, Véronique; Rizo, Philippe; Vinet, Françoise; Texier, Isabelle

    2009-02-01

    Fluorescence imaging (FLI) allows the in vivo monitoring of biological events associated with disease and represents a new promising tool for drug discovery. In particular, it speeds up the development and assessment of new therapies in oncology, helps in diagnosis, and improves surgery by fluorescence-guided tumor resection. This technique is highly sensitive, non-ionizing, easy to use and relatively inexpensive. Nevertheless, the main limitation of FLI lies in the optical properties of biological tissues. Mainly because of haemoglobin and water absorption, only near-infrared (NIR) light is adapted to image tissues in depth. Using a contrasting agent absorbing and emitting in the NIR region is therefore necessary to improve the background signal ratio, and thus the image contrast. Among many commercially available NIR optical contrast agents, only indocyanine green (ICG), has been approved by the United State Food and Drug Administration (FDA) for various medical applications. However, its instability (photo-degradation, thermal-degradation and low aqueous solubility) limits its applications as a fluorescent probe for imaging purposes. In order to improve the effectiveness of ICG, we engineered ICG-doped lipid nanoparticles (LNP). In this communication, we will report the design of these novel fluorescent nanoparticle probes. These low cost nanocarriers have numerous advantages, including their high chemical stability and biocompatibility. The characterization of the optical properties of the nanoparticles entrapping ICG will also be discussed. Finally, the biodistribution in mice of ICG when delivered through nanoparticles in comparison to free ICG in solution is presented. It demonstrates the efficient accumulation of ICG-doped nanoparticles in the tumor site.

  20. Molecular Imaging Approaches to Understanding the Roles of Hydrogen Peroxide Biology in Stress and Development

    OpenAIRE

    Dickinson, Bryan Craig

    2010-01-01

    The production of hydrogen peroxide (H2O2) in biological systems is associated with a variety of pathologies including neurodegenerative diseases, cancer, and the general process of aging. However, a growing body of evidence suggests that the reactivity of this particular reactive oxygen species (ROS) is also harnessed for physiological processes. Molecular imaging using fluorescence microscopy offers a valuable approach for deciphering the multifaceted roles of H2O2 in biological processes. ...

  1. Setup for studying kinetics of fluorescence anisotropy decay in biological objects

    Energy Technology Data Exchange (ETDEWEB)

    Akimov, A.V.; Dem`yanov, G.V.; Kurek, N.K. [Lebedev Physics Institute, Moscow (Russian Federation)] [and others

    1995-02-01

    A setup for studying the decay of the fluorescence intensity and anisotropy in biological objects, brought into use at an S-60 FIAN synchrotron radiation source, is described. The setup allows one to extend investigations of a spatial structure of biological membranes and lipoproteins. 5 refs., 2 figs.

  2. Fluorescent and Luminescent Probes for Monitoring Hydroxyl Radical under Biological Conditions.

    Science.gov (United States)

    Żamojć, Krzysztof; Zdrowowicz, Magdalena; Jacewicz, Dagmara; Wyrzykowski, Dariusz; Chmurzyński, Lech

    2016-01-01

    Detection and quantitative determination in biological media of the hydroxyl radical are of great importance due to the role this radical plays in many physiological and pathological processes. This review focuses on the progress that has been made in recent years in the development of fluorescent and luminescent probes employed to monitor hydroxyl radical concentrations under biological conditions. PMID:26042844

  3. Direct comparison of soft x-ray images of organelles with optical fluorescence images

    International Nuclear Information System (INIS)

    Soft x-ray microscopes operating in the water window region are capable of imaging living hydrated cells. Up to now, we have been able to take some soft x-ray images of living cells by the use of a contact x-ray microscope system with laser produced plasma soft x-ray source. Since the soft x-ray images are different from the optical images obtained with an ordinary microscope, it is very important to identify what is seen in the x-ray images. Hence, we have demonstrated the direct comparison between the images of organelles obtained with a fluorescence microscope and those with a soft x-ray microscope. Comparing the soft x-ray images to the fluorescence images, the fine structures of the organelles could be identified and observed. (author)

  4. Picosecond fluorescence lifetime imaging microscope for imaging of living glioma cells

    Science.gov (United States)

    Fang, Qiyin; Wang, Jingjing; Sun, Yinghua; Vernier, Thomas; Papaioannou, Thanassis; Jo, Javier; Thu, Mya M.; Gundersen, Martin A.; Marcu, Laura

    2005-03-01

    In this communication, we report the imaging of living glioma cells using fluorescence lifetime imaging (FLIM) technique. The growing interests in developing novel techniques for diagnosis and minimally invasive therapy of brain tumor have led to microscopic studies of subcellular structures and intracellular processes in glioma cells. Fluorescence microscopy has been used with a number of exogenous molecular probes specific for certain intracellular structures such as mitochondria, peripheral benzodiazepine receptor (PBR), and calcium concentration. When probes with overlapping emission spectra being used, separate samples are required to image each probe individually under conventional fluorescence microscopy. We have developed a wide-field FLIM microscope that uses fluorescence lifetime as an additional contrast for resolving multiple markers in the same essay. The FLIM microscope consists of a violet diode laser and a nitrogen-pumped dye laser to provide tunable sub-nanosecond excitation from UV to NIR. The detection system is based on a time-gated ICCD camera with minimum 80 ps gate width. The performance of the system was evaluated using fluorescence dyes with reported lifetime values. Living rat glioma C6 cells were stained with JC-1 and Rhodamine 123. FLIM images were acquired and their lifetimes in living cells were found in good agreements with values measured in solutions by a time-domain fluorescence spectrometer. These results indicate that imaging of glioma cells using FLIM can resolve multiple spectrally-overlapping probes and provide quantitative functional information about the intracellular environment.

  5. Neural imaging in songbirds using fiber optic fluorescence microscopy

    Science.gov (United States)

    Nooshabadi, Fatemeh; Hearn, Gentry; Lints, Thierry; Maitland, Kristen C.

    2012-02-01

    The song control system of juvenile songbirds is an important model for studying the developmental acquisition and generation of complex learned vocal motor sequences, two processes that are fundamental to human speech and language. To understand the neural mechanisms underlying song production, it is critical to characterize the activity of identified neurons in the song control system when the bird is singing. Neural imaging in unrestrained singing birds, although technically challenging, will advance our understanding of neural ensemble coding mechanisms in this system. We are exploring the use of a fiber optic microscope for functional imaging in the brain of behaving and singing birds in order to better understand the contribution of a key brain nucleus (high vocal center nucleus; HVC) to temporal aspects of song motor control. We have constructed a fluorescence microscope with LED illumination, a fiber bundle for transmission of fluorescence excitation and emission light, a ~2x GRIN lens, and a CCD for image acquisition. The system has 2 μm resolution, 375 μm field of view, 200 μm working distance, and 1 mm outer diameter. As an initial characterization of this setup, neurons in HVC were imaged using the fiber optic microscope after injection of quantum dots or fluorescent retrograde tracers into different song nuclei. A Lucid Vivascope confocal microscope was used to confirm the imaging results. Long-term imaging of the activity of these neurons in juvenile birds during singing may lead us to a better understanding of the central motor codes for song and the central mechanism by which auditory experience modifies song motor commands to enable vocal learning and imitation.

  6. Development of Photoactivated Fluorescent N-Hydroxyoxindoles and Their Application for Cell-Selective Imaging.

    Science.gov (United States)

    Lai, Jinping; Yu, An; Yang, Letao; Zhang, Yixiao; Shah, Birju P; Lee, Ki Bum

    2016-04-25

    Photoactivatable fluorophores are essential tools for studying the dynamic molecular interactions within important biological systems with high spatiotemporal resolution. However, currently developed photoactivatable fluorophores based on conventional dyes have several limitations including reduced photoactivation efficiency, cytotoxicity, large molecular size, and complicated organic synthesis. To overcome these challenges, we herein report a class of photoactivatable fluorescent N-hydroxyoxindoles formed through the intramolecular photocyclization of substituted o-nitrophenyl ethanol (ONPE). These oxindole fluorophores afford excellent photoactivation efficiency with ultra-high fluorescence enhancement (up to 800-fold) and are small in size. Furthermore, the oxindole derivatives show exceptional biocompatibility by generating water as the only photolytic side product. Moreover, structure-activity relationship analysis clearly revealed the strong correlation between the fluorescent properties and the substituent groups, which can serve as a guideline for the further development of ONPE-based fluorescent probes with desired photophysical and biological properties. As a proof-of-concept, we demonstrated the capability of a new substituted ONPE that has an uncaging wavelength of 365-405 nm and an excitation/emission at 515 and 620 nm, for the selective imaging of a cancer cell line (Hela cells) and a human neural stem cell line (hNSCs). PMID:27004772

  7. Compact instrument for fluorescence image-guided surgery

    Science.gov (United States)

    Wang, Xinghua; Bhaumik, Srabani; Li, Qing; Staudinger, V. Paul; Yazdanfar, Siavash

    2010-03-01

    Fluorescence image-guided surgery (FIGS) is an emerging technique in oncology, neurology, and cardiology. To adapt intraoperative imaging for various surgical applications, increasingly flexible and compact FIGS instruments are necessary. We present a compact, portable FIGS system and demonstrate its use in cardiovascular mapping in a preclinical model of myocardial ischemia. Our system uses fiber optic delivery of laser diode excitation, custom optics with high collection efficiency, and compact consumer-grade cameras as a low-cost and compact alternative to open surgical FIGS systems. Dramatic size and weight reduction increases flexibility and access, and allows for handheld use or unobtrusive positioning over the surgical field.

  8. Elastic registration for auto-fluorescence image averaging.

    Science.gov (United States)

    Kubecka, Libor; Jan, Jiri; Kolar, Radim; Jirik, Radovan

    2006-01-01

    The paper describes restitution of geometrical distortions and improvement of signal-to-noise ratio of auto-fluorescence retinal images, finally aimed at segmentation and area estimation of the lipofuscin spots as one of the features to be included in glaucoma diagnosis. The main problems - geometrical and illumination incompatibility of frames in the image sequence and a non-negligible "shear" distortion in the individual frames - have been solved by the presented registration procedure. The concept and some details of the MI-based regularized registration, together with evaluation of test results form the core of the contribution. PMID:17945684

  9. Multifocal two-photon excitation fluorescence sampling imaging combining lifetime and spectrum resolutions

    Science.gov (United States)

    Liu, Lixin; Lin, Ziyang; Qu, Junle; Chen, Danni; Xu, Gaixia; Guo, Baoping; Niu, Hanben

    2006-09-01

    Multifocal multiphoton microscopy (MMM) is a more efficient and powerful method for three-dimensional (3-D) fluorescence imaging with reduced acquisition time compared with conventional confocal and two-photon excitation fluorescence microscopy. We present a novel multifocal two-photon excitation fluorescence sampling imaging technique that is based on a specially designed streak camera and combines fluorescence lifetime and spectrum resolutions. A proof-of-principle experiment is performed on a standard fluorescent dye solution (Rhodamine 6G in ethanol), Time- and spectrum-resolved sampled fluorescence image of Rhodamine 6G is obtained in a snapshot. The reconstructed two-dimensional (2-D) fluorescence image of a prepared plant slide is also obtained by moving the sample laterally. The capability of this system capable of performing simultaneous 2-D measurements of temporal and spectral information has many potential applications, e.g., multi-well imaging and spectrally resolved multifocal multiphoton fluorescence lifetime imaging etc.

  10. Detection of biological warfare agents using ultra violet-laser induced fluorescence LIDAR

    Science.gov (United States)

    Joshi, Deepti; Kumar, Deepak; Maini, Anil K.; Sharma, Ramesh C.

    This review has been written to highlight the threat of biological warfare agents, their types and detection. Bacterial biological agent Bacillus anthracis (bacteria causing the disease anthrax) which is most likely to be employed in biological warfare is being discussed in detail. Standoff detection of biological warfare agents in aerosol form using Ultra violet-Laser Induced Fluorescence (UV-LIF) spectroscopy method has been studied. Range-resolved detection and identification of biological aerosols by both nano-second and non-linear femto-second LIDAR is also discussed. Calculated received fluorescence signal for a cloud of typical biological agent Bacillus globigii (Simulants of B. anthracis) at a location of ˜5.0 km at different concentrations in presence of solar background radiation has been described. Overview of current research efforts in internationally available working UV-LIF LIDAR systems are also mentioned briefly.

  11. Drug quantification in turbid media by fluorescence imaging combined with light-absorption correction using white Monte Carlo simulations

    DEFF Research Database (Denmark)

    Xie, Haiyan; Liu, Haichun; Svenmarker, Pontus; Axelsson, Johan; Xu, Can T.; Gräfe, Susanna; Lundeman, Jesper Holm; Cheng, Haynes Pak Hay; Svanberg, Sune; Bendsoe, Niels; Andersen, Peter E.; Svanberg, Katarina; Andersson-Engels, Stefan

    2011-01-01

    light by turbid media, such as biological tissue, the detected fluorescence signal does not have a simple and unique dependence on the fluorophore concentration for different tissues, but depends in a complex way on other parameters as well. For this reason, little has been done on drug quantification...... in vivo by the fluorescence imaging technique. In this paper we present a novel approach to compensate for the light absorption in homogeneous turbid media both for the excitation and emission light, utilizing time-resolved fluorescence white Monte Carlo simulations combined with the Beer-Lambert law....... This method shows that the corrected fluorescence intensity is almost proportional to the absolute fluorophore concentration. The results on controllable tissue phantoms and murine tissues are presented and show good correlations between the evaluated fluorescence intensities after the light...

  12. Mesoscopic fluorescence tomography for in-vivo imaging of developing Drosophila.

    Science.gov (United States)

    Vinegoni, Claudio; Razansky, Daniel; Pitsouli, Chrysoula; Perrimon, Norbert; Ntziachristos, Vasilis; Weissleder, Ralph

    2009-01-01

    Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (>1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery. We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly

  13. Fluorescence and Cerenkov luminescence imaging. Applications in small animal research.

    Science.gov (United States)

    Schwenck, J; Fuchs, K; Eilenberger, S H L; Rolle, A-M; Castaneda Vega, S; Thaiss, W M; Maier, F C

    2016-04-12

    This review addresses small animal optical imaging (OI) applications in diverse fields of basic research. In the past, OI has proven to be cost- and time-effective, allows real-time imaging as well as high-throughput analysis and does not imply the usage of ionizing radiation (with the exception of Cerenkov imaging applications). Therefore, this technique is widely spread - not only geographically, but also among very different fields of basic research - and is represented by a large body of publications. Originally used in oncology research, OI is nowadays emerging in further areas like inflammation and infectious disease as well as neurology. Besides fluorescent probe-based contrast, the feasibility of Cerenkov luminescence imaging (CLI) has been recently shown in small animals and thus represents a new route for future applications. Thus, this review will focus on examples for OI applications in inflammation, infectious disease, cell tracking as well as neurology, and provides an overview over CLI. PMID:27067794

  14. Phenoplant: a web resource for the exploration of large chlorophyll fluorescence image datasets

    OpenAIRE

    Rousseau, Céline; Hunault, Gilles; Gaillard, Sylvain; Bourbeillon, Julie; Montiel, Gregory; Simier, Philippe; Campion, Claire; Jacques, Marie Agnes; Belin, Etienne; Boureau, Tristan

    2015-01-01

    Background Image analysis is increasingly used in plant phenotyping. Among the various imaging techniques that can be used in plant phenotyping, chlorophyll fluorescence imaging allows imaging of the impact of biotic or abiotic stresses on leaves. Numerous chlorophyll fluorescence parameters may be measured or calculated, but only a few can produce a contrast in a given condition. Therefore, automated procedures that help screening chlorophyll fluorescence image datasets are needed, especiall...

  15. Development of Fluorescence Imaging Lidar for Boat-Based Coral Observation

    Science.gov (United States)

    Sasano, Masahiko; Imasato, Motonobu; Yamano, Hiroya; Oguma, Hiroyuki

    2016-06-01

    A fluorescence imaging lidar system installed in a boat-towable buoy has been developed for the observation of reef-building corals. Long-range fluorescent images of the sea bed can be recorded in the daytime with this system. The viability of corals is clear in these fluorescent images because of the innate fluorescent proteins. In this study, the specifications and performance of the system are shown.

  16. Development of Fluorescence Imaging Lidar for Boat-Based Coral Observation

    Directory of Open Access Journals (Sweden)

    Sasano Masahiko

    2016-01-01

    Full Text Available A fluorescence imaging lidar system installed in a boat-towable buoy has been developed for the observation of reef-building corals. Long-range fluorescent images of the sea bed can be recorded in the daytime with this system. The viability of corals is clear in these fluorescent images because of the innate fluorescent proteins. In this study, the specifications and performance of the system are shown.

  17. Volumetric retinal fluorescence microscopic imaging with extended depth of field

    Science.gov (United States)

    Li, Zengzhuo; Fischer, Andrew; Li, Wei; Li, Guoqiang

    2016-03-01

    Wavefront-engineered microscope with greatly extended depth of field (EDoF) is designed and demonstrated for volumetric imaging with near-diffraction limited optical performance. A bright field infinity-corrected transmissive/reflective light microscope is built with Kohler illumination. A home-made phase mask is placed in between the objective lens and the tube lens for ease of use. General polynomial function is adopted in the design of the phase plate for robustness and custom merit function is used in Zemax for optimization. The resulting EDoF system achieves an engineered point spread function (PSF) that is much less sensitive to object depth variation than conventional systems and therefore 3D volumetric information can be acquired in a single frame with expanded tolerance of defocus. In Zemax simulation for a setup using 32X objective (NA = 0.6), the EDoF is 20μm whereas a conventional one has a DoF of 1.5μm, indicating a 13 times increase. In experiment, a 20X objective lens with NA = 0.4 was used and the corresponding phase plate was designed and fabricated. Retinal fluorescence images of the EDoF microscope using passive adaptive optical phase element illustrate a DoF around 100μm and it is able to recover the volumetric fluorescence images that are almost identical to in-focus images after post processing. The image obtained from the EDoF microscope is also better in resolution and contrast, and the retinal structure is better defined. Hence, due to its high tolerance of defocus and fine restored image quality, EDoF optical systems have promising potential in consumer portable medical imaging devices where user's ability to achieve focus is not optimal, and other medical imaging equipment where achieving best focus is not a necessary.

  18. Confocal supercritical angle fluorescence microscopy for cell membrane imaging

    CERN Document Server

    Sivankutty, Siddharth; Mayet, Céline; Dupuis, Guillaume; Fort, Emmanuel; Lévêque-Fort, Sandrine

    2013-01-01

    We demonstrate sub-wavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fuorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modi?cation on the detection channel. Confocal-SAF microscopy would be a powerful tool to achieve high resolution surface imaging, especially for membrane imaging in biological samples

  19. PhytoBeta imager: a positron imager for plant biology

    International Nuclear Information System (INIS)

    Several positron emitting radioisotopes such as 11C and 13N can be used in plant biology research. The 11CO2 tracer is used to facilitate plant biology research toward optimization of plant productivity, biofuel development and carbon sequestration in biomass. Positron emission tomography (PET) imaging has been used to study carbon transport in live plants using 11CO2. Because plants typically have very thin leaves, little medium is present for the emitted positrons to undergo an annihilation event. The emitted positrons from 11C (maximum energy 960 keV) could require up to approximately 4 mm of water equivalent material for positron annihilation. Thus many of the positrons do not annihilate inside the leaf, resulting in limited sensitivity for PET imaging. To address this problem we have developed a compact beta-positive, beta-minus particle imager (PhytoBeta imager) for 11CO2 leaf imaging. The detector is based on a Hamamatsu H8500 position sensitive photomultiplier tube optically coupled via optical grease to a 0.5 mm thick Eljen EJ-212 plastic scintillator. The detector is equipped with a flexible arm to allow its placement and orientation over or under the leaf to be studied while maintaining the leaf's original orientation. To test the utility of the system the detector was used to measure carbon translocation in a leaf of the spicebush (Lindera benzoin) under two transient light conditions. (paper)

  20. GBIQ: a non-arbitrary, non-biased method for quantification of fluorescent images

    Science.gov (United States)

    Ninomiya, Youichirou; Zhao, Wei; Saga, Yumiko

    2016-01-01

    Non-arbitrary and non-biased quantification of fluorescent images is an essential tool for the data-centric approach to biological systems. Typical application is high-content analysis, where various phenotypic changes in cellular components and/or morphology are measured from fluorescent image data. A standard protocol to detect cellular phenotypes is cell-segmentation, in which boundaries of cellular components, such as cell nucleus and plasma membrane, are first identified to define cell segments, then acquiring various phenotypic data of each segment. To achieve reliable outcome, cell-segmentation requires manual adjustments of many parameters; this requirement could hamper automated image processing in high-throughput workflow, whose quantification must be non-arbitrary and non-biased. As a practical alternative to the segmentation-based method, we developed GBIQ (Grid Based Image Quantification), which allows comparison of cellular information without identification of single cells. GBIQ divides an image with tiles of fixed size grids and records statistics of the grids with their location coordinates, minimizing arbitrary intervenes. GBIQ requires only one parameter (size of grid) to be set; nonetheless it robustly produces results suitable for further statistical evaluation. The simplicity of GBIQ allows it to be readily implemented in an automated high-throughput image analysis workflow. PMID:27211912

  1. A microcomputed tomography guided fluorescence tomography system for small animal molecular imaging

    International Nuclear Information System (INIS)

    A prototype small animal imaging system was created for coupling fluorescence tomography (FT) with x-ray microcomputed tomography (microCT). The FT system has the potential to provide synergistic information content resultant from using microCT images as prior spatial information and then allows overlay of the FT image onto the original microCT image. The FT system was designed to use single photon counting to provide maximal sensitivity measurements in a noncontact geometry. Five parallel detector locations are used, each allowing simultaneous sampling of the fluorescence and transmitted excitation signals through the tissue. The calibration and linearity range performance of the system are outlined in a series of basic performance tests and phantom studies. The ability to image protoporphyrin IX in mouse phantoms was assessed and the system is ready for in vivo use to study biological production of this endogenous marker of tumors. This multimodality imaging system will have a wide range of applications in preclinical cancer research ranging from studies of the tumor microenvironment and treatment efficacy for emerging cancer therapeutics.

  2. Green fluorescent protein is lighting up fungal biology

    Science.gov (United States)

    Lorang, J.M.; Tuori, R.P; Martinez, J.P; Sawyer, T.L.; Redman, R.S.; Rollins, J. A.; Wolpert, T.J.; Johnson, K.B.; Rodriguez, R.J.; Dickman, M. B.; Ciuffetti, L.M.

    2001-01-01

    Prasher (42) cloned a cDNA for the green fluorescent protein (GFP) gene from the jellyfishAequorea victoria in 1992. Shortly thereafter, to the amazement of many investigators, this gene or derivatives thereof were successfully expressed and conferred fluorescence to bacteria andCaenorhabditis elegans cells in culture (10,31), followed by yeast (24, 39), mammals (40), Drosophila (66),Dictyostelium(23, 30), plants (28,49), and filamentous fungi (54). The tremendous success of GFP as a reporter can be attributed to unique qualities of this 238-amino-acid, 27-kDa protein which absorbs light at maxima of 395 and 475 nm and emits light at a maximum of 508 nm. The fluorescence of GFP requires only UV or blue light and oxygen, and therefore, unlike the case with other reporters (β-glucuronidase, β-galacturonidase, chloramphenicol acetyltransferase, and firefly luciferase) that rely on cofactors or substrates for activity, in vivo observation ofgfp expression is possible with individual cells, with cell populations, or in whole organisms interacting with symbionts or environments in real time. Complications caused by destructive sampling, cell permeablization for substrates, or leakage of products do not occur. Furthermore, the GFP protein is extremely stable in vivo and has been fused to the C or N terminus of many cellular and extracellular proteins without a loss of activity, thereby permitting the tagging of proteins for gene regulation analysis, protein localization, or specific organelle labeling. The mature protein resists many proteases and is stable up to 65°C and at pH 5 to 11, in 1% sodium dodecyl sulfate or 6 M guanidinium chloride (reviewed in references 17and 67), and in tissue fixed with formaldehyde, methanol, or glutaraldehyde. However, GFP loses fluorescence in methanol-acetic acid (3:1) and can be masked by autofluorescent aldehyde groups in tissue fixed with glutaraldehyde. Fluorescence is optimal at pH 7.2 to 8.0 (67).

  3. Synthesis and evaluation of [{sup 14}C]-Labelled and fluorescent-Tagged paclitaxel derivatives as new biological probes

    Energy Technology Data Exchange (ETDEWEB)

    Rao, C.S.; Chu, J.-J.; Lai, Y.-K. [Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan (China); Liu, R.-S. [Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan (China)

    1998-11-01

    Our present report deals with the preparation of hitherto unreported 7-([carbonyl-{sup 14}C]-acetyl)paclitaxel 4 and two new bioactive 7-substituted fluorescent taxoids (FITC 9 and rhodamine 11), as well as evaluation towards their applications as biological probes. The results in this report demonstrate that (a) the new paclitaxel derivatives 4, 9, 11 could be prepared with good yields starting from paclitaxel; (b) the [{sup 14}C]acetylation step was found to be better by using [{sup 14}C]acetic anhydride rather than [{sup 14}C]sodium acetate; (c) the radiochemical purity of 4 was 96% and its specific activity was 48 mCi/mmol; (d) the cytotoxicity of 4 was close to that of paclitaxel whereas 9, 11 were far less active than paclitaxel, but these cytotoxic levels were good enough for their biological applications; (e) the drug-quantitation by flow cytometric analysis using 9 and 11 was proved to be equally efficient with respect to the radioactivity-based determination employing 4; (f) the intracellular fluorescence mapping by 9 and 11 was found to be effective and the microtubule network pattern was visible in both the cases; (g) the overall fluorescence imaging efficiency was better with 11 while the intensity of fluorescence was higher with 9; (h) staining of nucleolus was observed in fluorescence studies of both 9 and 11. Based on these results, the newly prepared paclitaxel derivatives can be considered as efficient biological probes and should find further use in relevant applications. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  4. Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe.

    Science.gov (United States)

    Ali, Haydar; Bhunia, Susanta Kumar; Dalal, Chumki; Jana, Nikhil R

    2016-04-13

    Fluorescent carbon nanoparticle-based probes with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. However, synthesis of red fluorescent carbon nanoparticles and their transformation into functional nanoparticles are very challenging. Here we report red fluorescent carbon nanoparticle-based nanobioconjugates of carbon nanoparticles are synthesized via high temperature colloid-chemical approach and transformed into water-soluble functional nanoparticles via coating with amphiphilic polymer followed by covalent linking with desired biomolecules. Following this approach, carbon nanoparticles are functionalized with polyethylene glycol, primary amine, glucose, arginine, histidine, biotin and folic acid. These functional nanoparticles can be excited with blue/green light (i.e., 400-550 nm) to capture their emission spanning from 550 to 750 nm. Arginine and folic acid functionalized nanoparticles have been demonstrated as fluorescent cell labels where blue and green excitation has been used for imaging of labeled cells. The presented method can be extended for the development of carbon nanoparticle-based other bioimaging probes. PMID:27011336

  5. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    Science.gov (United States)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

  6. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

    Science.gov (United States)

    Moffitt, Jeffrey R.; Zhuang, Xiaowei

    2016-01-01

    Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

  7. Small portable interchangeable imager of fluorescence for fluorescence guided surgery and research.

    Science.gov (United States)

    Okusanya, Olugbenga T; Madajewski, Brian; Segal, Erin; Judy, Brendan F; Venegas, Ollin G; Judy, Ryan P; Quatromoni, Jon G; Wang, May D; Nie, Shuming; Singhal, Sunil

    2015-04-01

    Fluorescence guided surgery (FGS) is a developing field of surgical and oncologic research. Practically, FGS has shown useful applications in urologic surgery, benign biliary surgery, colorectal cancer liver metastasis resection, and ovarian cancer debulking. Most notably in in cancer surgery, FGS allows for the clear delineation of cancerous tissue from benign tissue. FGS requires the utilization of a fluorescent contrast agent and an intraoperative fluorescence imaging device (IFID). Currently available IFIDs are expensive, unable to work with multiple fluorophores, and can be cumbersome. This study aims to describe the development and utility of a small, cost-efficient, and interchangeable IFID made from commercially available components. Extensive research was done to design and construct a light-weight, portable, and cost-effective IFID. We researched the capabilities, size, and cost of several camera types and eventually decided on a near-infrared (NIR) charged couple device (CCD) camera for its overall profile. The small portable interchangeable imager of fluorescence (SPIIF) is a "scout" IFID system for FGS. The main components of the SPIIF are a NIR CCD camera with an articulating light filter. These components and a LED light source with an attached heat sink are mounted on a small metal platform. The system is connected to a laptop by a USB 2.0 cable. Pixielink © software on the laptop runs the system by controlling exposure time, gain, and image capture. After developing the system, we evaluated its utility as an IFID. The system weighs less than two pounds and can cover a large area. Due to its small size, it is easily made sterile by covering it with any sterile plastic sheet. To determine the system's ability to detect fluorescent signal, we used the SPIIF to detect indocyanine green under ex and in-vivo conditions and fluorescein under ex-vivo conditions. We found the SPIIF was able to detect both ICG and fluorescein under different depths of a

  8. Experimental evaluation of a hyperspectral imager for near-infrared fluorescent contrast agent studies

    Science.gov (United States)

    Luthman, A. S.; Bohndiek, Sarah E.

    2015-03-01

    Hyperspectral imaging (HSI) systems have the potential to combine morphological and spectral information to provide detailed and high sensitivity readouts in biological and medical applications. As HSI enables simultaneous detection in several spectral bands, the technology has significant potential for use in real-time multiplexed contrast agent studies. Examples include tumor detection in intraoperative and endoscopic imaging as well as histopathology. A multiplexed readout from multiple disease targets, such as cell surface receptors overexpressed in cancer cells, could improve both sensitivity and specificity of tumor identification. Here, we evaluate a commercial, compact, near-infrared HSI sensor that has the potential to enable low cost, video rate HSI for multiplexed fluorescent contrast agent studies in biomedical applications. The hyperspectral imager, based on a monolithically integrated Fabry-Perot etalon, has 70 spectral bands between 600-900 nm, making it ideal for this application. Initial calibration of the imager was performed to determine wavelength band response, quantum efficiency and the effect of F-number on the spectral response. A platform for wide-field fluorescence imaging in reflectance using fluorophore specific LED excitation was then developed. The applicability of the imaging platform for simultaneous readout of multiple fluorophore signals was demonstrated using a dilution series of Alexa Fluor 594 and Alexa Fluor 647, showing that nanomolar fluorophore concentrations can be detected. Our results show that the HSI system can clearly resolve the emission spectra of the two fluorophores in mixtures of concentrations across several orders of magnitude, indicating a high dynamic range performance. We therefore conclude that the HSI sensor tested here is suitable for detecting fluorescence in biomedical imaging applications.

  9. Microneedles rollers as a potential device to increase ALA diffusion and PpIX production: evaluations by wide-field fluorescence imaging and fluorescence spectroscopy

    Science.gov (United States)

    Gracielli Sousa, R. Phamilla; de Menezes, Priscila F. C.; Fujita, Alessandra K. L.; Requena, Michelle B.; Govone, Angelo Biassi; Escobar, André; de Nardi, Andrigo B.; Kurachi, Cristina; Bagnato, Vanderlei Salvador

    2014-03-01

    One of the limitations of topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is the poor ability to penetrate biological barriers of skin and the recurrence rates in treatments. This study aimed to identify possible signs of increased diffusion of ALA-induced PpIX by fluorescence images and fluorescence spectroscopy. The research was done using in vivo porcine skin model. Before the cream application, microholes was performed with microneedles rollers in only one direction, afterward the ALA cream was applied at a 2.5cm2 area in triplicate and an occlusive dressing was placed. PpIX production was monitored using fluorescence spectroscopy collected at skin surface after 70, 100, 140, and 180 minutes of ALA incubation. About 100 fluorescence spectra of each treatment were collected, distributed by about five points for each site. Wide-field fluorescence imaging was made after 70, 90, and 170 minutes after treatment. The results obtained by imaging analysis indicated increase of the PpIX diffusion in the skin surface using the microneedles rollers (MNs) before ALA application. Circular regions of red fluorescence around the microholes were observed. In addition, the fluorescence spectra showed a greater intensity (2 times as many) in groups microneedles rollers associated. In conclusion, our data shown greater homogeneity and PpIX production in the groups pre-treated with microneedles indicating that the technique can be used to greater uniformity of PpIX production throughout the area to be treated reducing the chances of recurrent tumor as well as has potential for decreasing the time of therapy. (FUNDING SUPPORT:CAPES, CNPq and FAPESP)

  10. Image registration and averaging of low laser power two-photon fluorescence images of mouse retina.

    Science.gov (United States)

    Alexander, Nathan S; Palczewska, Grazyna; Stremplewski, Patrycjusz; Wojtkowski, Maciej; Kern, Timothy S; Palczewski, Krzysztof

    2016-07-01

    Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [Palczewska G., Nat Med.20, 785 (2014) Sharma R., Biomed. Opt. Express4, 1285 (2013)]. PMID:27446697

  11. Inorganic phosphate nanorods are a novel fluorescent label in cell biology

    Directory of Open Access Journals (Sweden)

    Mukherjee Priyabrata

    2006-10-01

    Full Text Available Abstract We report the first use of inorganic fluorescent lanthanide (europium and terbium ortho phosphate [LnPO4·H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC, 786-O cells, or renal carcinoma cells (RCC. The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS, differential interference contrast (DIC microscopy, confocal microscopy, and transmission electron microscopy (TEM. At concentrations up to 50 μg/ml, the use of [3H]-thymidine incorporation assays, apoptosis assays (TUNEL, and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes

  12. Measurement of Nanoparticle Magnetic Hyperthermia Using Fluorescent Microthermal Imaging

    Science.gov (United States)

    Zheng, Xiaowan; van Keuren, Edward

    Nanoparticle magnetic hyperthermia uses the application of an AC magnetic field to ferromagnetic nanoparticles to elevate the temperature of cancer cells. The principle of hyperthermia as a true cell-specific therapy is that tumor cells are more sensitive to high temperature, so it is of great importance to control the locality and magnitude of the temperature differences. One technique to measure temperature variations on microscopic length scales is fluorescent microthermal imaging (FMI). Since it is the local temperature that is measured in FMI, effects such as heating due to nearby field coils can be accounted for. A dye, the rare earth chelate europium thenoyltrifluoroacetonate (Eu:TTA), with a strong temperature-dependent fluorescence emission has been incorporated into magnetic nanoparticles dispersed in a polymer films. FMI experiments were carried out on these samples under an applied high frequency magnetic field. Preliminary results show that FMI is a promising technique for characterizing the local generation of heat in nanoparticle magnetic hyperthermia.

  13. Development of temperature imaging using two-line atomic fluorescence.

    Science.gov (United States)

    Medwell, Paul R; Chan, Qing N; Kalt, Peter A M; Alwahabi, Zeyad T; Dally, Bassam B; Nathan, Graham J

    2009-02-20

    This work aims to advance understanding of the coupling between temperature and soot. The ability to image temperature using the two-line atomic fluorescence (TLAF) technique is demonstrated. Previous TLAF theory is extended from linear excitation into the nonlinear fluence regime. Nonlinear regime two-line atomic fluorescence (NTLAF) provides superior signal and reduces single-shot uncertainty from 250 K for conventional TLAF down to 100 K. NTLAF is shown to resolve the temperature profile across the stoichiometric envelope for hydrogen, ethylene, and natural gas flames, with deviation from thermocouple measurements not exceeding 100 K, and typically ≲30 K. Measurements in flames containing soot demonstrate good capacity of NTLAF to exclude interferences that hamper most two-dimensional thermometry techniques. PMID:23567586

  14. Probabilistic mapping and image clustering for quantitative assessment of fluorescent protein localizations in Arabidopsis guard cells

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Takumi Higaki, Natsumaro Kutsuna & Seiichiro Hasezawa ### Abstract The protocol reported here describes a method to quantitatively evaluate fluorescently-tagged protein localizations from fluorescent microscopic images with a combination of probabilistic mapping and image clustering. We demonstrate the use of this protocol using kidney-shaped guard cells of plants. ### Introduction Microscopic assessment of protein localizations with fluorescent protein taggin...

  15. Fluorescent probes in biology and medicine: measurement of intracellular pH values in individual cells

    Science.gov (United States)

    Slavik, Jan; Cimprich, Petr; Gregor, Martin; Smetana, Karel, Jr.

    1997-12-01

    The application possibilities of fluorescent probes have increased dramatically in the last few years. The main areas are as follows (Slavik, 1994, 1996, 1998). Intracellular ionic cell composition: There are selective ion-sensitive dyes for H+, Ca2+, Mg2+, K+, Na+, Fe3+, Cl-, Zn2+, Cd2+, Hg2+, Pb2+, Ba2+, La3+. Membrane potential: Using the so-called slow (Nernstian dyes) or electrochromic dyes one can assess the value of the transmembrane potential. Membrane fluidity: Fluorescent probes inform about the freedom of rotational and translational movement of membrane proteins and lipids. Selective labeling: Almost any object of interest inside the cell or on its surface can be selectively fluorescently labeled. There are dyes specific for DNA, RNA, oligonucleotides (FISH), Golgi, endoplasmic reticulum, mitochondria, vacuoles, cytoskeleton, etc. Using fluorescent dyes specific receptors may be localized, their conformational changes followed and the polarity of corresponding binding sites accessed. The endocytic pathway may be followed, enzymes and their local enzymatic activity localized. For really selective labeling fluorescent labeled antibodies exist. Imaging: One of the main advantages of fluorescence imaging is its versatility. It allow choice among ratio imaging in excitation, ratio imaging in emission and lifetime imaging. These approaches can be applied to both the classical wide-field fluorescence microscopy and to the laser confocal fluorescence microscopy, one day possibly to the scanning near field optical microscopy. Simultaneous application of several fluorescent dyes: The technical progress in both excitation sources and in detectors allows to extend the excitation deeper in the blue and ultraviolet side and the detection further in the NIR and IR. Consequently, up to 6 peaks in excitation and up to 6 peaks in emission can be followed without any substantial difficulties. Application of dyes such with longer fluorescence lifetimes such as rare earth

  16. Fluorescence Imaging In Vivo up to 1700 nm

    CERN Document Server

    Diao, Shuo; Hong, Guosong; Antaris, Alexander L; Chang, Junlei; Wu, Justin Z; Zhang, Bo; Kuo, Calvin J; Dai, Hongjie

    2015-01-01

    Compared to visible and near-infrared regions below ~ 900 nm, imaging in the second near-infrared window beyond 1000 nm (NIR-II, 1000-1700 nm) is promising for deep-tissue high-resolution optical imaging in vivo owing to reduced scattering of photons traversing through tissues. Here, we succeeded fluorescence imaging in vivo in the long 1500-1700 nm (NIR-IIb) region using a novel, chemical separation enriched large-diameter semiconducting single-walled carbon nanotube material. Imaging in the 1500-1700 nm window resolved 3-4 um wide capillary blood vessels at ~ 3 millimeters depth through the intact body and brain of mice with the ability of blood-flow speed mapping in individual capillary vessels. Further, non-invasive single fluorophore imaging inside the tumor of a live mouse was achieved in the 1500-1700 nm window. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting up to 1700 nm for a new paradigm of high performance in vivo optical imaging.

  17. Compact wearable dual-mode imaging system for real-time fluorescence image-guided surgery

    Science.gov (United States)

    Zhu, Nan; Huang, Chih-Yu; Mondal, Suman; Gao, Shengkui; Huang, Chongyuan; Gruev, Viktor; Achilefu, Samuel; Liang, Rongguang

    2015-09-01

    A wearable all-plastic imaging system for real-time fluorescence image-guided surgery is presented. The compact size of the system is especially suitable for applications in the operating room. The system consists of a dual-mode imaging system, see-through goggle, autofocusing, and auto-contrast tuning modules. The paper will discuss the system design and demonstrate the system performance.

  18. Defining a superlens operating regime for imaging fluorescent molecules.

    Science.gov (United States)

    Elsayad, Kareem; Heinze, Katrin G

    2009-01-01

    It has been shown that thin metal-based films can at certain frequencies act as planar near-field lenses for certain polarization components. A desirable property of such "lenses" is that they can also enhance and focus some large transverse spatial frequency components which contain sub-diffraction limit details. Over the last decade there has been much work in optimizing designs to reduce effects (such as material losses and surface roughness) that are detrimental to image reconstruction. One design that can reduce some of these undesirable effects, and which has received a fair amount of attention recently, is the stacked metal-dielectric superlens. Here we theoretically explore the imaging ability of such a design for the specific purpose of imaging a fluorescent dye (the common bio-marker GFP) in the vicinity of the superlens surface. Our calculations take into consideration the interaction (damping) of an oscillating electric dipole with the metallic layers in the superlens. We also assume a Gaussian frequency distribution spectrum for the dipole. We treat the metallic-alloy and dielectric-alloy layers separately using an appropriate effective medium theory. The transmission properties are evaluated via Transfer matrix (-matrix) calculations that were performed in the MatLab and MathCad environments. Our study shows that it is in principle possible to image fluorescent molecules using a simple bilayer planar superlens. We find that optimal parameters for such a superlens occur when the peak dipole emission-frequency is slightly offset from the Surface Plasmon resonance frequency of the metal-dielectric interfaces. The best resolution is obtained when the fluorescent molecules are not too close (>/ approximately 10 nm) or too far (>/approximately 30 nm) from the superlens surface. The realization and application of a superlens with the specified design is possible using current nanofabrication techniques. When combined with e.g. a sub-wavelength grating

  19. A two-photon fluorescent probe for bio-imaging of formaldehyde in living cells and tissues.

    Science.gov (United States)

    Li, Jun-Bin; Wang, Qian-Qian; Yuan, Lin; Wu, Yong-Xiang; Hu, Xiao-Xiao; Zhang, Xiao-Bing; Tan, Weihong

    2016-05-23

    Formaldehyde (FA) plays an important role in living systems as a reactive carbonyl species (RCS). An abnormal degree of FA is known to induce neurodegeneration, cognitive decrease and memory loss owing to the formation of strong cross-link DNA and protein and other molecules. The development of efficient methods for biological FA detection is of great biomedical importance. Although a few one-photon FA fluorescent probes have been reported for imaging in living cells, probes excited by two photons are more suitable for bio-imaging due to their low background fluorescence, less photobleaching, and deep penetration depth. In this study, a two-photon fluorescent probe for FA detection and bio-imaging in living cells and tissues was reported. The detection is based on the 2-aza-Cope sigmatropic rearrangement followed by elimination to release the fluorophore, resulting in both one- and two-photon excited fluorescence increase. The probe showed a high sensitivity to FA with a detection limit of 0.2 μM. Moreover, enabled the two-photon bio-imaging of FA in live HEK-293 cells and tissues with tissue-imaging depths of 40-170 μm. Furthermore, could be applied for the monitoring of endogenous FA in live MCF-7 cells, presaging its practical applications in biological systems. PMID:27137921

  20. Spatial Filter Based Bessel-Like Beam for Improved Penetration Depth Imaging in Fluorescence Microscopy

    Science.gov (United States)

    Purnapatra, Subhajit B.; Bera, Sampa; Mondal, Partha Pratim

    2012-09-01

    Monitoring and visualizing specimens at a large penetration depth is a challenge. At depths of hundreds of microns, several physical effects (such as, scattering, PSF distortion and noise) deteriorate the image quality and prohibit a detailed study of key biological phenomena. In this study, we use a Bessel-like beam in-conjugation with an orthogonal detection system to achieve depth imaging. A Bessel-like penetrating diffractionless beam is generated by engineering the back-aperture of the excitation objective. The proposed excitation scheme allows continuous scanning by simply translating the detection PSF. This type of imaging system is beneficial for obtaining depth information from any desired specimen layer, including nano-particle tracking in thick tissue. As demonstrated by imaging the fluorescent polymer-tagged-CaCO3 particles and yeast cells in a tissue-like gel-matrix, the system offers a penetration depth that extends up to 650 µm. This achievement will advance the field of fluorescence imaging and deep nano-particle tracking.

  1. Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil

    Directory of Open Access Journals (Sweden)

    F. Boschi

    2011-06-01

    Full Text Available Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical properties of several compounds. Luminescent properties of essential oils have not been extensively investigated. We evaluated the luminescent and fluorescent emissions of several essential oils, in order to detect them in living organisms by exploiting their optical properties. Some fluorescent emission data were high enough to be detected in dermal treatments. Consequently, we demonstrated how the fluorescent signal can be monitored for at least three hours on the skin of living mice treated with wild chamomile oil. The results encourage development of this technique to investigate the properties of drugs and cosmetics containing essential oils.

  2. Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil.

    Science.gov (United States)

    Boschi, F; Fontanella, M; Calderan, L; Sbarbati, A

    2011-01-01

    Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical properties of several compounds. Luminescent properties of essential oils have not been extensively investigated. We evaluated the luminescent and fluorescent emissions of several essential oils, in order to detect them in living organisms by exploiting their optical properties. Some fluorescent emission data were high enough to be detected in dermal treatments. Consequently, we demonstrated how the fluorescent signal can be monitored for at least three hours on the skin of living mice treated with wild chamomile oil. The results encourage development of this technique to investigate the properties of drugs and cosmetics containing essential oils. PMID:22193298

  3. A parallel adaptive finite element simplified spherical harmonics approximation solver for frequency domain fluorescence molecular imaging

    OpenAIRE

    Lu, Yujie; Zhu, Banghe; Shen, Haiou; Rasmussen, John C.; WANG, GE; Sevick-Muraca, Eva M.

    2010-01-01

    Fluorescence molecular imaging/tomography may play an important future role in preclinical research and clinical diagnostics. Time- and frequency-domain fluorescence imaging can acquire more measurement information than the continuous wave (CW) counterpart, improving the image quality of fluorescence molecular tomography. Although diffusion approximation (DA) theory has been extensively applied in optical molecular imaging, high-order photon migration models need to be further investigated to...

  4. Chamber bioaerosol study: human emissions of size-resolved fluorescent biological aerosol particles

    OpenAIRE

    Bhangar, Seema; Adams, Rachel I.; Pasut, Wilmer; Huffman, Alex; Arens, Edward A.; Taylor, John W; Bruns, Tom D; Nazaroff, William W

    2015-01-01

    Humans are a prominent source of airborne biological particles in occupied indoor spaces, but few studies have quantified human bioaerosol emissions. The chamber investigation reported here employs a fluorescence-based technique to evaluate bioaerosols with high temporal and particle size resolution. In a 75-m3 chamber, occupant emission rates of coarse (2.5–10 μm) fluorescent biological aerosol particles (FBAPs) under seated, simulated office-work conditions averaged 0.9 ± 0.3 million partic...

  5. Determination of uranium in seawater, biological samples and sediments using laser induced fluorescence spectrometry

    International Nuclear Information System (INIS)

    Uranium has been determined in seawater, biological samples and sediments using laser induced fluorescence spectrometry (LIFS). The biological samples and sediments are digested with a mixture of HNO3, HClO4 and HF. The conductivity of the seawater should be below 5.0 mS and the pH of the sample should be in the range 6.5-9.0. The volume of the reagent used to enhance the fluorescence intensity was 0.5 ml. Comparison with other methods was favorable, LIFS being rapid, simple and sensitive, and well suited to environmental monitoring. (author)

  6. Correlations between nuclear and fluorescent Imaging of mammary tumors in mice

    Science.gov (United States)

    Carroll, Robin; Stone, John; Blue, Eric; Bradley, Eric; Qian, Jianguo; Saha, Margaret; Welsh, Robert

    2008-10-01

    Progress with new imaging technologies permits the study of biological processes both in vivo and noninvasively. Two systems, a position-sensitive gamma camera and a cooled-CCD camera have been applied in this work. A C3H strain of mouse carrying the Mouse Mammary Tumor Virus (MMTV) was imaged using 800 nm Q-tracker fluorescent dots conjugated to a peptide targeting integrin αυβ C a mammary marker for angiogenesis. We subsequently imaged with the gamma camera to detect low levels of ^125I distribution, and hence, the activity of a trans-membrane protein called the sodium iodide symporter (NIS) responsible for iodine transport. Preliminary results indicate that the biodistribution of the tagged Q-tracker dots and ^125I co-localize very early in seemingly normal mammary glands of infected MMTV mice, while in larger palpable tumors the Q-dot signals are less apparent in comparison with the^125I signal.

  7. Two-photon fluorescence imaging through multicore fiber with digital phase conjugation

    Science.gov (United States)

    Stasio, Nicolino; Conkey, Donald B.; Moser, Christophe; Psaltis, Demetri

    2016-03-01

    We present near diffraction limited two photon fluorescence (TPF) imaging through a lensless, multi-core fiber (MCF) endoscope utilizing digital phase conjugation. The ultra-small size of MCFs make them desirable tools for imaging deep into the body. TPF imaging enables optical sectioning and is widely used in brain and biological imaging and is a desired modality for fiber endoscopes. Previous implementations of TPF imaging through MCFs focus and scan the light from individual cores for image formation. In such systems the resolution is limited by the MCF core spacing, although a lens may be used to improve the resolution at the expense of the field of view. Other, more recent work has improved the resolution limitation using custom built MCFs for focusing and scanning of ultrafast pulses using wavefront shaping. Here we present digital phase conjugation for ultrafast pulse focusing through a MCF for an imaging resolution independent of the MCF core spacing. Furthermore, the phase conjugation technique does not require the use of a lens at the fiber end for focus formation and is compatible with commercially available MCFs with a large number of cores. Here, we present a 3000 core MCF endoscope and demonstrate ultrafast pulse focusing with sufficient focus spot contrast and power for TPF endoscope imaging. We construct TPF images by digital scanning of the phase conjugated focus on the target object and collection of the emitted fluorescence through the MCF. This work demonstrates the viability of digital conjugation combined with commercially available MCFs for higher resolution lensless, two photon endoscopy.

  8. Potential applications of near infrared auto-fluorescence spectral polarized imaging for assessment of food quality

    Science.gov (United States)

    Zhou, Kenneth J.; Chen, Jun

    2016-03-01

    The current growing of food industry for low production costs and high efficiency needs for maintenance of high-quality standards and assurance of food safety while avoiding liability issues. Quality and safety of food depend on physical (texture, color, tenderness etc.), chemical (fat content, moisture, protein content, pH, etc.), and biological (total bacterial count etc.) features. There is a need for a rapid (less than a few minutes) and accurate detection system in order to optimize quality and assure safety of food. However, the fluorescence ranges for known fluorophores are limited to ultraviolet emission bands, which are not in the tissue near infrared (NIR) "optical window". Biological tissues excited by far-red or NIR light would exhibit strong emission in spectral range of 650-1,100 nm although no characteristic peaks show the emission from which known fluorophores. The characteristics of the auto-fluorescence emission of different types of tissues were found to be different between different tissue components such as fat, high quality muscle food. In this paper, NIR auto-fluorescence emission from different types of muscle food and fat was measured. The differences of fluorescence intensities of the different types of muscle food and fat emissions were observed. These can be explained by the change of the microscopic structure of physical, chemical, and biological features in meat. The difference of emission intensities of fat and lean meat tissues was applied to monitor food quality and safety using spectral polarized imaging, which can be detect deep depth fat under the muscle food up to several centimeter.

  9. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2013-01-15

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  10. Modelling of microcracks image treated with fluorescent dye

    Science.gov (United States)

    Glebov, Victor; Lashmanov, Oleg U.

    2015-06-01

    The main reasons of catastrophes and accidents are high level of wear of equipment and violation of the production technology. The methods of nondestructive testing are designed to find out defects timely and to prevent break down of aggregates. These methods allow determining compliance of object parameters with technical requirements without destroying it. This work will discuss dye penetrant inspection or liquid penetrant inspection (DPI or LPI) methods and computer model of microcracks image treated with fluorescent dye. Usually cracks on image look like broken extended lines with small width (about 1 to 10 pixels) and ragged edges. The used method of inspection allows to detect microcracks with depth about 10 or more micrometers. During the work the mathematical model of image of randomly located microcracks treated with fluorescent dye was created in MATLAB environment. Background noises and distortions introduced by the optical systems are considered in the model. The factors that have influence on the image are listed below: 1. Background noise. Background noise is caused by the bright light from external sources and it reduces contrast on the objects edges. 2. Noises on the image sensor. Digital noise manifests itself in the form of randomly located points that are differing in their brightness and color. 3. Distortions caused by aberrations of optical system. After passing through the real optical system the homocentricity of the bundle of rays is violated or homocentricity remains but rays intersect at the point that doesn't coincide with the point of the ideal image. The stronger the influence of the above-listed factors, the worse the image quality and therefore the analysis of the image for control of the item finds difficulty. The mathematical model is created using the following algorithm: at the beginning the number of cracks that will be modeled is entered from keyboard. Then the point with random position is choosing on the matrix whose size is

  11. Fluorescence-Doped Particles for Simultaneous Temperature and Velocity Imaging

    Science.gov (United States)

    Danehy, Paul M.; Tiemsin, Pacita I.; Wohl, Chrostopher J.; Verkamp, Max; Lowe, T.; Maisto, P.; Byun, G.; Simpson, R.

    2012-01-01

    Polystyrene latex microspheres (PSLs) have been used for particle image velocimetry (PIV) and laser Doppler velocimetry (LDV) measurements for several decades. With advances in laser technologies, instrumentation, and data processing, the capability to collect more information about fluid flow beyond velocity is possible using new seed materials. To provide additional measurement capability, PSLs were synthesized with temperature-sensitive fluorescent dyes incorporated within the particle. These multifunctional PSLs would have the greatest impact if they could be used in large scale facilities with minimal modification to the facilities or the existing instrumentation. Consequently, several potential dyes were identified that were amenable to existing laser systems currently utilized in wind tunnels at NASA Langley Research Center as well as other wind and fluid (water) tunnels. PSLs incorporated with Rhodamine B, dichlorofluorescein (DCF, also known as fluorescein 548 or fluorescein 27) and other dyes were synthesized and characterized for morphology and spectral properties. The resulting particles were demonstrated to exhibit fluorescent emission, which would enable determination of both fluid velocity and temperature. They also would allow near-wall velocity measurements whereas laser scatter from surfaces currently prevents near-wall measurements using undoped seed materials. Preliminary results in a wind tunnel facility located at Virginia Polytechnic Institute and State University (Virginia Tech) have verified fluorescent signal detection and temperature sensitivity of fluorophore-doped PSLs.

  12. Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm.

    Science.gov (United States)

    Diao, Shuo; Blackburn, Jeffrey L; Hong, Guosong; Antaris, Alexander L; Chang, Junlei; Wu, Justin Z; Zhang, Bo; Cheng, Kai; Kuo, Calvin J; Dai, Hongjie

    2015-12-01

    Compared to imaging in the visible and near-infrared regions below 900 nm, imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is a promising method for deep-tissue high-resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long-wavelength NIR region (1500-1700 nm, NIR-IIb). With this imaging agent, 3-4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high-performance in vivo optical imaging. PMID:26460151

  13. Frequency Domain Fluorescent Molecular Tomography and Molecular Probes for Small Animal Imaging

    Science.gov (United States)

    Kujala, Naresh Gandhi

    Fluorescent molecular tomography (FMT) is a noninvasive biomedical optical imaging that enables 3-dimensional quantitative determination of fluorochromes distributed in biological tissues. There are three methods for imaging large volume tissues based on different light sources: (a) using a light source of constant intensity, through a continuous or constant wave, (b) using a light source that is intensity modulated with a radio frequency (RF), and (c) using ultrafast pulses in the femtosecond range. In this study, we have developed a frequency domain fluorescent molecular tomographic system based on the heterodyne technique, using a single source and detector pair that can be used for small animal imaging. In our system, the intensity of the laser source is modulated with a RF frequency to produce a diffuse photon density wave in the tissue. The phase of the diffuse photon density wave is measured by comparing the reference signal with the signal from the tissue using a phasemeter. The data acquisition was performed by using a Labview program. The results suggest that we can measure the phase change from the heterogeneous inside tissue. Combined with fiber optics and filter sets, the system can be used to sensitively image the targeted fluorescent molecular probes, allowing the detection of cancer at an early stage. We used the system to detect the tumor-targeting molecular probe Alexa Fluor 680 and Alexa Fluor 750 bombesin peptide conjugates in phantoms as well as mouse tissues. We also developed and evaluated fluorescent Bombesin (BBN) probes to target gastrin-releasing peptide (GRP) receptors for optical molecular imaging. GRP receptors are over-expressed in several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. BBN is a 14 amino acid peptide that is an analogue to human gastrin-releasing peptide that binds specifically to GRPr receptors. BBN conjugates are significant in cancer detection and therapy. The

  14. High Resolution Fluorescence Imaging of Cancers Using Lanthanide Ion-Doped Upconverting Nanocrystals

    International Nuclear Information System (INIS)

    During the last decade inorganic luminescent nanoparticles that emit visible light under near infrared (NIR) excitation (in the biological window) have played a relevant role for high resolution imaging of cancer. Indeed, semiconductor quantum dots (QDs) and metal nanoparticles, mostly gold nanorods (GNRs), are already commercially available for this purpose. In this work we review the role which is being played by a relatively new class of nanoparticles, based on lanthanide ion doped nanocrystals, to target and image cancer cells using upconversion fluorescence microscopy. These nanoparticles are insulating nanocrystals that are usually doped with small percentages of two different rare earth (lanthanide) ions: The excited donor ions (usually Yb3+ ion) that absorb the NIR excitation and the acceptor ions (usually Er3+, Ho3+ or Tm3+), that are responsible for the emitted visible (or also near infrared) radiation. The higher conversion efficiency of these nanoparticles in respect to those based on QDs and GNRs, as well as the almost independent excitation/emission properties from the particle size, make them particularly promising for fluorescence imaging. The different approaches of these novel nanoparticles devoted to “in vitro” and “in vivo” cancer imaging, selective targeting and treatment are examined in this review

  15. Stable J-aggregation enabled dual photoacoustic and fluorescence nanoparticles for intraoperative cancer imaging.

    Science.gov (United States)

    Shakiba, Mojdeh; Ng, Kenneth K; Huynh, Elizabeth; Chan, Harley; Charron, Danielle M; Chen, Juan; Muhanna, Nidal; Foster, F Stuart; Wilson, Brian C; Zheng, Gang

    2016-07-01

    J-aggregates display nanoscale optical properties which enable their use in fluorescence and photoacoustic imaging applications. However, control over their optical properties in an in vivo setting is hampered by the conformational lability of the J-aggregate structure in complex biological environments. J-aggregating nanoparticles (JNP) formed by self-assembly of bacteriopheophorbide-lipid (Bchl-lipid) in lipid nanovesicles represents a novel strategy to stabilize J-aggregates for in vivo bioimaging applications. We find that 15 mol% Bchl-lipid embedded within a saturated phospholipid bilayer vesicle was optimal in terms of maximizing Bchl-lipid dye loading, while maintaining a spherical nanoparticle morphology and retaining spectral properties characteristic of J-aggregates. The addition of cholesterol maintains the stability of the J-aggregate absorption band for up to 6 hours in the presence of 90% FBS. In a proof-of-concept experiment, we successfully applied JNPs as a fluorescence contrast agent for real-time intraoperative detection of metastatic lymph nodes in a rabbit head-and-neck cancer model. Lymph node metastasis delineation was further verified by visualizing the JNP within the excised lymph node using photoacoustic imaging. Using JNPs, we demonstrate the possibility of using J-aggregates as fluorescence and photoacoustic contrast agents and may potentially spur the development of other nanomaterials that can stably induce J-aggregation for in vivo cancer bioimaging applications. PMID:26731304

  16. Light-induced fluorescence endoscopy (LIFE) imaging system for early cancer detection

    Science.gov (United States)

    Zeng, Haishan; MacAulay, Calum E.; Lam, Stephen; Palcic, Branko

    1999-09-01

    This paper summarizes our experiences on the development of a Light Induced Fluorescence Endoscopy (LIFE) imaging system for early cancer detection in the respiratory and gastrointestinal tract. The system utilizes tissue autofluorescence to provide real time video imaging of the examined organ. No exogenous fluorescent tumor markers are needed. It is used by a physician in adjunct to conventional white-light endoscopy. Suspicious areas are identified in pseudo color to guide biopsy. A multi- center clinical trial has demonstrated that in the lung, the relative sensitivity of white-light imaging + LIFE imaging vs. white-light imaging alone was 6.3 for intraepithelial neoplastic lesion detection and 2.71 when invasive carcinomas were also included. The following issues will be discussed: (1) spectroscopy study design for imaging system development; (2) architecture of the imaging systems; (3) different imaging modalities (white-light imaging, dual channel fluorescence imaging, and combined fluorescence/reflectance imaging); and (4) clinical applications.

  17. Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics

    Science.gov (United States)

    Veetil, Jithesh V.; Jin, Sha; Ye, Kaiming

    2012-01-01

    Background One of the major hurdles in studying diabetes pathophysiology is the lack of adequate methodology that allows for direct and real-time determination of glucose transport and metabolism in cells and tissues. In this article, we present a new methodology that adopts frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) to visualize and quantify the dynamics of intracellular glucose within living cells using a biosensor protein based on fluorescence resonance energy transfer (FRET). Method The biosensor protein was developed by fusing a FRET pair, an AcGFP1 donor and a mCherry acceptor to N- and C- termini of a mutant glucose-binding protein (GBP), respectively. The probe was expressed and biosynthesized inside the cells, offering continuous monitoring of glucose dynamics in real time through fluorescence lifetime imaging microscopy (FLIM) measurement. Results We transfected the deoxyribonucleic acid of the AcGFP1-GBP-mCherry sensor into murine myoblast cells, C2C12, and continuously monitored the changes in intracellular glucose concentrations in response to the variation in extracellular glucose, from which we determined glucose uptake and clearance rates. The distribution of intracellular glucose concentration was also characterized. We detected a high glucose concentration in a region close to the cell membrane and a low glucose concentration in a region close to the nucleus. The monoexponential decay of AcGFP1 was distinguished using FD-FLIM. Conclusions This work enables continuous glucose monitoring (CGM) within living cells using FD-FLIM and a biosensor protein. The sensor protein developed offers a new means for quantitatively analyzing glucose homeostasis at the cellular level. Data accumulated from these studies will help increase our understanding of the pathology of diabetes. PMID:23294772

  18. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

    Science.gov (United States)

    Hammers, Matthew D; Taormina, Michael J; Cerda, Matthew M; Montoya, Leticia A; Seidenkranz, Daniel T; Parthasarathy, Raghuveer; Pluth, Michael D

    2015-08-19

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

  19. Hyperspectral fluorescence imaging coupled with multivariate image analysis techniques for contaminant screening of leafy greens

    Science.gov (United States)

    Everard, Colm D.; Kim, Moon S.; Lee, Hoyoung

    2014-05-01

    The production of contaminant free fresh fruit and vegetables is needed to reduce foodborne illnesses and related costs. Leafy greens grown in the field can be susceptible to fecal matter contamination from uncontrolled livestock and wild animals entering the field. Pathogenic bacteria can be transferred via fecal matter and several outbreaks of E.coli O157:H7 have been associated with the consumption of leafy greens. This study examines the use of hyperspectral fluorescence imaging coupled with multivariate image analysis to detect fecal contamination on Spinach leaves (Spinacia oleracea). Hyperspectral fluorescence images from 464 to 800 nm were captured; ultraviolet excitation was supplied by two LED-based line light sources at 370 nm. Key wavelengths and algorithms useful for a contaminant screening optical imaging device were identified and developed, respectively. A non-invasive screening device has the potential to reduce the harmful consequences of foodborne illnesses.

  20. Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device.

    Science.gov (United States)

    Schlimpert, Susan; Flärdh, Klas; Buttner, Mark

    2016-01-01

    Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series. PMID:26967231

  1. Precision analysis for standard deviation measurements of single fluorescent molecule images

    OpenAIRE

    DeSantis, Michael C.; DeCenzo, Shawn H.; Li, Je-Luen; Y. M. Wang

    2009-01-01

    Standard deviation measurements of intensity profiles of stationary single fluorescent molecules are useful for studying axial localization, molecular orientation, and a fluorescence imaging system's spatial resolution. Here we report on the analysis of the precision of standard deviation measurements of intensity profiles of single fluorescent molecules imaged using an EMCCD camera. We have developed an analytical expression for the standard deviation measurement error of a single image whic...

  2. Precision analysis for standard deviation measurements of immobile single fluorescent molecule images

    OpenAIRE

    DeSantis, Michael C.; DeCenzo, Shawn H.; Li, Je-Luen; Y. M. Wang

    2010-01-01

    Standard deviation measurements of intensity profiles of stationary single fluorescent molecules are useful for studying axial localization, molecular orientation, and a fluorescence imaging system’s spatial resolution. Here we report on the analysis of the precision of standard deviation measurements of intensity profiles of single fluorescent molecules imaged using an EMCCD camera. We have developed an analytical expression for the standard deviation measurement error of a single image whic...

  3. Motion corrected photoacoustic difference imaging of fluorescent contrast agents

    Science.gov (United States)

    Märk, Julia; Wagener, Asja; Pönick, Sarah; Grötzinger, Carsten; Zhang, Edward; Laufer, Jan

    2016-03-01

    In fluorophores, such as exogenous dyes and genetically expressed proteins, the excited state lifetime can be modulated using pump-probe excitation at wavelengths corresponding to the absorption and fluorescence spectra. Simultaneous pump-probe pulses induce stimulated emission (SE) which, in turn, modulates the thermalized energy, and hence the photoacoustic (PA) signal amplitude. For time-delayed pulses, by contrast, SE is suppressed. Since this is not observed in endogenous chromophores, the location of the fluorophore can be determined by subtracting images acquired using simultaneous and time-delayed pump-probe excitation. This simple experimental approach exploits a fluorophorespecific contrast mechanism, and has the potential to enable deep-tissue molecular imaging at fluences below the MPE. In this study, some of the challenges to its in vivo implementation are addressed. First, the PA signal amplitude generated in fluorophores in vivo is often much smaller than that in blood. Second, tissue motion can give rise to artifacts that correspond to endogenous chromophores in the difference image. This would not allow the unambiguous detection of fluorophores. A method to suppress motion artifacts based on fast switching between simultaneous and time-delayed pump-probe excitation was developed. This enables the acquisition of PA signals using the two excitation modes with minimal time delay (20 ms), thus minimizing the effects of tissue motion. The feasibility of this method is demonstrated by visualizing a fluorophore (Atto680) in tissue phantoms, which were moved during the image acquisition to mimic tissue motion.

  4. A review of imaging techniques for systems biology

    Directory of Open Access Journals (Sweden)

    Po Ming J

    2008-08-01

    Full Text Available Abstract This paper presents a review of imaging techniques and of their utility in system biology. During the last decade systems biology has matured into a distinct field and imaging has been increasingly used to enable the interplay of experimental and theoretical biology. In this review, we describe and compare the roles of microscopy, ultrasound, CT (Computed Tomography, MRI (Magnetic Resonance Imaging, PET (Positron Emission Tomography, and molecular probes such as quantum dots and nanoshells in systems biology. As a unified application area among these different imaging techniques, examples in cancer targeting are highlighted.

  5. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    International Nuclear Information System (INIS)

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses. (paper)

  6. Towards native-state imaging in biological context in the electron microscope

    OpenAIRE

    Weston, Anne E.; Armer, Hannah E. J.; Collinson, Lucy M.

    2009-01-01

    Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to comp...

  7. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    Science.gov (United States)

    Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J.

    2015-09-01

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min-1 with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels-1.

  8. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    International Nuclear Information System (INIS)

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min−1 with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels−1

  9. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization.

    Science.gov (United States)

    Hutcheson, Joshua A; Majid, Aneeka A; Powless, Amy J; Muldoon, Timothy J

    2015-09-01

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min(-1) with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels(-1). PMID:26429450

  10. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    Energy Technology Data Exchange (ETDEWEB)

    Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J., E-mail: tmuldoon@uark.edu [Department of Biomedical Engineering, University of Arkansas, 120 Engineering Hall, Fayetteville, Arkansas 72701 (United States)

    2015-09-15

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min{sup −1} with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels{sup −1}.

  11. Monomeric red fluorescent protein variants used for imaging studies in different species

    NARCIS (Netherlands)

    Mueller-Taubenberger, Annette; Vos, Michel J.; Boettger, Angelika; Lasi, Margherita; Lai, Frank P. L.; Fischer, Markus; Rottner, Klemens

    2006-01-01

    Fluorescent proteins have proven to be excellent tools for live-cell imaging studies. In addition to green fluorescent protein (GFP) and its variants, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation

  12. Quantitative imaging of cell-permeable magnetic resonance contrast agents using x-ray fluorescence.

    Science.gov (United States)

    Endres, Paul J; Macrenaris, Keith W; Vogt, Stefan; Allen, Matthew J; Meade, Thomas J

    2006-01-01

    The inability to transduce cellular membranes is a limitation of current magnetic resonance imaging probes used in biologic and clinical settings. This constraint confines contrast agents to extracellular and vascular regions of the body, drastically reducing their viability for investigating processes and cycles in developmental biology. Conversely, a contrast agent with the ability to permeate cell membranes could be used in visualizing cell patterning, cell fate mapping, gene therapy, and, eventually, noninvasive cancer diagnosis. Therefore, we describe the synthesis and quantitative imaging of four contrast agents with the capability to cross cell membranes in sufficient quantity for detection. Each agent is based on the conjugation of a Gd(III) chelator with a cellular transduction moiety. Specifically, we coupled Gd(III)-diethylenetriaminepentaacetic acid DTPA and Gd(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid with an 8-amino acid polyarginine oligomer and an amphipathic stilbene molecule, 4-amino-4'-(N,N-dimethylamino)stilbene. The imaging modality that provided the best sensitivity and spatial resolution for direct detection of the contrast agents is synchrotron radiation x-ray fluorescence (SR-XRF). Unlike optical microscopy, SR-XRF provides two-dimensional images with resolution 10(3) better than (153)Gd gamma counting, without altering the agent by organic fluorophore conjugation. The transduction efficiency of the intracellular agents was evaluated by T(1) analysis and inductively coupled plasma mass spectrometry to determine the efficacy of each chelate-transporter combination. PMID:17150161

  13. Fluorescent Cy5 silica nanoparticles for cancer cell imaging

    Science.gov (United States)

    O'Connell, Claire; Nooney, Robert I.; Glynn, MacDara; Ducree, Jens; McDonagh, Colette

    2015-08-01

    Cancer is a leading cause of death worldwide, with metastasis responsible for the majority of cancer-related deaths. Circulating tumour cells (CTCs) play a central role in metastasis. Fluorescent silica particles (NPs), of diameter ~50 nm which contain a large concentration of Cy5 dye molecules and are extremely bright, have been developed to detect these rare CTCs. Due to this brightness, the particles have superior performance compared to single Cy5 dye molecule labels, for detecting cancer cells. Fluorescence measurements show that the NPs are almost 100 times brighter than the free dye. They do not photo bleach as readily and, due to the biocompatible silica surface, they can be chemically modified, layer-by-layer, in order to bind to cells. The choice of these chemical layers, in particular the NP to antibody linker, along with the incubation period and type of media used in the incubation, has a strong influence on the specific binding abilities of the NPs. In this work, NPs have been shown to selectively bind to the MCF-7 cell line by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane by conjugating anti-EpCAM antibody to the NP surface. Results have shown a high signal to noise ratio for this cell line in comparison to a HeLa control line. NP attachment to cells was verified qualitatively with the use of fluorescence microscopy and quantitatively using image analysis methods. Once the system has been optimised, other dyes will be doped into the silica NPs and their use in multiplexing will be investigated.

  14. Upconversion fluorescent nanoparticles as a potential tool for in-depth imaging

    International Nuclear Information System (INIS)

    Upconversion nanoparticles (UCNs) are nanoparticles that are excited in the near infrared (NIR) region with emission in the visible or NIR regions. This makes these particles attractive for use in biological imaging as the NIR light can penetrate the tissue better with minimal absorption/scattering. This paper discusses the study of the depth to which cells can be imaged using these nanoparticles. UCNs with NaYF4 nanocrystals doped with Yb3+, Er3+ (visible emission)/Yb3+, Tm3+ (NIR emission) were synthesized and modified with silica enabling their dispersion in water and conjugation of biomolecules to their surface. The size of the sample was characterized using transmission electron microscopy and the fluorescence measured using a fluorescence spectrometer at an excitation of 980 nm. Tissue phantoms were prepared by reported methods to mimic skin/muscle tissue and it was observed that the cells could be imaged up to a depth of 3 mm using the NIR emitting UCNs. Further, the depth of detection was evaluated for UCNs targeted to gap junctions formed between cardiac cells.

  15. Visualization of Polarized Membrane Type 1 Matrix Metalloproteinase Activity in Live Cells by Fluorescence Resonance Energy Transfer Imaging*S⃞

    OpenAIRE

    Ouyang, Mingxing; Lu, Shaoying; Li, Xiao-Yan; Xu, Jing; Seong, Jihye; Giepmans, Ben N. G.; Shyy, John Y.-J.; Weiss, Stephen J.; Wang, Yingxiao

    2008-01-01

    Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a critical role in cancer cell biology by proteolytically remodeling the extracellular matrix. Utilizing fluorescence resonance energy transfer (FRET) imaging, we have developed a novel biosensor, with its sensing element anchoring at the extracellular surface of cell membrane, to visualize MT1-MMP activity dynamically in live cells with subcellular resolution. Epidermal growth factor (EGF) induced significant FR...

  16. Near-infrared (NIR) fluorescence imaging of head and neck squamous cell carcinoma for fluorescence-guided surgery (Conference Presentation)

    Science.gov (United States)

    Moore, Lindsay; Warram, Jason M.; de Boer, Esther; Carroll, William R.; Morlandt, Anthony; Withrow, Kirk P.; Rosenthal, Eben L.

    2016-03-01

    During fluorescence-guided surgery, a cancer-specific optical probe is injected and visualized using a compatible device intraoperatively to provide visual contrast between diseased and normal tissues to maximize resection of cancer and minimize the resection of precious adjacent normal tissues. Six patients with squamous cell carcinomas of the head and neck region (oral cavity (n=4) or cutaneous (n=2)) were injected with an EGFR-targeting antibody (Cetuximab) conjugated to a near-infrared (NIR) fluorescent dye (IRDye800) 3, 4, or 7 days prior to surgical resection of the cancer. Each patient's tumor was then imaged using a commercially available, open-field NIR fluorescence imaging device each day prior to surgery, intraoperatively, and post-operatively. The mean fluorescence intensity (MFI) of the tumor was calculated for each specimen at each imaging time point. Adjacent normal tissue served as an internal anatomic control for each patient to establish a patient-matched "background" fluorescence. Resected tissues were also imaged using a closed-field NIR imaging device. Tumor to background ratios (TBRs) were calculated for each patient using both devices. Fluorescence histology was correlated with traditional pathology assessment to verify the specificity of antibody-dye conjugate binding. Peak TBRs using the open-field device ranged from 2.2 to 11.3, with an average TBR of 4.9. Peak TBRs were achieved between days 1 and 4. This study demonstrated that a commercially available NIR imaging device suited for intraoperative and clinical use can successfully be used with a fluorescently-labeled dye to delineate between diseased and normal tissue in this single cohort human study, illuminated the potential for its use in fluoresence-guided surgery.

  17. Time-Domain Fluorescence Lifetime Imaging Techniques Suitable for Solid-State Imaging Sensor Arrays

    Directory of Open Access Journals (Sweden)

    Robert K. Henderson

    2012-05-01

    Full Text Available We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD-based cameras for fluorescence lifetime imaging microscopy (FLIM by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 μm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast.

  18. A 3-D fluorescence imaging system incorporating structured illumination technology

    Science.gov (United States)

    Antos, L.; Emord, P.; Luquette, B.; McGee, B.; Nguyen, D.; Phipps, A.; Phillips, D.; Helguera, M.

    2010-02-01

    A currently available 2-D high-resolution, optical molecular imaging system was modified by the addition of a structured illumination source, OptigridTM, to investigate the feasibility of providing depth resolution along the optical axis. The modification involved the insertion of the OptigridTM and a lens in the path between the light source and the image plane, as well as control and signal processing software. Projection of the OptigridTM onto the imaging surface at an angle, was resolved applying the Scheimpflug principle. The illumination system implements modulation of the light source and provides a framework for capturing depth resolved mages. The system is capable of in-focus projection of the OptigridTM at different spatial frequencies, and supports the use of different lenses. A calibration process was developed for the system to achieve consistent phase shifts of the OptigridTM. Post-processing extracted depth information using depth modulation analysis using a phantom block with fluorescent sheets at different depths. An important aspect of this effort was that it was carried out by a multidisciplinary team of engineering and science students as part of a capstone senior design program. The disciplines represented are mechanical engineering, electrical engineering and imaging science. The project was sponsored by a financial grant from New York State with equipment support from two industrial concerns. The students were provided with a basic imaging concept and charged with developing, implementing, testing and validating a feasible proof-of-concept prototype system that was returned to the originator of the concept for further evaluation and characterization.

  19. Facile and green approach to prepare fluorescent carbon dots: Emergent nanomaterial for cell imaging and detection of vitamin B2.

    Science.gov (United States)

    Kundu, Aniruddha; Nandi, Sudipta; Das, Pradip; Nandi, Arun K

    2016-04-15

    Carbon dots (CDs) are a new representative in carbonaceous family and have initiated remarkable research interests over the past one decade in a large variety of fields. Herein, we have utilized a facile, one-step carbonization method to prepare fluorescent carbon dots using poly(vinyl alcohol) (PVA) both as a carbon source and as a surface passivating agent. The as prepared CDs emit bright blue fluorescence under ultraviolet illumination. The structure and optical properties of the CDs are thoroughly investigated by several methods such as high-resolution transmission electron microscopy; dynamic light scattering; UV-vis, fluorescence and Fourier transform infrared spectroscopy. The CDs exhibit excellent water solubility and demonstrate average hydrodynamic diameter of 11.3 nm, holding great promise for biological applications. The biocompatibility evaluation and in vitro imaging study reveals that the synthesized CDs can be used as effective fluorescent probes in bio-imaging without noticeable cytotoxicity. In addition, a unique sensor for the detection of vitamin B2 in aqueous solution is proposed on the basis of spontaneous fluorescence resonance energy transfer from CD to vitamin B2. These findings therefore suggest that the CDs can find potential applications in cellular imaging along with sensing of vitamin B2. PMID:26852351

  20. Inspection of fecal contamination on strawberries using fluorescence imaging

    Science.gov (United States)

    Chuang, Yung-Kun; Yang, Chun-Chieh; Kim, Moon S.; Delwiche, Stephen R.; Lo, Y. Martin; Chen, Suming; Chan, Diane E.

    2013-05-01

    Fecal contamination of produce is a food safety issue associated with pathogens such as Escherichia coli that can easily pollute agricultural products via animal and human fecal matters. Outbreaks of foodborne illnesses associated with consuming raw fruits and vegetables have occurred more frequently in recent years in the United States. Among fruits, strawberry is one high-potential vector of fecal contamination and foodborne illnesses since the fruit is often consumed raw and with minimal processing. In the present study, line-scan LED-induced fluorescence imaging techniques were applied for inspection of fecal material on strawberries, and the spectral characteristics and specific wavebands of strawberries were determined by detection algorithms. The results would improve the safety and quality of produce consumed by the public.

  1. Lateral width of shower image in the Auger fluorescence detector

    CERN Document Server

    Barbosa, H; Dobrigkeit, C; Engel, R; Gora, D; Heck, D; Homola, P; Klages, H; Medina-Tanco, G; Ortiz, J A; Pekala, J; Risse, M; Wilczynska, B; Wilczynski, H

    2005-01-01

    The impact of the lateral distribution of light in extensive air showers on the detection and reconstruction of shower profiles is investigated for the Auger fluorescence telescopes. Based on three-dimensional simulations, the capability of the Auger telescopes to measure the lateral distribution of light is evaluated. The ability to infer the actual lateral distribution is confirmed by the comparison of detailed simulations with real data. The contribution of pixels located far from the axis of the shower image is calculated and the accepted signal is rescaled in order to reconstruct a correct shower profile. The analysis presented here shows that: (a) the Auger telescopes are able to observe the lateral distribution of showers and (b) the energy corrections to account for the signal in outlying pixels can exceed 10%, depending on shower geometry.

  2. A Biologically Inspired CMOS Image Sensor

    CERN Document Server

    Sarkar, Mukul

    2013-01-01

    Biological systems are a source of inspiration in the development of small autonomous sensor nodes. The two major types of optical vision systems found in nature are the single aperture human eye and the compound eye of insects. The latter are among the most compact and smallest vision sensors. The eye is a compound of individual lenses with their own photoreceptor arrays.  The visual system of insects allows them to fly with a limited intelligence and brain processing power. A CMOS image sensor replicating the perception of vision in insects is discussed and designed in this book for industrial (machine vision) and medical applications. The CMOS metal layer is used to create an embedded micro-polarizer able to sense polarization information. This polarization information is shown to be useful in applications like real time material classification and autonomous agent navigation. Further the sensor is equipped with in pixel analog and digital memories which allow variation of the dynamic range and in-pixel b...

  3. Two-photon fluorescent sensor for K+ imaging in live cells (Conference Presentation)

    Science.gov (United States)

    Sui, Binglin; Yue, Xiling; Kim, Bosung; Belfield, Kevin D.

    2016-03-01

    It is difficult to overstate the physiological importance of potassium for life as its indispensable roles in a variety of biological processes are widely known. As a result, efficient methods for determining physiological levels of potassium are of paramount importance. Despite this, relatively few K+ fluorescence sensors have been reported, with only one being commercially available. A new two-photon excited fluorescent K+ sensor is reported. The sensor is comprised of three moieties, a highly selective K+ chelator as the K+ recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K+ sensing in living cells.

  4. FITC Doped Rattle-Type Silica Colloidal Particle-Based Ratiometric Fluorescent Sensor for Biosensing and Imaging of Superoxide Anion.

    Science.gov (United States)

    Zhou, Ying; Ding, Jie; Liang, Tingxizi; Abdel-Halim, E S; Jiang, Liping; Zhu, Jun-Jie

    2016-03-16

    Fluorescent nanosensors have been widely applied in recognition and imaging of bioactive small molecules; however, the complicated surface modification process and background interference limit their applications in practical biological samples. Here, a simple, universal method was developed for ratiometric fluorescent determination of general small molecules. Taking superoxide anion (O2(•-)) as an example, the designed sensor was composed of three main moieties: probe carrier, rattle-type silica colloidal particles (mSiO2@hmSiO2 NPs); reference fluorophore doped into the core of NPs, fluorescein isothiocyanate (FITC); fluorescent probe for superoxide anion, hydroethidine (HE). In the absence of O2(•-), the sensor just emitted green fluorescence of FITC at 518 nm. When released HE was oxidized by O2(•-), the oxidation product exhibited red fluorescence at 570 nm and the intensity was linearly associated with the concentration of O2(•-), while that of reference element remained constant. Accordingly, ratiometric determination of O2(•-) was sensitively and selectively achieved with a linear range of 0.2-20 μM, and the detection limit was calculated as low as 80 nM. Besides, the technique was also successfully applied for dual-emission imaging of O2(•-) in live cells and realized visual recognition with obvious fluorescence color change in normal conditions or under oxidative stress. As long as appropriate reference dyes and sensing probes are selected, ratiometric biosensing and imaging of bioactive small molecules would be achieved. Therefore, the design could provide a simple, accurate, universal platform for biological applications. PMID:26910878

  5. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

    Science.gov (United States)

    Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

    2009-09-01

    Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

  6. Autofluorescence of atmospheric bioaerosols - fluorescent biomolecules, biological standard particles and potential interferences

    Science.gov (United States)

    Pöhlker, C.; Huffmann, J. A.; Pöschl, U.

    2012-04-01

    Primary biological aerosol particles (PBAP) such as pollen, fungal spores, bacteria, biogenic polymers and debris from larger organisms are known to influence atmospheric chemistry and physics, the biosphere and public health. PBAP account for up to ~30% of fine and up to ~70% of coarse particulate matter in urban, rural and pristine environment and are released with estimated emission rates of up to ~1000 Tg/a [1]. Continuous measurements of the abundance, variability and diversity of PBAP have been difficult until recently, however. The application of on-line instruments able to detect autofluorescence from biological particles in real-time has been a promising development for the measurement of PBAP concentrations and fluxes in different environments [2,3]. The detected fluorescent biological aerosol particles (FBAP) can be regarded as a subset of PBAP, although the exact relationship between PBAP and FBAP is still being investigated. Autofluorescence of FBAP is usually a superposition of fluorescence from a mixture of individual fluorescent molecules (fluorophores). Numerous biogenic fluorophores such as amino acids (e.g., tryptophan, tyrosine), coenzymes (e.g., NAD(P)H, riboflavin) and biopolymers (e.g., cellulose) emit fluorescent light due to heterocyclic aromatic rings or conjugated double bonds within their molecular structures. The tryptophan emission peak is a common feature of most bioparticles because the amino acid is a constituent of many proteins and peptides. The influence of the coenzymes NAD(P)H and riboflavin on the autofluorescence of bacteria can be regarded as an indicator for bacterial metabolism and has been utilized to discriminate between viable and non-viable organisms [4]. However, very little information is available about other essential biofluorophores in fungal spores and pollen. In order to better understand the autofluorescence behavior of FBAP, we have used fluorescence spectroscopy and fluorescence microscopy to analyze standard

  7. Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

    NARCIS (Netherlands)

    Uzunbajakava, Natalia; Otto, Cees

    2003-01-01

    We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Ram

  8. In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue.

    Science.gov (United States)

    Kantelhardt, Sven R; Kalasauskas, Darius; König, Karsten; Kim, Ella; Weinigel, Martin; Uchugonova, Aisada; Giese, Alf

    2016-05-01

    High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining. PMID:26830089

  9. An image analysis system for near-infrared (NIR) fluorescence lymph imaging

    Science.gov (United States)

    Zhang, Jingdan; Zhou, Shaohua Kevin; Xiang, Xiaoyan; Rasmussen, John C.; Sevick-Muraca, Eva M.

    2011-03-01

    Quantitative analysis of lymphatic function is crucial for understanding the lymphatic system and diagnosing the associated diseases. Recently, a near-infrared (NIR) fluorescence imaging system is developed for real-time imaging lymphatic propulsion by intradermal injection of microdose of a NIR fluorophore distal to the lymphatics of interest. However, the previous analysis software3, 4 is underdeveloped, requiring extensive time and effort to analyze a NIR image sequence. In this paper, we develop a number of image processing techniques to automate the data analysis workflow, including an object tracking algorithm to stabilize the subject and remove the motion artifacts, an image representation named flow map to characterize lymphatic flow more reliably, and an automatic algorithm to compute lymph velocity and frequency of propulsion. By integrating all these techniques to a system, the analysis workflow significantly reduces the amount of required user interaction and improves the reliability of the measurement.

  10. Focused fluorescence excitation with time-reversed ultrasonically encoded light and imaging in thick scattering media

    International Nuclear Information System (INIS)

    Scattering dominates light propagation in biological tissue, and therefore restricts both resolution and penetration depth in optical imaging within thick tissue. As photons travel into the diffusive regime, typically 1 mm beneath human skin, their trajectories transition from ballistic to diffusive due to the increased number of scattering events, which makes it impossible to focus, much less track, photon paths. Consequently, imaging methods that rely on controlled light illumination are ineffective in deep tissue. This problem has recently been addressed by a novel method capable of dynamically focusing light in thick scattering media via time reversal of ultrasonically encoded (TRUE) diffused light. Here, using photorefractive materials as phase conjugate mirrors, we show a direct visualization and dynamic control of optical focusing with this light delivery method, and demonstrate its application for focused fluorescence excitation and imaging in thick turbid media. These abilities are increasingly critical for understanding the dynamic interactions of light with biological matter and processes at different system levels, as well as their applications for biomedical diagnosis and therapy. (letter)

  11. Focused fluorescence excitation with time-reversed ultrasonically encoded light and imaging in thick scattering media

    Science.gov (United States)

    Lai, Puxiang; Suzuki, Yuta; Xu, Xiao; Wang, Lihong V.

    2013-07-01

    Scattering dominates light propagation in biological tissue, and therefore restricts both resolution and penetration depth in optical imaging within thick tissue. As photons travel into the diffusive regime, typically 1 mm beneath human skin, their trajectories transition from ballistic to diffusive due to the increased number of scattering events, which makes it impossible to focus, much less track, photon paths. Consequently, imaging methods that rely on controlled light illumination are ineffective in deep tissue. This problem has recently been addressed by a novel method capable of dynamically focusing light in thick scattering media via time reversal of ultrasonically encoded (TRUE) diffused light. Here, using photorefractive materials as phase conjugate mirrors, we show a direct visualization and dynamic control of optical focusing with this light delivery method, and demonstrate its application for focused fluorescence excitation and imaging in thick turbid media. These abilities are increasingly critical for understanding the dynamic interactions of light with biological matter and processes at different system levels, as well as their applications for biomedical diagnosis and therapy.

  12. Development of a rapid method for the automatic classification of biological agents' fluorescence spectral signatures

    Science.gov (United States)

    Carestia, Mariachiara; Pizzoferrato, Roberto; Gelfusa, Michela; Cenciarelli, Orlando; Ludovici, Gian Marco; Gabriele, Jessica; Malizia, Andrea; Murari, Andrea; Vega, Jesus; Gaudio, Pasquale

    2015-11-01

    Biosecurity and biosafety are key concerns of modern society. Although nanomaterials are improving the capacities of point detectors, standoff detection still appears to be an open issue. Laser-induced fluorescence of biological agents (BAs) has proved to be one of the most promising optical techniques to achieve early standoff detection, but its strengths and weaknesses are still to be fully investigated. In particular, different BAs tend to have similar fluorescence spectra due to the ubiquity of biological endogenous fluorophores producing a signal in the UV range, making data analysis extremely challenging. The Universal Multi Event Locator (UMEL), a general method based on support vector regression, is commonly used to identify characteristic structures in arrays of data. In the first part of this work, we investigate fluorescence emission spectra of different simulants of BAs and apply UMEL for their automatic classification. In the second part of this work, we elaborate a strategy for the application of UMEL to the discrimination of different BAs' simulants spectra. Through this strategy, it has been possible to discriminate between these BAs' simulants despite the high similarity of their fluorescence spectra. These preliminary results support the use of SVR methods to classify BAs' spectral signatures.

  13. Phenotypic charactheristics of fluorescent pseudomonss, biological control agent of lincat disease of temanggung tobacco

    Directory of Open Access Journals (Sweden)

    NINING NURUL AZIZAH

    2007-04-01

    Full Text Available Fluorescent pseudomonass isolated from local plants-rishosphere in temanggung controlled lincat disease of tobacco. This report describe phenotypic charactheristics of the bacteria in order to be used as a base for the development of the bacteria as a biological control agent of lincat disease. Phenotypic charactheristics of six isolates of fluorescent Pseudomonass which controlled lincat disease in the field were determined in the laboratory of Plant Bacteriology, Faculty of Agriculture, Gadjah Mada University. Plant pathogenicity tests were conducted by hypersensitive reaction into tobacco leaf and inoculation to tobacco plants. Antagonism test between fluorescent Pseudomonass and other candidate of biological control agents were also conducted. The results indicated that the bacteria were rod shape, Gram negative, positive reaction in catalase and oxidase tests. Nitrate reduce to nitrite, arginine was hydrolysed, fluorescent pigment were produced on King’s B medium, levan formation positive and all bacteria denitrifiy. The bacteria used urea, tween 80 and amylum were not hydrolised, poly--hydroxybutyrate was not accumulated in the cells. Negative reactions were observed for lysine decarboxylation, indol production, VP/MR reaction, and gelatn liquefation. Some compounds could be used as solely carbon sources. All isolates grew on the medium containing 2% NaCl. The best pH for growth was 6-7 and all isolates grew at 20-41C. Negative result were obtained for hypersensitive reaction and pathogenicity tests.

  14. Imaging and the new biology: What's wrong with this picture?

    Science.gov (United States)

    Vannier, Michael W.

    2004-05-01

    The Human Genome has been defined, giving us one part of the equation that stems from the central dogma of molecular biology. Despite this awesome scientific achievement, the correspondence between genomics and imaging is weak, since we cannot predict an organism's phenotype from even perfect knowledge of its genetic complement. Biological knowledge comes in several forms, and the genome is perhaps the best known and most completely understood type. Imaging creates another form of biological information, providing the ability to study morphology, growth and development, metabolic processes, and diseases in vitro and in vivo at many levels of scale. The principal challenge in biomedical imaging for the future lies in the need to reconcile the data provided by one or multiple modalities with other forms of biological knowledge, most importantly the genome, proteome, physiome, and other "-ome's." To date, the imaging science community has not set a high priority on the unification of their results with genomics, proteomics, and physiological functions in most published work. Images are relatively isolated from other forms of biological data, impairing our ability to conceive and address many fundamental questions in research and clinical practice. This presentation will explain the challenge of biological knowledge integration in basic research and clinical applications from the standpoint of imaging and image processing. The impediments to progress, isolation of the imaging community, and mainstream of new and future biological science will be identified, so the critical and immediate need for change can be highlighted.

  15. Application of X-ray fluorescence analytical techniques in phytoremediation and plant biology studies

    Energy Technology Data Exchange (ETDEWEB)

    Necemer, Marijan [Jozef Stefan Institute, Jamova 39, 1000 Ljubljana (Slovenia)], E-mail: marijan.necemer@ijs.si; Kump, Peter; Scancar, Janez; Jacimovic, Radojko; Simcic, Jurij; Pelicon, Primoz; Budnar, Milos; Jeran, Zvonka [Jozef Stefan Institute, Jamova 39, 1000 Ljubljana (Slovenia); Pongrac, Paula; Regvar, Marjana; Vogel-Mikus, Katarina [Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, SI-1000 Ljubljana (Slovenia)

    2008-11-15

    Phytoremediation is an emerging technology that employs the use of higher plants for the clean-up of contaminated environments. Progress in the field is however handicapped by limited knowledge of the biological processes involved in plant metal uptake, translocation, tolerance and plant-microbe-soil interactions; therefore a better understanding of the basic biological mechanisms involved in plant/microbe/soil/contaminant interactions would allow further optimization of phytoremediation technologies. In view of the needs of global environmental protection, it is important that in phytoremediation and plant biology studies the analytical procedures for elemental determination in plant tissues and soil should be fast and cheap, with simple sample preparation, and of adequate accuracy and reproducibility. The aim of this study was therefore to present the main characteristics, sample preparation protocols and applications of X-ray fluorescence-based analytical techniques (energy dispersive X-ray fluorescence spectrometry-EDXRF, total reflection X-ray fluorescence spectrometry-TXRF and micro-proton induced X-ray emission-micro-PIXE). Element concentrations in plant leaves from metal polluted and non-polluted sites, as well as standard reference materials, were analyzed by the mentioned techniques, and additionally by instrumental neutron activation analysis (INAA) and atomic absorption spectrometry (AAS). The results were compared and critically evaluated in order to assess the performance and capability of X-ray fluorescence-based techniques in phytoremediation and plant biology studies. It is the EDXRF, which is recommended as suitable to be used in the analyses of a large number of samples, because it is multi-elemental, requires only simple preparation of sample material, and it is analytically comparable to the most frequently used instrumental chemical techniques. The TXRF is compatible to FAAS in sample preparation, but relative to AAS it is fast, sensitive and

  16. Application of X-ray fluorescence analytical techniques in phytoremediation and plant biology studies

    International Nuclear Information System (INIS)

    Phytoremediation is an emerging technology that employs the use of higher plants for the clean-up of contaminated environments. Progress in the field is however handicapped by limited knowledge of the biological processes involved in plant metal uptake, translocation, tolerance and plant-microbe-soil interactions; therefore a better understanding of the basic biological mechanisms involved in plant/microbe/soil/contaminant interactions would allow further optimization of phytoremediation technologies. In view of the needs of global environmental protection, it is important that in phytoremediation and plant biology studies the analytical procedures for elemental determination in plant tissues and soil should be fast and cheap, with simple sample preparation, and of adequate accuracy and reproducibility. The aim of this study was therefore to present the main characteristics, sample preparation protocols and applications of X-ray fluorescence-based analytical techniques (energy dispersive X-ray fluorescence spectrometry-EDXRF, total reflection X-ray fluorescence spectrometry-TXRF and micro-proton induced X-ray emission-micro-PIXE). Element concentrations in plant leaves from metal polluted and non-polluted sites, as well as standard reference materials, were analyzed by the mentioned techniques, and additionally by instrumental neutron activation analysis (INAA) and atomic absorption spectrometry (AAS). The results were compared and critically evaluated in order to assess the performance and capability of X-ray fluorescence-based techniques in phytoremediation and plant biology studies. It is the EDXRF, which is recommended as suitable to be used in the analyses of a large number of samples, because it is multi-elemental, requires only simple preparation of sample material, and it is analytically comparable to the most frequently used instrumental chemical techniques. The TXRF is compatible to FAAS in sample preparation, but relative to AAS it is fast, sensitive and

  17. Application of X-ray fluorescence analytical techniques in phytoremediation and plant biology studies

    Science.gov (United States)

    Nečemer, Marijan; Kump, Peter; Ščančar, Janez; Jaćimović, Radojko; Simčič, Jurij; Pelicon, Primož; Budnar, Miloš; Jeran, Zvonka; Pongrac, Paula; Regvar, Marjana; Vogel-Mikuš, Katarina

    2008-11-01

    Phytoremediation is an emerging technology that employs the use of higher plants for the clean-up of contaminated environments. Progress in the field is however handicapped by limited knowledge of the biological processes involved in plant metal uptake, translocation, tolerance and plant-microbe-soil interactions; therefore a better understanding of the basic biological mechanisms involved in plant/microbe/soil/contaminant interactions would allow further optimization of phytoremediation technologies. In view of the needs of global environmental protection, it is important that in phytoremediation and plant biology studies the analytical procedures for elemental determination in plant tissues and soil should be fast and cheap, with simple sample preparation, and of adequate accuracy and reproducibility. The aim of this study was therefore to present the main characteristics, sample preparation protocols and applications of X-ray fluorescence-based analytical techniques (energy dispersive X-ray fluorescence spectrometry—EDXRF, total reflection X-ray fluorescence spectrometry—TXRF and micro-proton induced X-ray emission—micro-PIXE). Element concentrations in plant leaves from metal polluted and non-polluted sites, as well as standard reference materials, were analyzed by the mentioned techniques, and additionally by instrumental neutron activation analysis (INAA) and atomic absorption spectrometry (AAS). The results were compared and critically evaluated in order to assess the performance and capability of X-ray fluorescence-based techniques in phytoremediation and plant biology studies. It is the EDXRF, which is recommended as suitable to be used in the analyses of a large number of samples, because it is multi-elemental, requires only simple preparation of sample material, and it is analytically comparable to the most frequently used instrumental chemical techniques. The TXRF is compatible to FAAS in sample preparation, but relative to AAS it is fast

  18. Biological synthesis of fluorescent nanoparticles by cadmium and tellurite resistant Antarctic bacteria: exploring novel natural nanofactories

    OpenAIRE

    Plaza, D. O.; Gallardo, C.; Straub, Y. D.; Bravo, D.; Pérez-Donoso, J. M.

    2016-01-01

    Background Fluorescent nanoparticles or quantum dots (QDs) have been intensely studied for basic and applied research due to their unique size-dependent properties. There is an increasing interest in developing ecofriendly methods to synthesize these nanoparticles since they improve biocompatibility and avoid the generation of toxic byproducts. The use of biological systems, particularly prokaryotes, has emerged as a promising alternative. Recent studies indicate that QDs biosynthesis is rela...

  19. Diversity oriented fluorescence library approach (DOFLA) for live cell imaging probe development.

    Science.gov (United States)

    Yun, Seong-Wook; Kang, Nam-Young; Park, Sung-Jin; Ha, Hyung-Ho; Kim, Yun Kyung; Lee, Jun-Seok; Chang, Young-Tae

    2014-04-15

    A cell is the smallest functional unit of life. All forms of life rely on cellular processes to maintain normal functions, and changes in cell function induced by metabolic disturbances, physicochemical damage, infection, or abnormal gene expression may cause disease. To understand basic biology and to develop therapeutics for diseases, researchers need to study live cells. Along with advances in fluorescence microscopy and in vitro cell culture, live-cell imaging has become an essential tool in modern biology for the study of molecular and cellular events. Although researchers have often used fluorescent proteins to visualize cell-type-specific markers, this method requires genetic manipulations, which may not be appropriate in nontransgenic cells. Immunodetection of cellular markers requires the use of xenogenic antibodies, which may not detect intracellular markers in live cells. One option for overcoming these problems is the use of fluorescent small molecules targeted to specific cell types, which can enter live cells and interact with molecules of interest. We have used combinatorial chemistry to develop a large number of fluorescent small molecules as new imaging probes even without prior information about the probes' binding targets and mechanism, a strategy that we call the diversity oriented fluorescence library approach (DOFLA). We have used DOFLA to produce novel sensors and probes that detect a variety of biological and chemical molecules in vivo as well as in vitro. In this Account, we describe a series of fluorescent small molecules developed using DOFLA that bind specifically to particular cell types. These molecules provide new ways to detect and isolate these cells. The fluorescent probes CDy1, CDg4, and CDb8 tag embryonic stem cells and induced pluripotent stem cells but not fibroblasts or germ-line cells. CDr3 binds to an intracellular neural stem cell marker, fatty acid binding protein 7, which allows researchers to separate neural stem cells

  20. Nanometer fluorescent hybrid silica particle as ultrasensitive and photostable biological labels.

    Science.gov (United States)

    Yang, Huang-Hao; Qu, Hui-Ying; Lin, Peng; Li, Shun-Hua; Ding, Ma-Tai; Xu, Jin-Gou

    2003-05-01

    Nanometer-sized fluorescent hybrid silica (NFHS) particles were prepared for use as sensitive and photostable fluorescent probes in biological staining and diagnostics. The first step of the synthesis involves the covalent modification of 3-aminopropyltrimethoxysilane with an organic fluorophore, such as fluorescein isothiocyanate, under N2 atmosphere for getting a fluorescent silica precursor. Then the NFHS particles, with a diameter of well below 40 nm, were prepared by controlled hydrolysis of the fluorescent silica precursor with tetramethoxysilane (TMOS) using the reverse micelle technique. The fluorophores are dispersed homogeneously in the silica network of the NFHS particles and well protected from the environmental oxygen. Furthermore, since the fluorophores are covalently bound to the silica network, there is no migration, aggregation and leakage of the fluorophores. In comparison with common single organic fluorophores, these particle probes are brighter, more stable against photobleaching and do not suffer from intermittent on/off light emission (blinking). We have used these newly developed NFHS particles as a fluorescent marker to label antibodies, using silica immobilization method, for the immunoassay of human alpha-fetoprotein (AFP). The detection limit of this method was down to 0.05 ng mL(-1) under our current experimental conditions. We think this material would attract much attention and be applied widely in biotechnology. PMID:12790198

  1. Fluorescence guided lymph node biopsy in large animals using direct image projection device

    Science.gov (United States)

    Ringhausen, Elizabeth; Wang, Tylon; Pitts, Jonathan; Akers, Walter J.

    2016-03-01

    The use of fluorescence imaging for aiding oncologic surgery is a fast growing field in biomedical imaging, revolutionizing open and minimally invasive surgery practices. We have designed, constructed, and tested a system for fluorescence image acquisition and direct display on the surgical field for fluorescence guided surgery. The system uses a near-infrared sensitive CMOS camera for image acquisition, a near-infra LED light source for excitation, and DLP digital projector for projection of fluorescence image data onto the operating field in real time. Instrument control was implemented in Matlab for image capture, processing of acquired data and alignment of image parameters with the projected pattern. Accuracy of alignment was evaluated statistically to demonstrate sensitivity to small objects and alignment throughout the imaging field. After verification of accurate alignment, feasibility for clinical application was demonstrated in large animal models of sentinel lymph node biopsy. Indocyanine green was injected subcutaneously in Yorkshire pigs at various locations to model sentinel lymph node biopsy in gynecologic cancers, head and neck cancer, and melanoma. Fluorescence was detected by the camera system during operations and projected onto the imaging field, accurately identifying tissues containing the fluorescent tracer at up to 15 frames per second. Fluorescence information was projected as binary green regions after thresholding and denoising raw intensity data. Promising results with this initial clinical scale prototype provided encouraging results for the feasibility of optical projection of acquired luminescence during open oncologic surgeries.

  2. Enhancing contrast and quantitation by spatial frequency domain fluorescence molecular imaging

    Science.gov (United States)

    Sun, Jessica; Hathi, Deep; Zhou, Haiying; Shokeen, Monica; Akers, Walter J.

    2016-03-01

    Optical imaging with fluorescent contrast agents is highly sensitive for molecular imaging but is limited in depth to a few centimeters below the skin. Planar fluorescence imaging with full-field, uniform illumination and scientific camera image capture provides a portable and robust configuration for real-time, sensitive fluorescence detection with scalable resolution, but is inherently surface weighted and therefore limited in depth to a few millimeters. At the NIR region (700-1000 nm), tissue absorption and autofluorescence are relatively reduced, increasing depth penetration and reducing background signal, respectively. Optical imaging resolution scales with depth, limiting microscopic resolution with multiphoton microscopy and optical coherence tomography to skin and peri-tumoral tissues are not uniform, varying in thickness and color, complicating subsurface fluorescence measurements. Diffuse optical imaging methods have been developed that better quantify optical signals relative to faster full-field planar reflectance imaging, but require long scan times, complex instrumentation, and reconstruction algorithms. Here we report a novel strategy for rapid measurement of subsurface fluorescence using structured light illumination to improve quantitation of deep-seated fluorescence molecular probe accumulation. This technique, in combination with highly specific, tumor-avid fluorescent molecular probes, will easily integrate noninvasive diagnostics for superficial cancers and fluorescence guided surgery.

  3. A Highly Sensitive Fluorescent Sensor for Palladium and Direct Imaging of Its Ecotoxicity in Living Model Organisms.

    Science.gov (United States)

    Liu, Fei; Du, Juan; Xu, Meiying; Sun, Guoping

    2016-01-01

    Rhodamine is an ideal platform for fluorescence probes owing to its spiro-lactam framework and excellent photochemical properties. Herein, a novel rhodamine-based palladium fluorescent chemosensor, Rd-Eb, showing a fast response time (3 min), high sensitivity for palladium species over other ions, and a low detection limit (1.91×10(-7)  m), was synthesized. It can act as an obvious colorimetric as well as a fluorescent "off/on" sensor for Pd(2+) . In addition, it is also an excellent sensor for in vivo imaging of Pd(2+) in zebra fish and Daphnia magna, illuminating the impact of palladium on organisms at different growth stages with respect to biological toxicology. PMID:26419633

  4. Conjugated Polymer Nanoparticles for Fluorescence Imaging and Sensing of Neurotransmitter Dopamine in Living Cells and the Brains of Zebrafish Larvae.

    Science.gov (United States)

    Qian, Cheng-Gen; Zhu, Sha; Feng, Pei-Jian; Chen, Yu-Lei; Yu, Ji-Cheng; Tang, Xin; Liu, Yun; Shen, Qun-Dong

    2015-08-26

    Nanoscale materials are now attracting a great deal of attention for biomedical applications. Conjugated polymer nanoparticles have remarkable photophysical properties that make them highly advantageous for biological fluorescence imaging. We report on conjugated polymer nanoparticles with phenylboronic acid tags on the surface for fluorescence detection of neurotransmitter dopamine in both living PC12 cells and brain of zebrafish larvae. The selective enrichment of dopamine and fluorescence signal amplification characteristics of the nanoparticles show rapid and high-sensitive probing such neurotransmitter with the detection limit of 38.8 nM, and minimum interference from other endogenous molecules. It demonstrates the potential of nanomaterials as a multifunctional nanoplatform for targeting, diagnosis, and therapy of dopamine-relative disease. PMID:26238670

  5. Fluorescence life-time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles.

    Science.gov (United States)

    Schwartz, Shmulik; Fixler, Dror; Popovtzer, Rachela; Shefi, Orit

    2015-11-01

    Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right). PMID:25755202

  6. Fluorescent screens and image processing for the APS linac test stand

    International Nuclear Information System (INIS)

    A fluorescent screen was used to monitor relative beam position and spot size of a 56-MeV electron beam in the linac test stand. A chromium doped alumina ceramic screen inserted into the beam was monitored by a video camera. The resulting image was captured using a frame grabber and stored into memory. Reconstruction and analysis of the stored image was performed using PV-WAVE. This paper will discuss the hardware and software implementation of the fluorescent screen and imaging system. Proposed improvements for the APS linac fluorescent screens and image processing will also be discussed

  7. Fluorescent screens and image processing for the APS linac test stand

    International Nuclear Information System (INIS)

    A fluorescent screen was used to monitor relative beam position and spot size of a 56-MeV electron beam in the linac test stand. A chromium doped alumina ceramic screen inserted into the beam was monitored by a video camera. The resulting image was captured using a frame grabber and stored into memory. Reconstruction and analysis of the stored image was performed using PV-WAVE. This paper will discuss the hardware and software implementation of the fluorescent screen and imaging system. Proposed improvements for the APS linac fluorescent screens and image

  8. Optical gating in fluorescence imaging through a tissue-like medium

    International Nuclear Information System (INIS)

    Full text: In this paper, Monte Carlo simulation of light propagation through turbid media is used to study fluorescence imaging under a confocal microscope. Optical gating methods, such as a pinhole gating method, have been investigated. The results reveal that the pinhole gating method is efficient in 1-photon fluorescence imaging, but is limited in 2-photon fluorescence imaging. The effect of other system parameters in a microscope such as the numerical aperture of an objective is also studied. The results show that an image with a higher numerical aperture objective gives a stronger signal while retains equal or higher resolution. Copyright (1999) Australian Optical Society

  9. Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system.

    Science.gov (United States)

    Luo, Yuan; Gelsinger-Austin, Paul J; Watson, Jonathan M; Barbastathis, George; Barton, Jennifer K; Kostuk, Raymond K

    2008-09-15

    A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral transmittance filtering properties that enable obtaining spatial-spectral information within an object. We demonstrate this imaging system's ability to obtain multiple depth-resolved fluorescence images simultaneously. PMID:18794943

  10. High-contrast fluorescence imaging in fixed and living cells using optimized optical switches.

    Directory of Open Access Journals (Sweden)

    Liangxing Wu

    Full Text Available We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID imaging microscopy. A triazole-substituted BIPS (TzBIPS is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP and a far-red absorbing merocyanine (MC state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C₁₂-TzBIPS is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.

  11. Analytic expression of fluorescence ratio detection correlates with depth in multi-spectral sub-surface imaging

    Energy Technology Data Exchange (ETDEWEB)

    Leblond, F; Ovanesyan, Z; Davis, S C; Valdes, P A; Hartov, A; Pogue, B W; Paulsen, K D [Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755 (United States); Kim, A; Wilson, B C [Division of Biophysics and Bio-imaging, Ontario Cancer Institute, 610 University Avenue, Toronto, ON M5B 2K3 (Canada); Roberts, D W, E-mail: Frederic.Leblond@dartmouth.edu [Section of Neurosurgery, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States)

    2011-11-07

    Here we derived analytical solutions to diffuse light transport in biological tissue based on spectral deformation of diffused near-infrared measurements. These solutions provide a closed-form mathematical expression which predicts that the depth of a fluorescent molecule distribution is linearly related to the logarithm of the ratio of fluorescence at two different wavelengths. The slope and intercept values of the equation depend on the intrinsic values of absorption and reduced scattering of tissue. This linear behavior occurs if the following two conditions are satisfied: the depth is beyond a few millimeters and the tissue is relatively homogeneous. We present experimental measurements acquired with a broad-beam non-contact multi-spectral fluorescence imaging system using a hemoglobin-containing diffusive phantom. Preliminary results confirm that a significant correlation exists between the predicted depth of a distribution of protoporphyrin IX molecules and the measured ratio of fluorescence at two different wavelengths. These results suggest that depth assessment of fluorescence contrast can be achieved in fluorescence-guided surgery to allow improved intra-operative delineation of tumor margins.

  12. Analytic expression of fluorescence ratio detection correlates with depth in multi-spectral sub-surface imaging

    International Nuclear Information System (INIS)

    Here we derived analytical solutions to diffuse light transport in biological tissue based on spectral deformation of diffused near-infrared measurements. These solutions provide a closed-form mathematical expression which predicts that the depth of a fluorescent molecule distribution is linearly related to the logarithm of the ratio of fluorescence at two different wavelengths. The slope and intercept values of the equation depend on the intrinsic values of absorption and reduced scattering of tissue. This linear behavior occurs if the following two conditions are satisfied: the depth is beyond a few millimeters and the tissue is relatively homogeneous. We present experimental measurements acquired with a broad-beam non-contact multi-spectral fluorescence imaging system using a hemoglobin-containing diffusive phantom. Preliminary results confirm that a significant correlation exists between the predicted depth of a distribution of protoporphyrin IX molecules and the measured ratio of fluorescence at two different wavelengths. These results suggest that depth assessment of fluorescence contrast can be achieved in fluorescence-guided surgery to allow improved intra-operative delineation of tumor margins.

  13. A Combined Light Sheet Fluorescence and Differential Interference Contrast Microscope for Live Imaging of Multicellular Specimens

    Science.gov (United States)

    Baker, Ryan; Taormina, Michael; Jemielita, Matthew; Parthasarathy, Raghuveer

    2015-03-01

    We present a microscope capable of both light sheet fluorescence microscopy (LSFM) and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: LSFM provides high speed three-dimensional imaging of fluorescently labeled components of multicellular systems, large fields of view, and low phototoxicity, while DICM reveals the unlabeled neighborhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a shared detection path for both imaging modes enables simple integration of the two techniques in one microscope. To demonstrate the instrument's utility, we provide several examples which focus on the digestive tract of the larval zebrafish. We show that DICM can sometimes circumvent the need for fluorescent based techniques, augmenting the number of parameters obtainable per experiment when used alongside LSFM, and that DICM can be used to augment each experiment by imaging complementary features, such as non-fluorescent local environments near fluorescent samples (e.g. fluorescent enteric neurons imaged alongside the non-fluorescent gut wall), interactions between fluorescent and non-fluorescent samples (e.g. bacteria), and more. NSF Award 0922951, NIH Award 1P50 GM098911

  14. Iodinated oil-loaded, fluorescent mesoporous silica-coated iron oxide nanoparticles for magnetic resonance imaging/computed tomography/fluorescence trimodal imaging.

    Science.gov (United States)

    Xue, Sihan; Wang, Yao; Wang, Mengxing; Zhang, Lu; Du, Xiaoxia; Gu, Hongchen; Zhang, Chunfu

    2014-01-01

    In this study, a novel magnetic resonance imaging (MRI)/computed tomography (CT)/fluorescence trifunctional probe was prepared by loading iodinated oil into fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (i-fmSiO4@SPIONs). Fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (fmSiO4@SPIONs) were prepared by growing fluorescent dye-doped silica onto superparamagnetic iron oxide nanoparticles (SPIONs) directed by a cetyltrimethylammonium bromide template. As prepared, fmSiO4@SPIONs had a uniform size, a large surface area, and a large pore volume, which demonstrated high efficiency for iodinated oil loading. Iodinated oil loading did not change the sizes of fmSiO4@SPIONs, but they reduced the MRI T2 relaxivity (r2) markedly. I-fmSiO4@SPIONs were stable in their physical condition and did not demonstrate cytotoxic effects under the conditions investigated. In vitro studies indicated that the contrast enhancement of MRI and CT, and the fluorescence signal intensity of i-fmSiO4@SPION aqueous suspensions and macrophages, were intensified with increased i-fmSiO4@SPION concentrations in suspension and cell culture media. Moreover, for the in vivo study, the accumulation of i-fmSiO4@SPIONs in the liver could also be detected by MRI, CT, and fluorescence imaging. Our study demonstrated that i-fmSiO4@SPIONs had great potential for MRI/CT/fluorescence trimodal imaging. PMID:24904212

  15. Biological imaging in radiation therapy: role of positron emission tomography

    International Nuclear Information System (INIS)

    In radiation therapy (RT), staging, treatment planning, monitoring and evaluation of response are traditionally based on computed tomography (CT) and magnetic resonance imaging (MRI). These radiological investigations have the significant advantage to show the anatomy with a high resolution, being also called anatomical imaging. In recent years, so called biological imaging methods which visualize metabolic pathways have been developed. These methods offer complementary imaging of various aspects of tumour biology. To date, the most prominent biological imaging system in use is positron emission tomography (PET), whose diagnostic properties have clinically been evaluated for years. The aim of this review is to discuss the valences and implications of PET in RT. We will focus our evaluation on the following topics: the role of biological imaging for tumour tissue detection/delineation of the gross tumour volume (GTV) and for the visualization of heterogeneous tumour biology. We will discuss the role of fluorodeoxyglucose-PET in lung and head and neck cancer and the impact of amino acids (AA)-PET in target volume delineation of brain gliomas. Furthermore, we summarize the data of the literature about tumour hypoxia and proliferation visualized by PET. We conclude that, regarding treatment planning in radiotherapy, PET offers advantages in terms of tumour delineation and the description of biological processes. However, to define the real impact of biological imaging on clinical outcome after radiotherapy, further experimental, clinical and cost/benefit analyses are required. (topical review)

  16. Stable J-aggregation enabled dual photoacoustic and fluorescence nanoparticles for intraoperative cancer imaging

    Science.gov (United States)

    Shakiba, Mojdeh; Ng, Kenneth K.; Huynh, Elizabeth; Chan, Harley; Charron, Danielle M.; Chen, Juan; Muhanna, Nidal; Foster, F. Stuart; Wilson, Brian C.; Zheng, Gang

    2016-06-01

    J-aggregates display nanoscale optical properties which enable their use in fluorescence and photoacoustic imaging applications. However, control over their optical properties in an in vivo setting is hampered by the conformational lability of the J-aggregate structure in complex biological environments. J-aggregating nanoparticles (JNP) formed by self-assembly of bacteriopheophorbide-lipid (Bchl-lipid) in lipid nanovesicles represents a novel strategy to stabilize J-aggregates for in vivo bioimaging applications. We find that 15 mol% Bchl-lipid embedded within a saturated phospholipid bilayer vesicle was optimal in terms of maximizing Bchl-lipid dye loading, while maintaining a spherical nanoparticle morphology and retaining spectral properties characteristic of J-aggregates. The addition of cholesterol maintains the stability of the J-aggregate absorption band for up to 6 hours in the presence of 90% FBS. In a proof-of-concept experiment, we successfully applied JNPs as a fluorescence contrast agent for real-time intraoperative detection of metastatic lymph nodes in a rabbit head-and-neck cancer model. Lymph node metastasis delineation was further verified by visualizing the JNP within the excised lymph node using photoacoustic imaging. Using JNPs, we demonstrate the possibility of using J-aggregates as fluorescence and photoacoustic contrast agents and may potentially spur the development of other nanomaterials that can stably induce J-aggregation for in vivo cancer bioimaging applications.J-aggregates display nanoscale optical properties which enable their use in fluorescence and photoacoustic imaging applications. However, control over their optical properties in an in vivo setting is hampered by the conformational lability of the J-aggregate structure in complex biological environments. J-aggregating nanoparticles (JNP) formed by self-assembly of bacteriopheophorbide-lipid (Bchl-lipid) in lipid nanovesicles represents a novel strategy to stabilize J

  17. Precise diagnosis in different scenarios using photoacoustic and fluorescence imaging with dual-modality nanoparticles

    Science.gov (United States)

    Peng, Dong; Du, Yang; Shi, Yiwen; Mao, Duo; Jia, Xiaohua; Li, Hui; Zhu, Yukun; Wang, Kun; Tian, Jie

    2016-07-01

    Photoacoustic imaging and fluorescence molecular imaging are emerging as important research tools for biomedical studies. Photoacoustic imaging offers both strong optical absorption contrast and high ultrasonic resolution, and fluorescence molecular imaging provides excellent superficial resolution, high sensitivity, high throughput, and the ability for real-time imaging. Therefore, combining the imaging information of both modalities can provide comprehensive in vivo physiological and pathological information. However, currently there are limited probes available that can realize both fluorescence and photoacoustic imaging, and advanced biomedical applications for applying this dual-modality imaging approach remain underexplored. In this study, we developed a dual-modality photoacoustic-fluorescence imaging nanoprobe, ICG-loaded Au@SiO2, which was uniquely designed, consisting of gold nanorod cores and indocyanine green with silica shell spacer layers to overcome fluorophore quenching. This nanoprobe was examined by both PAI and FMI for in vivo imaging on tumor and ischemia mouse models. Our results demonstrated that the nanoparticles can specifically accumulate at the tumor and ischemic areas and be detected by both imaging modalities. Moreover, this dual-modality imaging strategy exhibited superior advantages for a precise diagnosis in different scenarios. The new nanoprobe with the dual-modality imaging approach holds great potential for diagnosis and stage classification of tumor and ischemia related diseases.Photoacoustic imaging and fluorescence molecular imaging are emerging as important research tools for biomedical studies. Photoacoustic imaging offers both strong optical absorption contrast and high ultrasonic resolution, and fluorescence molecular imaging provides excellent superficial resolution, high sensitivity, high throughput, and the ability for real-time imaging. Therefore, combining the imaging information of both modalities can provide

  18. Multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology.

    Science.gov (United States)

    Crane, Lucia M A; Themelis, George; Buddingh, K Tim; Buddingh, Tim; Harlaar, Niels J; Pleijhuis, Rick G; Sarantopoulos, Athanasios; van der Zee, Ate G J; Ntziachristos, Vasilis; van Dam, Gooitzen M

    2010-01-01

    The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases. Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema. As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor. In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix. The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid. Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion. It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used. The aim of this video article is to demonstrate the detection of

  19. Segmenting Intracellular Distribution Images Derived by Fluorescent Dyes Using a Potts Model Hamiltonian

    CERN Document Server

    Hu, Dandan; Ronhovde, Peter; Bloch, Sharon; Achilefu, Samuel; Nussinov, Zohar

    2012-01-01

    We apply a multiresolution community detection algorithm to perform unsupervised segmentation of complex intracellular signals derived using fluorescent dyes. In our earlier work, when applying our method to benchmarks, our algorithm was shown to be one of the best and to be especially suited to the detection of camouflage images. In the current manuscript, we have explored this algorithm in a more complex scenario. The current image processing problem is framed as identifying clusters with respective average fluorescent lifetimes (FLTs) against a background or "solvent" in fluorescence lifetime imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the proposed algorithm from different starting points. Our method is more efficient than a well-known image segmentation...

  20. Near-Infrared Fluorescence Enhanced (NIR-FE) Molecular Imaging of Live Cells on Gold Substrates

    CERN Document Server

    Hong, Guosong; Welsher, Kevin; Chen, Zhuo; Robinson, Joshua T; Wang, Hailiang; Zhang, Bo; Dai, Hongjie

    2011-01-01

    Low quantum yields of near infrared (NIR) fluorophores have limited their capabilities as imaging probes in a transparent, low background imaging window. Here for the first time we reported near-infrared fluorescence enhance (NIR-FE) cell imaging using nanostructured Au substrate, which was employed as a general platform for both single-walled carbon nanotubes (SWNTs) and organic fluorescent labels in the NIR region. Fluorescence intensity, as well as cell targeting specificity, was greatly improved by this novel imaging technique. With NIR-FE imaging, we were able to image SWNT-stained cells at short exposure time of 300ms, and push the detectable limit of SWNT staining of cells down to an ultralow concentration of ~50 pM. Further, different degrees of fluorescence enhancement for endocytosed, intracellular SWNTs vs. nanotubes on the cell membrane at the cell/gold interface were observed, suggesting the possibility of using this technique to track the transmembrane behavior of NIR fluorophores.

  1. Low Light CMOS Contact Imager with an Integrated Poly-Acrylic Emission Filter for Fluorescence Detection

    Directory of Open Access Journals (Sweden)

    Yonathan Dattner

    2010-05-01

    Full Text Available This study presents the fabrication of a low cost poly-acrylic acid (PAA based emission filter integrated with a low light CMOS contact imager for fluorescence detection. The process involves the use of PAA as an adhesive for the emission filter. The poly-acrylic solution was chosen due its optical transparent properties, adhesive properties, miscibility with polar protic solvents and most importantly its bio-compatibility with a biological environment. The emission filter, also known as an absorption filter, involves dissolving an absorbing specimen in a polar protic solvent and mixing it with the PAA to uniformly bond the absorbing specimen and harden the filter. The PAA is optically transparent in solid form and therefore does not contribute to the absorbance of light in the visible spectrum. Many combinations of absorbing specimen and polar protic solvents can be derived, yielding different filter characteristics in different parts of the spectrum. We report a specific combination as a first example of implementation of our technology. The filter reported has excitation in the green spectrum and emission in the red spectrum, utilizing the increased quantum efficiency of the photo sensitive sensor array. The thickness of the filter (20 μm was chosen by calculating the desired SNR using Beer-Lambert’s law for liquids, Quantum Yield of the fluorophore and the Quantum Efficiency of the sensor array. The filters promising characteristics make it suitable for low light fluorescence detection. The filter was integrated with a fully functional low noise, low light CMOS contact imager and experimental results using fluorescence polystyrene micro-spheres are presented.

  2. Deployment of a Fully-Automated Green Fluorescent Protein Imaging System in a High Arctic Autonomous Greenhouse

    Directory of Open Access Journals (Sweden)

    Alain Berinstain

    2013-03-01

    Full Text Available Higher plants are an integral part of strategies for sustained human presence in space. Space-based greenhouses have the potential to provide closed-loop recycling of oxygen, water and food. Plant monitoring systems with the capacity to remotely observe the condition of crops in real-time within these systems would permit operators to take immediate action to ensure optimum system yield and reliability. One such plant health monitoring technique involves the use of reporter genes driving fluorescent proteins as biological sensors of plant stress. In 2006 an initial prototype green fluorescent protein imager system was deployed at the Arthur Clarke Mars Greenhouse located in the Canadian High Arctic. This prototype demonstrated the advantageous of this biosensor technology and underscored the challenges in collecting and managing telemetric data from exigent environments. We present here the design and deployment of a second prototype imaging system deployed within and connected to the infrastructure of the Arthur Clarke Mars Greenhouse. This is the first imager to run autonomously for one year in the un-crewed greenhouse with command and control conducted through the greenhouse satellite control system. Images were saved locally in high resolution and sent telemetrically in low resolution. Imager hardware is described, including the custom designed LED growth light and fluorescent excitation light boards, filters, data acquisition and control system, and basic sensing and environmental control. Several critical lessons learned related to the hardware of small plant growth payloads are also elaborated.

  3. Preparation and Characterization of Highly Fluorescent, Glutathione-coated Near Infrared Quantum Dots for in Vivo Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Yoshichika Yoshioka

    2008-10-01

    Full Text Available Fluorescent probes that emit in the near-infrared (NIR, 700-1,300 nm region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4. The GSH-QDs (800 nm emission were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer, and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM, the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIRfluorescence imaging of a lymph node in a mouse is presented.

  4. Fluorescence imaging under background light with a self-reset complementary metal–oxide–semiconductor image sensor

    Directory of Open Access Journals (Sweden)

    Takahiro Yamaguchi

    2015-11-01

    Full Text Available The authors propose and demonstrate the fluorescence imaging of green fluorescence protein (GFP expressed in a brain slice with a self-reset complementary metal–oxide–semiconductor image sensor under background light. By using a self-reset function to avoid pixel saturation, the weak fluorescence of GFP was successfully observed, even under background light. The result suggests that the sensor can be applied to in vivo imaging of laboratory animals during light–dark cycles, so that they can observe the different responses to bright and dark environments.

  5. Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.

    Science.gov (United States)

    Burnette, Dylan T; Sengupta, Prabuddha; Dai, Yuhai; Lippincott-Schwartz, Jennifer; Kachar, Bechara

    2011-12-27

    Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes. PMID:22167805

  6. Multimodal fluorescence mediated tomography and SPECT/CT for small animals imaging

    OpenAIRE

    Solomon, Metasebya; Nothdruft, Ralph E.; Akers, Walter; Edwards, W. Barry; Liang, Kexian; Xu, Baogang; Suddlow, Gail P.; Deghani, Hamid; Tai, Yuan-Chuan; Eggebrecht, Adam T.; Achilefu, Samuel; Culver, Joseph P.

    2013-01-01

    Spatial and temporal co-registration of nuclear and optical images would enable the fusion of the information from theses complementary molecular imaging modalities. A critical challenge in integration is fitting optical hardware into the nuclear imaging platforms. Flexible fiber-based fluorescence mediated tomography (FMT) systems provide a viable solution because the various imaging bore sizes of small animal nuclear imaging systems can potentially accommodate the FMT fiber imaging arrays. ...

  7. New developments of X-ray fluorescence imaging techniques in laboratory

    Science.gov (United States)

    Tsuji, Kouichi; Matsuno, Tsuyoshi; Takimoto, Yuki; Yamanashi, Masaki; Kometani, Noritsugu; Sasaki, Yuji C.; Hasegawa, Takeshi; Kato, Shuichi; Yamada, Takashi; Shoji, Takashi; Kawahara, Naoki

    2015-11-01

    X-ray fluorescence (XRF) analysis is a well-established analytical technique with a long research history. Many applications have been reported in various fields, such as in the environmental, archeological, biological, and forensic sciences as well as in industry. This is because XRF has a unique advantage of being a nondestructive analytical tool with good precision for quantitative analysis. Recent advances in XRF analysis have been realized by the development of new x-ray optics and x-ray detectors. Advanced x-ray focusing optics enables the making of a micro x-ray beam, leading to micro-XRF analysis and XRF imaging. A confocal micro-XRF technique has been applied for the visualization of elemental distributions inside the samples. This technique was applied for liquid samples and for monitoring chemical reactions such as the metal corrosion of steel samples in the NaCl solutions. In addition, a principal component analysis was applied for reducing the background intensity in XRF spectra obtained during XRF mapping, leading to improved spatial resolution of confocal micro-XRF images. In parallel, the authors have proposed a wavelength dispersive XRF (WD-XRF) imaging spectrometer for a fast elemental imaging. A new two dimensional x-ray detector, the Pilatus detector was applied for WD-XRF imaging. Fast XRF imaging in 1 s or even less was demonstrated for Euro coins and industrial samples. In this review paper, these recent advances in laboratory-based XRF imaging, especially in a laboratory setting, will be introduced.

  8. Photoacoustic tomography imaging of biological tissues

    Science.gov (United States)

    Su, Yixiong; Wang, Ruikang K.; Xu, Kexin; Zhang, Fan; Yao, Jianquan

    2005-01-01

    Non-invasive laser-induced photoacoustic tomography is attracting more and more attentions in the biomedical optical imaging field. This imaging modality takes the advantages in that the tomography image has the optical contrast similar to the optical techniques while enjoying the high spatial resolution comparable to the ultrasound. Currently, its biomedical applications are mainly focused on breast cancer diagnosis and small animal imaging. In this paper, we report in detail a photoacoustic tomography experiment system constructed in our laboratory. In our system, a Q-switched ND:YAG pulse laser operated at 532nm with a 10ns pulse width is employed to generate photoacoustic signal. A tissue-mimicking phantom was built to test the system. When imaged, the phantom and detectors were immersed in a water tank to facilitate the acoustic detection. Based on filtered back-projection process of photoacoustic imaging, the two-dimension distribution of optical absorption in tissue phantom was reconstructed.

  9. Imaging of Flow Patterns with Fluorescent Molecular Rotors

    OpenAIRE

    Mustafic, Adnan; Huang, Hsuan-Ming; Theodorakis, Emmanuel A.; Haidekker, Mark A

    2010-01-01

    Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer states (TICT) upon photoexcitation. Some classes of molecular rotors, among them those that are built on the benzylidene malononitrile motif, return to the ground state either by nonradiative intramolecular rotation or by fluorescence emission. In low-viscosity solvents, intramolecular rotation dominates, and the fluorescence quantum yield is low. Higher solvent viscosities reduce the intram...

  10. Chamber bioaerosol study: human emissions of size-resolved fluorescent biological aerosol particles.

    Science.gov (United States)

    Bhangar, S; Adams, R I; Pasut, W; Huffman, J A; Arens, E A; Taylor, J W; Bruns, T D; Nazaroff, W W

    2016-04-01

    Humans are a prominent source of airborne biological particles in occupied indoor spaces, but few studies have quantified human bioaerosol emissions. The chamber investigation reported here employs a fluorescence-based technique to evaluate bioaerosols with high temporal and particle size resolution. In a 75-m(3) chamber, occupant emission rates of coarse (2.5-10 μm) fluorescent biological aerosol particles (FBAPs) under seated, simulated office-work conditions averaged 0.9 ± 0.3 million particles per person-h. Walking was associated with a 5-6× increase in the emission rate. During both walking and sitting, 60-70% or more of emissions originated from the floor. The increase in emissions during walking (vs. while sitting) was mainly attributable to release of particles from the floor; the associated increased vigor of upper body movements also contributed. Clothing, or its frictional interaction with human skin, was demonstrated to be a source of coarse particles, and especially of the highly fluorescent fraction. Emission rates of FBAPs previously reported for lecture classes were well bounded by the experimental results obtained in this chamber study. In both settings, the size distribution of occupant FBAP emissions had a dominant mode in the 3-5 μm diameter range. PMID:25704637

  11. Enhanced fluorescence imaging performance of hydrophobic colloidal ZnO nanoparticles by a facile method

    International Nuclear Information System (INIS)

    Highlights: • A dual phase hydrothermal method was developed to synthesize ZnO nanoparticles. • ZnO nanoparticles show a stability and solubility in the aqueous environment. • ZnO nanoparticles with a blue emission wavelength at around 420 nm and small size (30 nm). • ZnO nanoparticles as biological labeling agent was also shown. - Abstract: A facile synthesis method for the formation of ZnO nanoparticles by using a double-phase reaction was demonstrated in this paper. The morphology of the synthesized ZnO nanoparticles shows a flower-shape. Hydrogen peroxide was used as a unique oxygenic source to promote the formation of ZnO in the presence of organic zinc precursor. The as-synthesized ZnO nanoparticles also show a stability and solubility in the aqueous environment. The structure and properties of ZnO nanoparticles were investigated by the transmission electron microscopy (TEM) and X-ray diffraction (XRD) as well as UV–vis and photoluminescence spectroscopy. The as-prepared hydrophobic colloidal ZnO nanoparticles could be modified to become water-soluble via ligand exchange with amineothanethiol⋅HCl while retaining the photoluminescence properties. In addition, the potential application for biological label of water-soluble ZnO nanoparticles were also demonstrated. These results not only have applications towards using colloidal ZnO nanoparticles effectively in biological fluorescence imaging, but also promote its application in the field of targeted drug delivery

  12. A two-photon fluorescent probe with a large turn-on signal for imaging hydrogen sulfide in living tissues

    International Nuclear Information System (INIS)

    Highlights: • A two-photon fluorescent probe for sensing H2S was developed. • The probe shows a large turn on signal (120-fold enhancement). • The probe is suitable for fluorescence imaging of H2S in living cells and tissues. • The probe was capable of detecting H2S up to 170 μm depth in live tissues. - Abstract: A two-photon fluorescence turn-on H2S probe GCTPOC–H2S based on a two-photon platform with a large cross-section, GCTPOC, and a sensitive H2S recognition site, dinitrophenyl ether was constructed. The probe GCTPOC–H2S exhibits desirable properties such as high sensitivity, high selectivity, functioning well at physiological pH and low cytotoxicity. In particular, the probe shows a 120-fold enhancement in the presence of Na2S (500 μM), which is larger than the reported two-photon fluorescent H2S probes. The large fluorescence enhancement of the two-photon probe GCTPOC–H2S renders it attractive for imaging H2S in living tissues with deep tissue penetration. Significantly, we have demonstrated that the probe GCTPOC–H2S is suitable for fluorescence imaging of H2S in living tissues with deep penetration by using two-photon microscopy. The further application of the two-photon probe for the investigation of biological functions and pathological roles of H2S in living systems is under progress

  13. Performance evaluation of CCD- and mobile-phone-based near-infrared fluorescence imaging systems with molded and 3D-printed phantoms

    Science.gov (United States)

    Wang, Bohan; Ghassemi, Pejhman; Wang, Jianting; Wang, Quanzeng; Chen, Yu; Pfefer, Joshua

    2016-03-01

    Increasing numbers of devices are emerging which involve biophotonic imaging on a mobile platform. Therefore, effective test methods are needed to ensure that these devices provide a high level of image quality. We have developed novel phantoms for performance assessment of near infrared fluorescence (NIRF) imaging devices. Resin molding and 3D printing techniques were applied for phantom fabrication. Comparisons between two imaging approaches - a CCD-based scientific camera and an NIR-enabled mobile phone - were made based on evaluation of the contrast transfer function and penetration depth. Optical properties of the phantoms were evaluated, including absorption and scattering spectra and fluorescence excitation-emission matrices. The potential viability of contrastenhanced biological NIRF imaging with a mobile phone is demonstrated, and color-channel-specific variations in image quality are documented. Our results provide evidence of the utility of novel phantom-based test methods for quantifying image quality in emerging NIRF devices.

  14. Observations of fluorescent and biological aerosol at a high-altitude site in central France

    Directory of Open Access Journals (Sweden)

    A. M. Gabey

    2013-08-01

    Full Text Available Total bacteria, fungal spore and yeast counts were compared with ultraviolet-light-induced fluorescence (UV-LIF measurements of ambient aerosol at the summit of the Puy de Dôme (PdD mountain in central France (1465 m a.s.l, which represents a background elevated site. Bacteria, fungal spores and yeast were enumerated by epifluorescence microscopy (EFM and found to number 2.2 to 23 L−1 and 0.8 to 2 L−1, respectively. Bacteria counts on two successive nights were an order of magnitude larger than in the intervening day. A wide issue bioaerosol spectrometer, version 3 (WIBS-3 was used to perform UV-LIF measurements on ambient aerosol sized 0.8 to 20 μm. Mean total number concentration was 270 L−1 (σ = 66 L−1, found predominantly in a size mode at 2 μm for most of the campaign. Total concentration (fluorescent + non-fluorescent aerosol peaked at 500 L−1 with a size mode at 1 μm because of a change in air mass origin lasting around 48 h. The WIBS-3 features two excitation and fluorescence detection wavelengths corresponding to different biological molecules, although non-biological interferents also contribute. The mean fluorescent particle concentration after short-wave (280 nm; associated with tryptophan excitation was 12 L−1 (σ = 6 L−1, and did not vary much throughout the campaign. In contrast, the mean concentration of particles fluorescent after long-wave (370 nm; associated with NADH excitation was 95 L−1 (σ = 25 L−1, and a nightly rise and subsequent fall of up to 100 L−1 formed a strong diurnal cycle in the latter. The two fluorescent populations exhibited size modes at 3 μm and 2 to 3 μm, respectively. A hierarchical agglomerative cluster analysis algorithm was applied to the data and used to extract different particle factors. A cluster concentration time series representative of bacteria was identified. This was found to exhibit a diurnal cycle with a maximum peak appearing during the day. Analysis of organic

  15. Influence of angle's ranges for recording an X-ray fluorescence hologram on reconstructed atomic images

    Institute of Scientific and Technical Information of China (English)

    XIE Hong-Lan; CHEN Jian-Wen; GAO Hong-Yi; ZHU Hua-Feng; LI Ru-Xin; XU Zhi-Zhan

    2004-01-01

    X-ray fluorescence holography (XFH) is a novel method for three-dimensional (3D) imaging of atomic structure. Theoretically, in an XFH experiment, one has to measure the fluorescence energy on a spherical surface to get well-resolved 3D images of atoms. But in practice, the experimental system arrangement does not allow the measurement of the fluorescent intensity oscillations in the full sphere. The holographic information losses because of the limited sampling range (less than 4π) will directly result in defective reconstructed atomic images. In this work, the atomic image of a Fe single crystal (001) was reconstructed by numerically simulating X-ray fluorescence holograms of the crystal at different recording angle's ranges and step lengths. Influences of the ranges of azimuth angles and polar angles and the step length of polar angles on the reconstructed atomic images were discussed.

  16. Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging

    Science.gov (United States)

    Husvogt, Lennart; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Hornegger, Joachim; Connolly, James L.; Fujimoto, James G.

    2016-01-01

    We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems. PMID:27500636

  17. First X-ray fluorescence CT experimental results at the SSRF X-ray imaging beamline

    Institute of Scientific and Technical Information of China (English)

    DENG Biao; YANG Qun; XIE Hong-Lan; DU Guo-Hao; XIAO Wi-Qiao

    2011-01-01

    X-ray fluorescence CT is a non-destructive technique for detecting elemental composition and distribution inside a specimen. In this paper, the first experimental results of X-ray fluorescence CT obtained at the SSRF X-ray imaging beamline (BL13W1) are described. The test samples were investigated and the 2D elemental image was reconstructed using a filtered back-projection algorithm. In the sample the element Cd was observed. Up to now, the X-ray fluorescence CT could be carried out at the SSRF X-ray imaging beamline.

  18. Simplified and optimized multispectral imaging for 5-ALA-based fluorescence diagnosis of malignant lesions.

    Science.gov (United States)

    Minamikawa, Takeo; Matsuo, Hisataka; Kato, Yoshiyuki; Harada, Yoshinori; Otsuji, Eigo; Yanagisawa, Akio; Tanaka, Hideo; Takamatsu, Tetsuro

    2016-01-01

    5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is now clinically applied for accurate and ultrarapid diagnosis of malignant lesions such as lymph node metastasis during surgery. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA, protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue chromophores, such as collagen and flavins. In this study, we demonstrated PPIX fluorescence estimation with autofluorescence elimination for 5-ALA-based fluorescence diagnosis of malignant lesions by simplified and optimized multispectral imaging. We computationally optimized observation wavelength regions for the estimation of PPIX fluorescence in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen and flavins. By using the fluorescence intensities of the optimized wavelength regions, we verified quantitative detection of PPIX fluorescence by using chemical mixtures of PPIX, flavins, and collagen. Furthermore, we demonstrated detection capability by using metastatic and non-metastatic lymph nodes of colorectal cancer patients. These results suggest the potential and usefulness of the background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions, and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis. PMID:27149301

  19. Random laser in biological tissues impregnated with a fluorescent anticancer drug

    Science.gov (United States)

    Lahoz, F.; Martín, I. R.; Urgellés, M.; Marrero-Alonso, J.; Marín, R.; Saavedra, C. J.; Boto, A.; Díaz, M.

    2015-04-01

    We have demonstrated that chemically modified anticancer drugs can provide random laser (RL) when infiltrated in a biological tissue. A fluorescent biomarker has been covalently bound to tamoxifen, which is one of the most frequently used drugs for breast cancer therapy. The light emitted by the drug-dye composite is scattered in tissue, which acts as a gain medium. Both non-coherent and coherent RL regimes have been observed. Moreover, the analysis of power Fourier transforms of coherent RL spectra indicates that the tissues show a dominant random laser cavity length of about 18 µm, similar to the average size of single cells. These results show that RL could be obtained from other drugs, if properly marked with a fluorescent tag, which could be appealing for new forms of combined opto-chemical therapies.

  20. In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells

    OpenAIRE

    Vermaas, Wim F. J.; Timlin, Jerilyn A.; Jones, Howland D. T.; Sinclair, Michael B.; Nieman, Linda T.; Hamad, Sawsan W.; Melgaard, David K.; Haaland, David M.

    2008-01-01

    Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (≈0.03-μm3) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exc...

  1. Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

    Directory of Open Access Journals (Sweden)

    Yoko Hayashi-Takanaka

    Full Text Available To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph and acetylated H3K9 (H3K9ac. These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green, Cy3 (red, and Cy5 or CF640 (far-red.

  2. Lipophilic porphyrin microparticles induced by AOT reverse micelles: a fluorescence lifetime imaging study.

    Science.gov (United States)

    Togashi, Denisio M; Costa, Sílvia M B; Sobral, Abílio J F N

    2006-01-20

    Fluorescence Lifetime Imaging Microscopy (FLIM) technique was applied to investigate the fluorescence dynamics and structural features of large colloidal aggregates of meso-tetra(N-dodecyl-4-amino sulfonyl-phenyl)porphyrin (PC12) induced by Sodium 1,4-bis(2-ethyl hexyl)sulfosuccinate (AOT) reverse micelles. The aggregate's particle sizes (down to 1 microm) obtained from the confocal fluorescence images matched with the particle sizes measured in the images obtained from Scanning Electron Microscopy (SEM). The fluorescence decays for those aggregates in the micro spatial domain show triexponential fluorescence lifetimes (tau1 approximately 12 ns, tau2 approximately 3 ns and tau3 approximately 1 ns) which are independent of the aggregate's size. PMID:16154681

  3. Imaging of multi-color fluorescence emission from leaf tissues

    Czech Academy of Sciences Publication Activity Database

    Nedbal, Ladislav

    2-3, č. 102 (2009), s. 169-175. ISSN 0166-8595 Institutional research plan: CEZ:AV0Z60870520 Keywords : Chlorophyll fluorescence * Blue-green fluorescence * Pyridine nucleotide Subject RIV: CE - Biochemistry Impact factor: 2.303, year: 2009

  4. Time-domain imaging with quench-based fluorescent contrast agents

    Science.gov (United States)

    Akers, Walter J.; Solomon, Metasebya; Sudlow, Gail P.; Berezin, Mikhail; Achilefu, Samuel

    2012-03-01

    Quench-based probes utilize unique characteristics of fluorescence resonance energy transfer (FRET) to enhance contrast upon de-quenching. This mechanism has been used in a variety of molecular probes for imaging of cancer related enzyme activity such as matrix metalloproteinases, cathepsins and caspases. While non-fluorescent upon administration, fluorescence can be restored by separation of donor and acceptor, resulting in higher intensity in the presence of activator. Along with decreased quantum yield, FRET also results in altered fluorescence lifetime. Time-domain imaging can further enhance contrast and information yield from quench-based probes. We present in vivo time-domain imaging for detecting activation of quench-based probes. Quench-based probes utilize unique characteristics of fluorescence resonance energy transfer (FRET) to enhance contrast upon de-quenching. This mechanism has been used in a variety of molecular probes for imaging of cancer related enzyme activity such as matrix metalloproteinases, cathepsins and caspases. While non-fluorescent upon administration, fluorescence can be restored by separation of donor and acceptor, resulting in higher intensity in the presence of activator. Along with decreased quantum yield, FRET also results in altered fluorescence lifetime. Time-domain imaging can further enhance contrast and information yield from quench-based probes. We present in vivo time-domain imaging for detecting activation of quench-based probes. Time-domain diffuse optical imaging was performed to assess the FRET and quenching in living mice with orthotopic breast cancer. Tumor contrast enhancement was accompanied by increased fluorescence lifetime after administration of quenched probes selective for matrix metalloproteinases while no significant change was observed for non-quenched probes for integrin receptors. These results demonstrate the utility of timedomain imaging for detection of cancer-related enzyme activity in vivo.

  5. Strategies for Biologic Image-Guided Dose Escalation: A Review

    International Nuclear Information System (INIS)

    There is increasing interest in how to incorporate functional and molecular information obtained by noninvasive, three-dimensional tumor imaging into radiotherapy. The key issues are to identify radioresistant regions that can be targeted for dose escalation, and to develop radiation dose prescription and delivery strategies providing optimal treatment for the individual patient. In the present work, we review the proposed strategies for biologic image-guided dose escalation with intensity-modulated radiation therapy. Biologic imaging modalities and the derived images are discussed, as are methods for target volume delineation. Different dose escalation strategies and techniques for treatment delivery and treatment plan evaluation are also addressed. Furthermore, we consider the need for response monitoring during treatment. We conclude with a summary of the current status of biologic image-based dose escalation and of areas where further work is needed for this strategy to become incorporated into clinical practice

  6. In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish

    Science.gov (United States)

    Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

    2014-03-01

    Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

  7. Precise diagnosis in different scenarios using photoacoustic and fluorescence imaging with dual-modality nanoparticles.

    Science.gov (United States)

    Peng, Dong; Du, Yang; Shi, Yiwen; Mao, Duo; Jia, Xiaohua; Li, Hui; Zhu, Yukun; Wang, Kun; Tian, Jie

    2016-08-14

    Photoacoustic imaging and fluorescence molecular imaging are emerging as important research tools for biomedical studies. Photoacoustic imaging offers both strong optical absorption contrast and high ultrasonic resolution, and fluorescence molecular imaging provides excellent superficial resolution, high sensitivity, high throughput, and the ability for real-time imaging. Therefore, combining the imaging information of both modalities can provide comprehensive in vivo physiological and pathological information. However, currently there are limited probes available that can realize both fluorescence and photoacoustic imaging, and advanced biomedical applications for applying this dual-modality imaging approach remain underexplored. In this study, we developed a dual-modality photoacoustic-fluorescence imaging nanoprobe, ICG-loaded Au@SiO2, which was uniquely designed, consisting of gold nanorod cores and indocyanine green with silica shell spacer layers to overcome fluorophore quenching. This nanoprobe was examined by both PAI and FMI for in vivo imaging on tumor and ischemia mouse models. Our results demonstrated that the nanoparticles can specifically accumulate at the tumor and ischemic areas and be detected by both imaging modalities. Moreover, this dual-modality imaging strategy exhibited superior advantages for a precise diagnosis in different scenarios. The new nanoprobe with the dual-modality imaging approach holds great potential for diagnosis and stage classification of tumor and ischemia related diseases. PMID:27406825

  8. Remote Detection of Biological Particles and Chemical Plumes Using UV Fluorescence Lidar

    Science.gov (United States)

    Tiee, J. J.; Hof, D. E.; Karl, R. R.; Martinez, R. J.; Quick, C. R.; Cooper, D. I.; Eichinger, W. E.; Holtkamp, D. B.

    1992-01-01

    A lidar system based on ultraviolet (UV) laser induced fluorescence (LIF) was developed for the remote detection of atmospherically dispersed biological particles and chemical vapors. This UV fluorescence lidar has many potential applications for monitoring environmental pollution, industrial waste emission, agricultural insect control, illicit chemical processing, and military defense operations. The general goal of this work is to investigate the research issues associated with the long range detection and identification of chemicals, e.g. aromatic solvents and chemical precursors, and biological materials, e.g. bacillus thuringiensis (BT) and bacillus globiggi (BG). In the detection of biological particulates, we are particularly interested in extending the detection range of an existing solar-blind 248-nm lidar system. We are investigating the use of longer excitation laser wavelengths (i.e. lambda greater than 280-nm to have more favorable atmospheric light transmission characteristics) for improving detection range to better than 10 km. In the detection of chemical plumes, our main research objectives are to determine how accurately and sensitively a chemical plume can be located at range, and how well spectrally the chemical species can be measured to allow their identification.

  9. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    International Nuclear Information System (INIS)

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals

  10. Wavefront sensorless approaches to adaptive optics for in vivo fluorescence imaging of mouse retina

    Science.gov (United States)

    Wahl, Daniel J.; Bonora, Stefano; Mata, Oscar S.; Haunerland, Bengt K.; Zawadzki, Robert J.; Sarunic, Marinko V.; Jian, Yifan

    2016-03-01

    Adaptive optics (AO) is necessary to correct aberrations when imaging the mouse eye with high numerical aperture. In order to obtain cellular resolution, we have implemented wavefront sensorless adaptive optics for in vivo fluorescence imaging of mouse retina. Our approach includes a lens-based system and MEMS deformable mirror for aberration correction. The AO system was constructed with a reflectance channel for structural images and fluorescence channel for functional images. The structural imaging was used in real-time for navigation on the retina using landmarks such as blood vessels. We have also implemented a tunable liquid lens to select the retinal layer of interest at which to perform the optimization. At the desired location on the mouse retina, the optimization algorithm used the fluorescence image data to drive a modal hill-climbing algorithm using an intensity or sharpness image quality metric. The optimization requires ~30 seconds to complete a search up to the 20th Zernike mode. In this report, we have demonstrated the AO performance for high-resolution images of the capillaries in a fluorescence angiography. We have also made progress on an approach to AO with pupil segmentation as a possible sensorless technique suitable for small animal retinal imaging. Pupil segmentation AO was implemented on the same ophthalmic system and imaging performance was demonstrated on fluorescent beads with induced aberrations.

  11. Image navigation as a means to expand the boundaries of fluorescence-guided surgery

    International Nuclear Information System (INIS)

    Hybrid tracers that are both radioactive and fluorescent help extend the use of fluorescence-guided surgery to deeper structures. Such hybrid tracers facilitate preoperative surgical planning using (3D) scintigraphic images and enable synchronous intraoperative radio- and fluorescence guidance. Nevertheless, we previously found that improved orientation during laparoscopic surgery remains desirable. Here we illustrate how intraoperative navigation based on optical tracking of a fluorescence endoscope may help further improve the accuracy of hybrid surgical guidance. After feeding SPECT/CT images with an optical fiducial as a reference target to the navigation system, optical tracking could be used to position the tip of the fluorescence endoscope relative to the preoperative 3D imaging data. This hybrid navigation approach allowed us to accurately identify marker seeds in a phantom setup. The multispectral nature of the fluorescence endoscope enabled stepwise visualization of the two clinically approved fluorescent dyes, fluorescein and indocyanine green. In addition, the approach was used to navigate toward the prostate in a patient undergoing robot-assisted prostatectomy. Navigation of the tracked fluorescence endoscope toward the target identified on SPECT/CT resulted in real-time gradual visualization of the fluorescent signal in the prostate, thus providing an intraoperative confirmation of the navigation accuracy. (paper)

  12. Image navigation as a means to expand the boundaries of fluorescence-guided surgery

    Science.gov (United States)

    Brouwer, Oscar R.; Buckle, Tessa; Bunschoten, Anton; Kuil, Joeri; Vahrmeijer, Alexander L.; Wendler, Thomas; Valdés-Olmos, Renato A.; van der Poel, Henk G.; van Leeuwen, Fijs W. B.

    2012-05-01

    Hybrid tracers that are both radioactive and fluorescent help extend the use of fluorescence-guided surgery to deeper structures. Such hybrid tracers facilitate preoperative surgical planning using (3D) scintigraphic images and enable synchronous intraoperative radio- and fluorescence guidance. Nevertheless, we previously found that improved orientation during laparoscopic surgery remains desirable. Here we illustrate how intraoperative navigation based on optical tracking of a fluorescence endoscope may help further improve the accuracy of hybrid surgical guidance. After feeding SPECT/CT images with an optical fiducial as a reference target to the navigation system, optical tracking could be used to position the tip of the fluorescence endoscope relative to the preoperative 3D imaging data. This hybrid navigation approach allowed us to accurately identify marker seeds in a phantom setup. The multispectral nature of the fluorescence endoscope enabled stepwise visualization of the two clinically approved fluorescent dyes, fluorescein and indocyanine green. In addition, the approach was used to navigate toward the prostate in a patient undergoing robot-assisted prostatectomy. Navigation of the tracked fluorescence endoscope toward the target identified on SPECT/CT resulted in real-time gradual visualization of the fluorescent signal in the prostate, thus providing an intraoperative confirmation of the navigation accuracy.

  13. Functional surface engineering of quantum dot hydrogels for selective fluorescence imaging of extracellular lactate release.

    Science.gov (United States)

    Zhang, Xiaomeng; Ding, Shushu; Cao, Sumei; Zhu, Anwei; Shi, Guoyue

    2016-06-15

    Selective and sensitive detection of extracellular lactate is of fundamental significance for studying the metabolic alterations in tumor progression. Here we report the rational design and synthesis of a quantum-dot-hydrogel-based fluorescent probe for biosensing and bioimaging the extracellular lactate. By surface engineering the destabilized quantum dot sol with Nile Blue, the destabilized Nile-Blue-functionalized quantum dot sol cannot only self-assemble forming quantum dot hydrogel but also monitor lactate in the presence of nicotinamide adenine dinucleotide cofactor and lactate dehydrogenase through fluorescence resonance energy transfer. Notably, the surface engineered quantum dot hydrogel show high selectivity toward lactate over common metal ions, amino acids and other small molecules that widely coexist in biological system. Moreover, the destabilized Nile-Blue-functionalized quantum dots can encapsulate isolated cancer cells when self-assembled into a hydrogel and thus specifically detect and image the extracellular lactate metabolism. By virtue of these properties, the functionalized quantum dot hydrogel was further successfully applied to monitor the effect of metabolic agents. PMID:26852200

  14. New instrumentation for sensitive image analysis of fluorescence in cells and tissues

    International Nuclear Information System (INIS)

    Fluorescence methods are increasingly applied in clinical cytometry. New instrumental developments have made this possible. A first example consists of the laser scanning microscope (LSM), which enables digital storage of an image of a scanned area in computer memory. The development of low-magnification superwide field microscopes, especially designed for image analysis, has contributed significantly to the analysis of weakly fluorescent images. These microscopes can combine fluorescence detection with conventional modes of microscopy, such as phase-contrast, absorbance microscopy, and reflectance microscopy. These new instruments make use of immersion objectives with maximal numerical aperture (NA) and superwide fields, and permit a relatively low magnification. This is obtained by direct projection of the image of the objective on a TV tube without using a conventional projective eyepiece. A third instrumental development combines scanning electron microscopy (SEM) with fluorescence light microscopy (LM). This has been achieved by incorporating an epi-fluorescence microscope into the vacuum chamber of a scanning electron microscope. Fluorescence exciting light can be guided onto the specimen in the vacuum chamber of the SEM and the fluorescence can be observed through the light microscope objective in the vacuum chamber. The image is transferred to the outside by a light pipe connection to an epi-fluorescence microscope. All three instrumental developments of the type described above permit a much wider use of quantitative methods in fluorescence cytochemistry and histochemistry. In contrast to the older microfluorometry equipment, the newer instruments permit an effective pattern recognition. Strongly fluorescent cells or cellular organelles can often be automatically isolated from the background by thresholding of the image

  15. Silica-porphyrin hybrid nanotubes for in vivo cell tracking by near-infrared fluorescence imaging.

    Science.gov (United States)

    Hayashi, Koichiro; Nakamura, Michihiro; Ishimura, Kazunori

    2012-04-21

    Near-infrared fluorescent silica-porphyrin hybrid nanotubes (HNTs) were successfully synthesized by π-π stacking, electrostatic interaction and a sol-gel reaction. The HNTs-labeled macrophages were detected in vivo, and the minimum detectable number of cells was 200. Furthermore, the biodistribution of HNTs-labeled macrophages was tracked by fluorescence imaging. PMID:22437325

  16. Multispectral fluorescence imaging for detection of bovine feces on Romaine lettuce and baby spinach leaves

    Science.gov (United States)

    Hyperspectral fluorescence imaging with ultraviolet-A excitation was used to evaluate the feasibility of two-waveband fluorescence algorithms for the detection of bovine fecal contaminants on the abaxial and adaxial surfaces of Romaine lettuce and baby spinach leaves. Correlation analysis was used t...

  17. Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

    Directory of Open Access Journals (Sweden)

    Davidson Michael W

    2008-03-01

    Full Text Available Abstract Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP, the expanding set of fluorescent protein (FP variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1, a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His. Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP. To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our

  18. Targeted Thiazole Orange Derivative with Folate: Synthesis, Fluorescence and in Vivo Fluorescence Imaging

    OpenAIRE

    Qingyang Meng; Baolian Zhang; Yiqi Wang; Xuening Fei; Yingchun Gu

    2010-01-01

    A Thiazole Orange conjugated with folate derivative was synthesized in two steps. Firstly, folate was coupled with 1-(3-aminopropyl)-4-methylquinolinium bromide to afford folate-methylquinolinium bromide, which then reacted with benzothiazolium to obtain the title folate-conjugated compound. The compound was evaluated by 1H-NMR MS, TG/DTA and fluorescence spectroscopic methods. The title compound could selectively target folate receptor expressing tumors according to the in vivo fluorescence ...

  19. Single-atom-resolved fluorescence imaging of an atomic Mott insulator

    DEFF Research Database (Denmark)

    Sherson, Jacob; Weitenberg, Christof; Andres, Manuel;

    2010-01-01

    situ images of a quantum fluid in which each underlying quantum particle is detected. Here we report fluorescence imaging of strongly interacting bosonic Mott insulators in an optical lattice with single-atom and single-site resolution. From our images, we fully reconstruct the atom distribution on the...

  20. Observations of fluorescent and biological aerosol at a high-altitude site in Central France

    Directory of Open Access Journals (Sweden)

    A. M. Gabey

    2013-01-01

    Full Text Available Total bacteria, fungal spore and yeast counts were compared with UV Light-Induced Fluorescence (UV-LIF measurements of ambient aerosol at the summit of the Puy de Dôme (pdD mountain in Central France (1465 m a.s.l, which represents a background elevated site. Bacteria, fungal spores and yeast were enumerated by epifluorescence microscopy (EFM and found to number 2.2 to 23 L−1 and 0.8 to 2 L−1, respectively. Bacteria counts on two successive nights were an order of magnitude larger than in the intervening day.

    A Wide Issue Bioaerosol Spectrometer, version 3 (WIBS-3 was used to perform UV-LIF measurements on ambient aerosol sized 0.8 to 20 μm. Mean total number concentration was 270 L−1 (σ = 66 L−1 found predominantly in a size mode at 2 μm for most of the campaign. Total concentration (fluorescent + non-fluorescent aerosol peaked at 500 L−1 with a size mode at 1 μm because of a change in air mass origin lasting around 48 h. The WIBS-3 features two excitation and fluorescence detection wavelengths corresponding to different biological molecules. The mean fluorescent particle concentration after short-wave (280 nm; Tryptophan excitation was 12 L−1 (σ = 6 L−1, and did not vary much through the campaign. In contrast the mean concentration of particles fluorescent after long-wave (370 nm; NADH excitation was 95 L−1 (σ = 25 L−1, and a nightly rise and subsequent fall of up to 100 L−1 formed a strong diurnal cycle in the latter. The fluorescent populations exhibited size modes at 3 μm and 2 to 3 μm, respectively. A hierarchical agglomerative cluster analysis algorithm was applied to the data and used to extract different particle factors. A cluster concentration time series representative of bacteria was identified. This was found to exhibit a diurnal cycle with a maximum peak appearing during the day.

  1. Early detection of tumor masses by in vivo hematoporphyrin-mediated fluorescence imaging

    Science.gov (United States)

    Autiero, Maddalena; Celentano, Luigi; Cozzolino, Rosanna; Laccetti, Paolo; Marotta, Marcello; Mettivier, Giovanni; Cristina Montesi, Maria; Quarto, Maria; Riccio, Patrizia; Roberti, Giuseppe; Russo, Paolo

    2007-02-01

    We investigated the capability of fluorescence reflectance imaging (FRI) for the early detection of surface tumors in mice. We used a hematoporphyrin (HP) compound (HP dichlorohydrate) as a red fluorescent marker and a low noise, high sensitivity, digital CCD camera for fluorescence imaging. In this preliminary study, highly malignant anaplastic human thyroid carcinoma cells were implanted subcutaneously in one mouse and their growth was monitored daily for 5 days by FRI. The selective HP uptake by the tumor tissues was successfully observed: we observed the fluorescence of tumor only 3 days after cancer cells injection, i.e. when the tumor mass was neither visible (to the naked eye) or palpable. These measurements indicate that FRI is a suitable technique to detect minute subcutaneous tumor masses. This FRI system will be coupled to a radionuclide imaging system based on a CdTe detector for in vivo multimodal imaging in mice.

  2. Cancer detection using NIR elastic light scattering and tissue fluorescence imaging

    International Nuclear Information System (INIS)

    Near infrared imaging using elastic light scattering and tissue fluorescence under long-wavelength laser excitation are explored for cancer detection. Various types of normal and malignant human tissue samples were utilized in this investigation

  3. Early detection of tumor masses by in vivo hematoporphyrin-mediated fluorescence imaging

    International Nuclear Information System (INIS)

    We investigated the capability of fluorescence reflectance imaging (FRI) for the early detection of surface tumors in mice. We used a hematoporphyrin (HP) compound (HP dichlorohydrate) as a red fluorescent marker and a low noise, high sensitivity, digital CCD camera for fluorescence imaging. In this preliminary study, highly malignant anaplastic human thyroid carcinoma cells were implanted subcutaneously in one mouse and their growth was monitored daily for 5 days by FRI. The selective HP uptake by the tumor tissues was successfully observed: we observed the fluorescence of tumor only 3 days after cancer cells injection, i.e. when the tumor mass was neither visible (to the naked eye) or palpable. These measurements indicate that FRI is a suitable technique to detect minute subcutaneous tumor masses. This FRI system will be coupled to a radionuclide imaging system based on a CdTe detector for in vivo multimodal imaging in mice

  4. Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

    Science.gov (United States)

    Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

    2015-10-01

    Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

  5. Time efficient methods for scanning a fluorescent membrane with a fluorescent microscopic imager for the quality assurance of food

    Science.gov (United States)

    Lerm, Steffen; Holder, Silvio; Schellhorn, Mathias; Brückner, Peter; Linß, Gerhard

    2013-05-01

    An important part of the quality assurance of meat is the estimation of germs in the meat exudes. The kind and the number of the germs in the meat affect the medical risk for the consumer of the meat. State-of-the-art analyses of meat are incubator test procedures. The main disadvantages of such incubator tests are the time consumption, the necessary equipment and the need of special skilled employees. These facts cause in high inspection cost. For this reason a new method for the quality assurance is necessary which combines low detection limits and less time consumption. One approach for such a new method is fluorescence microscopic imaging. The germs in the meat exude are caught in special membranes by body-antibody reactions. The germ typical signature could be enhanced with fluorescent chemical markers instead of reproduction of the germs. Each fluorescent marker connects with a free germ or run off the membrane. An image processing system is used to detect the number of fluorescent particles. Each fluorescent spot should be a marker which is connected with a germ. Caused by the small object sizes of germs, the image processing system needs a high optical magnification of the camera. However, this leads to a small field of view and a small depth of focus. For this reasons the whole area of the membrane has to be scanned in three dimensions. To minimize the time consumption, the optimal path has to be found. This optimization problem is influenced by features of the hardware and is presented in this paper. The traversing range in each direction, the step width, the velocity, the shape of the inspection volume and the field of view have influence on the optimal path to scan the membrane.

  6. Turn-off fluorescence sensor for the detection of ferric ion in water using green synthesized N-doped carbon dots and its bio-imaging.

    Science.gov (United States)

    Edison, Thomas Nesakumar Jebakumar Immanuel; Atchudan, Raji; Shim, Jae-Jin; Kalimuthu, Senthilkumar; Ahn, Byeong-Cheol; Lee, Yong Rok

    2016-05-01

    This paper reports turn-off fluorescence sensor for Fe(3+) ion in water using fluorescent N-doped carbon dots as a probe. A simple and efficient hydrothermal carbonization of Prunus avium fruit extract for the synthesis of fluorescent nitrogen-doped carbon dots (N-CDs) is described. This green approach proceeds quickly and provides good quality N-CDs. The mean size of synthesized N-CDs was approximately 7nm calculated from the high-resolution transmission electron microscopic images. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy revealed the presence of -OH, -NH2, -COOH, and -CO functional groups over the surface of CDs. The N-CDs showed excellent fluorescent properties, and emitted blue fluorescence at 411nm upon excitation at 310nm. The calculated quantum yield of the synthesized N-CDs is 13% against quinine sulfate as a reference fluorophore. The synthesized N-CDs were used as a fluorescent probe towards the selective and sensitive detection of biologically important Fe(3+) ions in water by fluorescence spectroscopy and for bio-imaging of MDA-MB-231 cells. The limit of detection (LOD) and the Stern-Volmer quenching constant for the synthesized N-CDs were 0.96μM and 2.0958×10(3)M of Fe(3+) ions. The green synthesized N-CDs are efficiently used as a promising candidate for the detection of Fe(3+) ions and bio-imaging. PMID:26994332

  7. Janus particles for biological imaging and sensing.

    Science.gov (United States)

    Yi, Yi; Sanchez, Lucero; Gao, Yuan; Yu, Yan

    2016-06-21

    Janus particles, named after the two-faced Roman god Janus, have different surface makeups, structures or compartments on two sides. This review highlights recent advances in employing Janus particles as novel analytical tools for live cell imaging and biosensing. Unlike conventional particles used in analytical science, two-faced Janus particles provide asymmetry and directionality, and can combine different or even incompatible properties within a single particle. The broken symmetry enables imaging and quantification of rotational dynamics, revealing information beyond what traditional measurements offer. The spatial segregation of molecules on the surface of a single particle also allows analytical functions that would otherwise interfere with each other to be decoupled, opening up opportunities for novel multimodal analytical methods. We summarize here the development of Janus particles, a few general methods for their fabrication and, more importantly, the emerging and novel applications of Janus particles as multi-functional imaging probes and sensors. PMID:27052001

  8. Polymerized LB films imaged with a combined atomic force microscope-fluorescence microscope

    OpenAIRE

    Putman, Constant A.J.; Hansma, Helen G.; Gaub, Hermann E.; Hansma, Paul K.

    1992-01-01

    The first results obtained with a new stand-alone atomic force microscope (AFM) integrated with a standard Zeiss optical fluorescence microscope are presented. The optical microscope allows location and selection of objects to be imaged with the high-resolution AFM. Furthermore, the combined microscope enables a direct comparison between features observed in the fluorescence microscope and those observed in the images obtained with the AFM, in air or under liquid. The cracks in polymerized La...

  9. In Vivo Imaging of the Actin Polymerization State with Two-Photon Fluorescence Anisotropy

    OpenAIRE

    Vishwasrao, Harshad D.; Trifilieff, Pierre; Kandel, Eric R.

    2012-01-01

    Using two-photon fluorescence anisotropy imaging of actin-GFP, we have developed a method for imaging the actin polymerization state that is applicable to a broad range of experimental systems extending from fixed cells to live animals. The incorporation of expressed actin-GFP monomers into endogenous actin polymers enables energy migration FRET (emFRET, or homoFRET) between neighboring actin-GFPs. This energy migration reduces the normally high polarization of the GFP fluorescence. We derive...

  10. Recent trends in total reflection X-ray fluorescence spectrometry for biological applications

    International Nuclear Information System (INIS)

    This review is focused on the application of total reflection X-ray fluorescence (TXRF) spectrometry in the field of biological research. In the last decade, most papers were published by authors who applied laboratory-scale TXRF equipments. The application of synchrotron radiation as excitation source (SR-TXRF) shows a slowly increasing tendency. In the cited papers the micro-, trace and multielement capability of these TXRF techniques was demonstrated in the clinical and medical laboratory practice, as well as in various plant physiological studies. For speciation of elements in biological matrices, the TXRF was used as element specific detector following an off-line separation step (e.g., thin layer chromatography, high performance liquid chromatography), however, these off-line methods are not competitive with the on-line coupled HPLC-inductively coupled plasma mass spectrometry

  11. Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

    Directory of Open Access Journals (Sweden)

    Kazuo Yamagata

    Full Text Available Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

  12. In-vivo optical detection of cancer using chlorin e6 – polyvinylpyrrolidone induced fluorescence imaging and spectroscopy

    International Nuclear Information System (INIS)

    Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6) formulated in polyvinylpyrrolidone (PVP) as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a patient. Fluorescence imaging was performed on MGH human bladder tumor xenografted on both the chick chorioallantoic membrane (CAM) and the murine model using a fluorescence endoscopy imaging system. In addition, fiber optic based fluorescence spectroscopy was performed on tumors and various normal organs in the same mice to validate the macroscopic images. In one patient, fluorescence imaging was performed on angiosarcoma lesions and normal skin in conjunction with fluorescence spectroscopy to validate Ce6-PVP induced fluorescence visual assessment of the lesions. Margins of tumor xenografts in the CAM model were clearly outlined under fluorescence imaging. Ce6-PVP-induced fluorescence imaging yielded a specificity of 83% on the CAM model. In mice, fluorescence intensity of Ce6-PVP was higher in bladder tumor compared to adjacent muscle and normal bladder. Clinical results confirmed that fluorescence imaging clearly captured the fluorescence of Ce6-PVP in angiosarcoma lesions and good correlation was found between fluorescence imaging and spectral measurement in the patient. Combination of Ce6-PVP induced fluorescence imaging and spectroscopy could allow for optical detection and discrimination between cancer and the surrounding normal tissues. Ce6-PVP seems to be a promising fluorophore for fluorescence diagnosis of cancer

  13. Evaluation of chicory seeds maturity by chlorophyll fluorescence imaging

    OpenAIRE

    Ooms, David; Destain, Marie-France

    2011-01-01

    Chicory (Cichorium intybus L.) seed production includes sorting to remove foreign materials and non-viable seeds. A machine vision system was developed to monitor the fluorescence in order to detect the immature chicory seeds. It comprised a monochromatic light source, a highpass filter and a monochromatic CCD camera sensitive to red and infrared. With this device, blue light reflected by the seeds was blocked whilst red fluorescence was measured by the camera. A segmentation a...

  14. Complementarity of ultrasound and fluorescence imaging in an orthotopic mouse model of pancreatic cancer

    International Nuclear Information System (INIS)

    Pancreatic cancer is a devastating disease characterized by dismal 5-year survival rates and limited treatment options. In an effort to provide useful models for preclinical evaluation of new experimental therapeutics, we and others have developed orthotopic mouse models of pancreatic cancer. The utility of these models for pre-clinical testing is dependent upon quantitative, noninvasive methods for monitoring in vivo tumor progression in real time. Toward this goal, we performed whole-body fluorescence imaging and ultrasound imaging to evaluate and to compare these noninvasive imaging modalities for assessing tumor burden and tumor progression in an orthotopic mouse model of pancreatic cancer. The human pancreatic cancer cell line XPA-1, engineered for stable, high-level expression of red fluorescent protein (RFP), was implanted into the pancreas of nude mice using orthotopic implantation. The tumors were allowed to grow over a period of one to several weeks during which time the mice were imaged using both fluorescence imaging and ultrasound imaging to measure tumor burden and to monitor tumor growth. Whole-body fluorescence imaging and ultrasound imaging both allowed for the visualization and measurement of orthotopic pancreatic tumor implants in vivo. The imaging sessions were well-tolerated by the mice and yielded data which correlated well in the quantitative assessment of tumor burden. Whole-body fluorescence and two-dimensional ultrasound imaging showed a strong correlation for measurement of tumor size over a range of tumor sizes (R2 = 0.6627, P = 0.003 for an exposure time of 67 msec and R2 = 0.6553, P = 0.003 for an exposure time of 120 msec). Our findings suggest a complementary role for fluorescence imaging and ultrasound imaging in assessing tumor burden and tumor progression in orthotopic mouse models of human cancer

  15. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

    Science.gov (United States)

    Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

    2016-02-01

    Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

  16. In vivo tomographic imaging with fluorescence and MRI using tumor-targeted dual-labeled nanoparticles

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2013-12-01

    Full Text Available Yue Zhang,1 Bin Zhang,1 Fei Liu,1,2 Jianwen Luo,1,3 Jing Bai1 1Department of Biomedical Engineering, School of Medicine, 2Tsinghua-Peking Center for Life Sciences, 3Center for Biomedical Imaging Research, Tsinghua University, Beijing, People's Republic of China Abstract: Dual-modality imaging combines the complementary advantages of different modalities, and offers the prospect of improved preclinical research. The combination of fluorescence imaging and magnetic resonance imaging (MRI provides cross-validated information and direct comparison between these modalities. Here, we report on the application of a novel tumor-targeted, dual-labeled nanoparticle (NP, utilizing iron oxide as the MRI contrast agent and near infrared (NIR dye Cy5.5 as the fluorescent agent. Results of in vitro experiments verified the specificity of the NP to tumor cells. In vivo tumor targeting and uptake of the NPs in a mouse model were visualized by fluorescence and MR imaging collected at different time points. Quantitative analysis was carried out to evaluate the efficacy of MRI contrast enhancement. Furthermore, tomographic images were also acquired using both imaging modalities and cross-validated information of tumor location and size between these two modalities was revealed. The results demonstrate that the use of dual-labeled NPs can facilitate the dual-modal detection of tumors, information cross-validation, and direct comparison by combing fluorescence molecular tomography (FMT and MRI. Keywords: dual-modality, fluorescence molecular tomography (FMT, magnetic resonance imaging (MRI, nanoparticle

  17. Biosynthesis of fluorescent gold nanoclusters for in vitro and in vivo tumor imaging

    Science.gov (United States)

    Li, Linlin; Liu, Xi; Fu, Changhui; Tan, Longfei; Liu, Huiyu

    2015-11-01

    Recently, fluorescent metallic nanoclusters with sizes in the few-nanometer range have showed great potentials in biomedical applications for their stable and tailorable fluorescence. Although many studies have focused on fabricating these kinds of materials with chemical methods, there has been little focus on the biosynthesis of gold nanoclusters with green and facile methods. In this study, a facile, scalable, cost-effective and environmentally benign biosynthesis approach was developed to produce fluorescent gold nanoclusters (AuNCs). Biomasses including egg white, egg yolk and serums were used as both capping agents and reductants in the biosynthesis of AuNCs. As a new kind of fluorescent imaging agent, they were used for in vitro and in vivo tumor imaging that can efficiently track cancer cells with excellent biocompatibility. This work provides new insight into green biosynthesis and biomedical applications of fluorescent metallic nanoclusters.

  18. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    Directory of Open Access Journals (Sweden)

    Eriksen Jens

    2009-01-01

    Full Text Available Abstract DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

  19. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    Directory of Open Access Journals (Sweden)

    Hojman Pernille

    2009-04-01

    Full Text Available Abstract DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

  20. Automated parasite faecal egg counting using fluorescence labelling, smartphone image capture and computational image analysis.

    Science.gov (United States)

    Slusarewicz, Paul; Pagano, Stefanie; Mills, Christopher; Popa, Gabriel; Chow, K Martin; Mendenhall, Michael; Rodgers, David W; Nielsen, Martin K

    2016-07-01

    Intestinal parasites are a concern in veterinary medicine worldwide and for human health in the developing world. Infections are identified by microscopic visualisation of parasite eggs in faeces, which is time-consuming, requires technical expertise and is impractical for use on-site. For these reasons, recommendations for parasite surveillance are not widely adopted and parasite control is based on administration of rote prophylactic treatments with anthelmintic drugs. This approach is known to promote anthelmintic resistance, so there is a pronounced need for a convenient egg counting assay to promote good clinical practice. Using a fluorescent chitin-binding protein, we show that this structural carbohydrate is present and accessible in shells of ova of strongyle, ascarid, trichurid and coccidian parasites. Furthermore, we show that a cellular smartphone can be used as an inexpensive device to image fluorescent eggs and, by harnessing the computational power of the phone, to perform image analysis to count the eggs. Strongyle egg counts generated by the smartphone system had a significant linear correlation with manual McMaster counts (R(2)=0.98), but with a significantly lower coefficient of variation (P=0.0177). Furthermore, the system was capable of differentiating equine strongyle and ascarid eggs similar to the McMaster method, but with significantly lower coefficients of variation (Psmartphones as relatively sophisticated, inexpensive and portable medical diagnostic devices. PMID:27025771

  1. A new indicator in early drought diagnosis of cucumber with chlorophyll fluorescence imaging

    Science.gov (United States)

    Wang, Heng; Li, Haifeng; Xu, Liang; Liu, Xu

    2015-05-01

    Crop population growth information can more fully reflect the state of crop growth, eliminate individual differences, and reduce error in judgment. We have built a suitable plant population growth information online monitoring system with the plant chlorophyll fluorescence and spectral scanning imaging to get the crop growth status. On the basis of the fluorescence image detection, we have studied the early drought diagnosis of cucumber. The typical chlorophyll fluorescence parameters can not reflect the drought degree significantly. We define a new indication parameter (DI). With the drought deepening, DI declines. DI can enlarge the early manifestation of cucumber drought (3-5 days), indicate more significantly in the early drought diagnosis of cucumber.

  2. Aminopeptidase N/CD13 targeting fluorescent probes: synthesis and application to tumor cell imaging.

    Science.gov (United States)

    Zhang, Zhouen; Harada, Hiroshi; Tanabe, Kazuhito; Hatta, Hiroshi; Hiraoka, Masahiro; Nishimoto, Sei-ichi

    2005-11-01

    A family of fluorescein-peptide conjugates (CNP1-3) for aminopeptidase N (APN/CD13) targeting fluorescent probes were designed and synthesized. Among the three conjugates, CNP1 bearing tumor-homing cyclic peptide CNGRC, could selectively label APN/CD13 over-expressing on the surface of tumor cells of HT-1080, as identified by means of fluorescent microscopic cell imaging. CNP1 was shown to be a promising fluorescent probe applicable to tumor-targeting molecular imaging. PMID:15885853

  3. Lensless wide-field fluorescent imaging on a chip using compressive decoding of sparse objects

    OpenAIRE

    Coskun, Ahmet F; Sencan, Ikbal; Su, Ting-Wei; Ozcan, Aydogan

    2010-01-01

    We demonstrate the use of a compressive sampling algorithm for on-chip fluorescent imaging of sparse objects over an ultra-large field-of-view (>8 cm2) without the need for any lenses or mechanical scanning. In this lensfree imaging technique, fluorescent samples placed on a chip are excited through a prism interface, where the pump light is filtered out by total internal reflection after exciting the entire sample volume. The emitted fluorescent light from the specimen is collected through a...

  4. Pattern recognition software and techniques for biological image analysis.

    Directory of Open Access Journals (Sweden)

    Lior Shamir

    Full Text Available The increasing prevalence of automated image acquisition systems is enabling new types of microscopy experiments that generate large image datasets. However, there is a perceived lack of robust image analysis systems required to process these diverse datasets. Most automated image analysis systems are tailored for specific types of microscopy, contrast methods, probes, and even cell types. This imposes significant constraints on experimental design, limiting their application to the narrow set of imaging methods for which they were designed. One of the approaches to address these limitations is pattern recognition, which was originally developed for remote sensing, and is increasingly being applied to the biology domain. This approach relies on training a computer to recognize patterns in images rather than developing algorithms or tuning parameters for specific image processing tasks. The generality of this approach promises to enable data mining in extensive image repositories, and provide objective and quantitative imaging assays for routine use. Here, we provide a brief overview of the technologies behind pattern recognition and its use in computer vision for biological and biomedical imaging. We list available software tools that can be used by biologists and suggest practical experimental considerations to make the best use of pattern recognition techniques for imaging assays.

  5. Nucleic Acid-Based Nanodevices in Biological Imaging.

    Science.gov (United States)

    Chakraborty, Kasturi; Veetil, Aneesh T; Jaffrey, Samie R; Krishnan, Yamuna

    2016-06-01

    The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand. PMID:27294440

  6. Fluorescence-enhanced optical tomography and nuclear imaging system for small animals

    Science.gov (United States)

    Tan, I.-Chih; Lu, Yujie; Darne, Chinmay; Rasmussen, John C.; Zhu, Banghe; Azhdarinia, Ali; Yan, Shikui; Smith, Anne M.; Sevick-Muraca, Eva M.

    2012-03-01

    Near-infrared (NIR) fluorescence is an alternative modality for molecular imaging that has been demonstrated in animals and recently in humans. Fluorescence-enhanced optical tomography (FEOT) using continuous wave or frequency domain photon migration techniques could be used to provide quantitative molecular imaging in vivo if it could be validated against "gold-standard," nuclear imaging modalities, using dual-labeled imaging agents. Unfortunately, developed FEOT systems are not suitable for incorporation with CT/PET/SPECT scanners because they utilize benchtop devices and require a large footprint. In this work, we developed a miniaturized fluorescence imaging system installed in the gantry of the Siemens Inveon PET/CT scanner to enable NIR transillumination measurements. The system consists of a CCD camera equipped with NIR sensitive intensifier, a diode laser controlled by a single board compact controller, a 2-axis galvanometer, and RF circuit modules for homodyne detection of the phase and amplitude of fluorescence signals. The performance of the FEOT system was tested and characterized. A mouse-shaped solid phantom of uniform optical properties with a fluorescent inclusion was scanned using CT, and NIR fluorescence images at several projections were collected. The method of high-order approximation to the radioactive transfer equation was then used to reconstruct the optical images. Dual-labeled agents were also used on a tumor bearing mouse to validate the results of the FEOT against PET/CT image. The results showed that the location of the fluorophore obtained from the FEOT matches the location of tumor obtained from the PET/CT images. Besides validation of FEOT, this hybrid system could allow multimodal molecular imaging (FEOT/PET/CT) for small animal imaging.

  7. Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.

    Science.gov (United States)

    Lee, Sungmoo; Piao, Hong Hua; Sepheri-Rad, Masoud; Jung, Arong; Sung, Uhna; Song, Yoon-Kyu; Baker, Bradley J

    2016-01-01

    Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera. PMID:26890551

  8. A compact gamma camera for biological imaging

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, E L; Cella, J; Majewski, S; Popov, V; Qian, Jianguo; Saha, M S; Smith, M F; Weisenberger, A G; Welsh, R E

    2006-02-01

    A compact detector, sized particularly for imaging a mouse, is described. The active area of the detector is approximately 46 mm; spl times/ 96 mm. Two flat-panel Hamamatsu H8500 position-sensitive photomultiplier tubes (PSPMTs) are coupled to a pixellated NaI(Tl) scintillator which views the animal through a copper-beryllium (CuBe) parallel-hole collimator specially designed for {sup 125}I. Although the PSPMTs have insensitive areas at their edges and there is a physical gap, corrections for scintillation light collection at the junction between the two tubes results in a uniform response across the entire rectangular area of the detector. The system described has been developed to optimize both sensitivity and resolution for in-vivo imaging of small animals injected with iodinated compounds. We demonstrate an in-vivo application of this detector, particularly to SPECT, by imaging mice injected with approximately 10-15; spl mu/Ci of {sup 125}I.

  9. Fluorescence Modified Chitosan-Coated Magnetic Nanoparticles for High-Efficient Cellular Imaging

    Directory of Open Access Journals (Sweden)

    Nie Fang

    2009-01-01

    Full Text Available Abstract Labeling of cells with nanoparticles for living detection is of interest to various biomedical applications. In this study, novel fluorescent/magnetic nanoparticles were prepared and used in high-efficient cellular imaging. The nanoparticles coated with the modified chitosan possessed a magnetic oxide core and a covalently attached fluorescent dye. We evaluated the feasibility and efficiency in labeling cancer cells (SMMC-7721 with the nanoparticles. The nanoparticles exhibited a high affinity to cells, which was demonstrated by flow cytometry and magnetic resonance imaging. The results showed that cell-labeling efficiency of the nanoparticles was dependent on the incubation time and nanoparticles’ concentration. The minimum detected number of labeled cells was around 104by using a clinical 1.5-T MRI imager. Fluorescence and transmission electron microscopy instruments were used to monitor the localization patterns of the magnetic nanoparticles in cells. These new magneto-fluorescent nanoagents have demonstrated the potential for future medical use.

  10. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P [Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Zhu, R; Mayer, B [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F [Agilent Technologies Austria GmbH, Aubrunnerweg 11, A-4040 Linz (Austria); Salio, M; Shepherd, D; Polzella, P; Cerundolo, V [Cancer Research UK Tumor Immunology Group, Weatherall Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS (United Kingdom); Dieudonne, M, E-mail: ferry_kienberger@agilent.com [Agilent Technologies Belgium, Wingepark 51, Rotselaar, AN B-3110 (Belgium)

    2010-03-19

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on {alpha}-galactosylceramide ({alpha}GalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from {approx} 25 to {approx} 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  11. Spectral and fluorescence imaging of immune system and tissue response to an immunogenic agent

    Science.gov (United States)

    Choe, Se-woon; Acharya, Abhinav; Keselowsky, Benjamin G.; Sorg, Brian S.

    2009-05-01

    Imaging of immune system and tissue response to immunogenic agents can be important to the development of new biomaterials. Additionally, quantitative functional imaging can be useful for testing and evaluation of methods to alter or control the immune system response to implanted materials. In this preliminary study, we employ spectral imaging and fluorescence imaging to measure immune system and tissue response to implanted immunogenic agents. Poly (D,L lactide-co-glycolide) (PLGA) with a 50:50 composition was used to create immunogenic microparticles (MPs). Lipopolysaccharide (LPS) encapsulated in the MPs was used to provoke a tissue immune response in mice and encapsulated fluorescein isothiocyanate (FITC) was used to fluorescently label the MPs for imaging. Control MPs did not contain LPS. The MPs were delivered at 50 particles/μL in a total volume of 20μL by subcutaneous injection in the skin of a nude mouse in a dorsal skin-fold window chamber preparation. Cultured immune cells from a mouse leukemic monocyte macrophage cell line were exogenously labeled with the fluorescent dye DiD in solution at a concentration of 8000cells/μL. Immediately after window chamber surgery and implantation of the MPs, 100μL of the fluorescent macrophage solution was administered via the tail vein. Fluorescence imaging was used to track MPs and macrophages while spectral imaging was used for imaging and measurement of hemoglobin saturation in the tissue microvasculature. Imaging was performed periodically over about three days. The spectral and fluorescence imaging combination enabled detailed observations of the macrophage response and functional effects on the tissue.

  12. Clinical multi-colour fluorescence imaging of malignant tumours - initial experience

    Energy Technology Data Exchange (ETDEWEB)

    Svanberg, K.; Wang, I. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Oncology; Colleen, S. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Urology; Idvall, I. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Pathology; Ingvar, C. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Surgery; Rydell, R. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Univ. Hospital (Sweden). Dept. of Oto-Rhino-Laryngology; Jocham, D. [Luebeck Univ. (Germany). Abt. fuer Urologie; Diddens, H. [Luebeck Univ. (Germany). Medizinisches Laser Zentrum; Bown, S.; Gregory, G. [National Medical Laser Centre, Dept. of Surgery, Rayne Inst., London (United Kingdom); Montan, S. [Spectraphos AB, Ideon, Lund (Sweden); Andersson-Engels, S.; Svanberg, S. [Lund Univ. Hospital (Sweden). Lund Medical Laser Centre]|[Lund Inst. of Technology (Sweden). Dept. of Physics

    1998-01-01

    The purpose of this study was to present a new technique for non-invasive tumour detection based on tissue fluorescence imaging. A clinically adapted multi-colour fluorescence system was employed in the real-time imaging of malignant tumours of the skin, breast, head and neck region, and urinary bladder. Tumour detection was based on the contrast displayed in fluorescence between normal and malignant tissue, related to the selective uptake of tumour-marking agents and natural chromophore differences between various tissues. In order to demarcate basal cell carcinomas of the skin, ALA was applied topically 4-6 h before the fluorescence investigation. For urinary bladder tumour visualisation, ALA was instilled into the bladder 1-2 h prior to the study. Malignant and premalignant lesions in the head and neck region were imaged after i.v. injection of HPD (Photofrin). The tumour imaging system was coupled to an endoscope. Fluorescence light emission from the tissue surface was induced with 100-ns-long optical pulses at 390 nm, generated from a frequency-doubled alexandrite laser. With the use of special image-splitting optics, the tumour fluorescence, intensified in a micro-channel plate, was imaged in 3 selected wavelength bands. These 3 images were processed together to form a new optimised-contrast image of the tumour. This image, updated at a rate of about 3 frames/s was mixed with a normal colour video image of the tissue. A clear demarcation from normal surrounding tissue was found during in vivo measurements of superficial bladder carcinoma, basal cell carcinoma of the skin, and leukoplakia with dysplasia of the lip, and in vitro investigations of resected breast cancer. (orig./MG).

  13. Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

    Directory of Open Access Journals (Sweden)

    Karolina Jahn

    Full Text Available For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR and Asante Calcium Green (ACG for two-photon (2P-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+-free and Ca(2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+-dependent way, unraveling in vitro dissociation constants K(D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns and long (2.44 ns decay time components attributable to the Ca(2+-free and Ca(2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D of 180 nM was determined. Thus, quantitative [Ca(2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

  14. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    Energy Technology Data Exchange (ETDEWEB)

    Cuneo, Kyle C. [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Mito, Jeffrey K.; Javid, Melodi P. [Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States); Ferrer, Jorge M. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Kim, Yongbaek [Department of Clinical Pathology, College of Veterinary Medicine, Seoul National University, Seoul (Korea, Republic of); Lee, W. David [The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Bawendi, Moungi G. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Brigman, Brian E. [Department of Orthopedic Surgery, Duke University School of Medicine, Durham, North Carolina (United States); Kirsch, David G., E-mail: david.kirsch@duke.edu [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States)

    2013-05-01

    Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

  15. Quantitative method to assess caries via fluorescence imaging from the perspective of autofluorescence spectral analysis

    Science.gov (United States)

    Chen, Q. G.; Zhu, H. H.; Xu, Y.; Lin, B.; Chen, H.

    2015-08-01

    A quantitative method to discriminate caries lesions for a fluorescence imaging system is proposed in this paper. The autofluorescence spectral investigation of 39 teeth samples classified by the International Caries Detection and Assessment System levels was performed at 405 nm excitation. The major differences in the different caries lesions focused on the relative spectral intensity range of 565-750 nm. The spectral parameter, defined as the ratio of wavebands at 565-750 nm to the whole spectral range, was calculated. The image component ratio R/(G + B) of color components was statistically computed by considering the spectral parameters (e.g. autofluorescence, optical filter, and spectral sensitivity) in our fluorescence color imaging system. Results showed that the spectral parameter and image component ratio presented a linear relation. Therefore, the image component ratio was graded as 1.62 to quantitatively classify sound, early decay, established decay, and severe decay tissues, respectively. Finally, the fluorescence images of caries were experimentally obtained, and the corresponding image component ratio distribution was compared with the classification result. A method to determine the numerical grades of caries using a fluorescence imaging system was proposed. This method can be applied to similar imaging systems.

  16. Increased Depth of Cellular Imaging in the Intact Lung Using Far-Red and Near-Infrared Fluorescent Probes

    Directory of Open Access Journals (Sweden)

    Brian D. Gray

    2006-03-01

    Full Text Available Scattering of shorter-wavelength visible light limits the fluorescence imaging depth of thick specimens such as whole organs. In this study, we report the use of four newly synthesized near-infrared and far-red fluorescence probes (excitation/emission, in nm: 644/670; 683/707; 786/814; 824/834 to image tumor cells in the subpleural vasculature of the intact rat lungs. Transpelural imaging of tumor cells labeled with long-wavelength probes and expressing green fluorescent protein (GFP; excitation/emission 488/507 nm was done in the intact rat lung after perfusate administration or intravenous injection. Our results show that the average optimum imaging depth for the long-wavelength probes is higher (27.8±0.7 μm than for GFP (20±0.5 μm; p=0.008; n=50, corresponding to a 40% increase in the volume of tissue accessible for high-resolution imaging. The maximum depth of cell visualization was significantly improved with the novel dyes (36.4±1 μm from the pleural surface compared with GFP (30.1±0.5 μm; p=0.01; n=50. Stable binding of the long-wavelength vital dyes to the plasma membrane also permitted in vivo tracking of injected tumor cells in the pulmonary vasculature. These probes offer a significant improvement in the imaging quality of in situ biological processes in the deeper regions of intact lungs.

  17. Increased Depth of Cellular Imaging in the Intact Lung Using Far-Red and Near-Infrared Fluorescent Probes

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available Scattering of shorter-wavelength visible light limits the fluorescence imaging depth of thick specimens such as whole organs. In this study, we report the use of four newly synthesized near-infrared and far-red fluorescence probes (excitation/emission, in nm: 644/670; 683/707; 786/814; 824/834 to image tumor cells in the subpleural vasculature of the intact rat lungs. Transpelural imaging of tumor cells labeled with long-wavelength probes and expressing green fluorescent protein (GFP; excitation/emission 488/507 nm was done in the intact rat lung after perfusate administration or intravenous injection. Our results show that the average optimum imaging depth for the long-wavelength probes is higher ( 27.8±0.7 μ m than for GFP ( 20±0.5 μ m; p=0.008 ; n=50 , corresponding to a 40% increase in the volume of tissue accessible for high-resolution imaging. The maximum depth of cell visualization was significantly improved with the novel dyes ( 36.4±1 μ m from the pleural surface compared with GFP ( 30.1±0.5 μ m; p=0.01 ; n=50 . Stable binding of the long-wavelength vital dyes to the plasma membrane also permitted in vivo tracking of injected tumor cells in the pulmonary vasculature. These probes offer a significant improvement in the imaging quality of in situ biological processes in the deeper regions of intact lungs.

  18. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, B-M.; Stoller, P.; Reiser, K.; Eichler, J.; Yan, M.; Rubenchik, A.; Da Silva, L.

    2000-03-06

    A confocal microscopy imaging system was devised to selectively detect Second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.

  19. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    International Nuclear Information System (INIS)

    A confocal microscopy imaging system was devised to selectively detect Second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing

  20. Stepwise multi-photon activation fluorescence reveals a new method of melanoma imaging for dermatologists

    Science.gov (United States)

    Lai, Zhenhua; Lian, Christine; Ma, Jie; Yu, Jingyi; Gu, Zetong; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2014-02-01

    Previous research has shown that the stepwise multi-photon activated fluorescence (SMPAF) of melanin, activated by a continuous-wave (CW) mode near infrared (NIR) laser, is a low cost and reliable method of detecting melanin. SMPAF images of melanin in a mouse hair and a formalin fixed mouse melanoma were compared with conventional multiphoton fluorescence microscopy (MPFM) images and confocal reflectance microscopy (CRM) images, all of which were acquired at an excitation wavelength of 920 nm, to further prove the effectiveness of SMPAF in detecting melanin. SMPAF images add specificity for melanin detection to MPFM images and CRM images. Melanin SMPAF can be a promising technology to enable melanoma imaging for dermatologists.

  1. Preliminary experiment of fluorescent X-ray computed tomography to detect dual agents for biological study.

    Science.gov (United States)

    Yu, Q; Takeda, T; Yuasa, T; Hasegawa, Y; Wu, J; Thet-Thet-Lwin; Hyodo, K; Dilmanian, F A; Itai, Y; Akatsuka, T

    2001-05-01

    The simultaneous observation of various information, such as blood flow, tissue metabolism and distribution of receptors, is quite important in order to understand the functional state of biomedical objects. The simultaneous detectability of contrast agents by fluorescent X-ray computed tomography (FXCT) with synchrotron radiation is examined in this study. The system consisted of a silicon (111) double-crystal monochromator, an X-ray slit system, a scanning table, a PIN diode, a highly purified germanium detector and an X-ray charge-coupled device (CCD) camera. The monochromatic X-ray beam energy was adjusted to 37.0 keV and collimated into a pencil beam of 1 x 1 mm. The fluorescent spectra of the K alpha lines for iodine and xenon were detected simultaneously. FXCT could image the distribution of both iodine and xenon agents in a phantom clearly and the contrast ratio was significantly better than that of transmission X-ray computed tomography images. PMID:11486409

  2. Membrane Permeable Esterase–Activated Fluorescent Imaging Probe

    OpenAIRE

    Kim, Youngmi; Choi, Yongdoo; Weissleder, Ralph; Tung, Ching-Hsuan

    2007-01-01

    An esterase-triggered probe 2 derived from a cyanine-based pH sensitive dye was developed for cell labeling. Permeation of probe 2 into cells and subsequent hydrolytic activation by cellular esterases result in a bright fluorescent intracellular signal.

  3. Histological image classification using biologically interpretable shape-based features

    International Nuclear Information System (INIS)

    Automatic cancer diagnostic systems based on histological image classification are important for improving therapeutic decisions. Previous studies propose textural and morphological features for such systems. These features capture patterns in histological images that are useful for both cancer grading and subtyping. However, because many of these features lack a clear biological interpretation, pathologists may be reluctant to adopt these features for clinical diagnosis. We examine the utility of biologically interpretable shape-based features for classification of histological renal tumor images. Using Fourier shape descriptors, we extract shape-based features that capture the distribution of stain-enhanced cellular and tissue structures in each image and evaluate these features using a multi-class prediction model. We compare the predictive performance of the shape-based diagnostic model to that of traditional models, i.e., using textural, morphological and topological features. The shape-based model, with an average accuracy of 77%, outperforms or complements traditional models. We identify the most informative shapes for each renal tumor subtype from the top-selected features. Results suggest that these shapes are not only accurate diagnostic features, but also correlate with known biological characteristics of renal tumors. Shape-based analysis of histological renal tumor images accurately classifies disease subtypes and reveals biologically insightful discriminatory features. This method for shape-based analysis can be extended to other histological datasets to aid pathologists in diagnostic and therapeutic decisions

  4. Detecting the barium daughter in 136Xe 0-νββ decay using single-molecule fluorescence imaging techniques

    Science.gov (United States)

    Nygren, David R.

    2015-11-01

    Single-molecule fluorescent imaging may provide an avenue to efficiently detect the Ba++ daughter atom in the decay 136Xe → Ba + 2e-, and, unambiguously associate the birth point in space within the electron trajectories of the decay event. Chelation of doubly-charged alkaline earth elements such as calcium and barium by certain precursor molecules converts the resulting complex from a non-fluorescent to a fluorescent state. Repeated photo-excitation of a single fluorescent complex reveals both presence and location with high precision. This technique, widespread now in biochemistry, biophysics and biology, may permit a similar discriminating response in a large high-pressure xenon gas TPC for the Ba++ ion from xenon double-beta decay. The TPC measures the event time and energy of the two nascent electrons, as well as topology and position in 3-D from their trajectories in the gas. Measurement of the 2-D location of the molecular ion after arrival at the cathode plane permits an association of ion with the event. Demonstration of an efficient, highly specific detection of the barium daughter would provide a long-sought pathway to a background-free result in the search for this decay mode, of central importance for determining the nature of the neutrino.

  5. Chromosome translocations as biological dosimeter identified by in situ fluorescent hybridization (FISH)

    International Nuclear Information System (INIS)

    Chromosomal aberration analysis in lymphocytes is used for dose assessment by workers overexposed to ionizing radiation. The conventional method is based on the dicentric metaphase chromosomes and/or micronucleus scoring, which gives thoroughly satisfactory results for accidental and recent exposures. Nevertheless, when evaluating acute doses occurring in the past or accidents where the time elapsed between exposure and analysis is unknown, the translocations scoring could be the method to use. This is because translocations retain the cell's reproductive potential so that they persist over time. For a long time the detection of translocations for dosimetric purposes was impractical as a routine method until situ fluorescent hybridization (FISH) techniques appeared. This method arising from molecular biology and applied to biological dosimetry has greatly simplified detection and although it is expensive, requiring special infrastructure, its availability is a sign of the state of development for these techniques in the region. The use of translocations scoring for biological dosimetry, as for the dicentric scoring, requires calibration curves for the different types of ionizing radiation. Therefore, the aim of this work was to prepare a dose-effect curve for Co-60 γ rays by chromosome translocations analysis. The study material was peripheral lymphocytes from a clinically healthy donor, irradiated and cultured in vitro and the detection of translocations in chromosomes 1, 2 and 4. The results showed a dose effect curve linear quadratic, accordingly with other authors

  6. Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

    Directory of Open Access Journals (Sweden)

    Haibiao Gong

    Full Text Available Fluorescence in the near-infrared (NIR spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP(f is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG substrate, leading to covalent attachment of the fluorescent dye to the SNAP(f. This property makes SNAP(f a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAP(f-Beta-2 adrenergic receptor (SNAP(f-ADRβ2 fusion protein were created. The ADRβ2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP(f-ADRβ2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAP(f-ADRβ2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAP(f-ADRβ2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAP(f-ADRβ2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.

  7. Red to far-red multispectral fluorescence image fusion for detection of fecal contamination on apples

    Science.gov (United States)

    This research developed a multispectral algorithm derived from hyperspectral line-scan fluorescence imaging under violet/blue LED excitation for detection of fecal contamination on Golden Delicious apples. Using a hyperspectral line-scan imaging system consisting of an EMCCD camera, spectrograph, an...

  8. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    International Nuclear Information System (INIS)

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 106-3 x 108 labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 106 cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells. (orig.)

  9. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daldrup-Link, Heike E. [Department of Radiology, UCSF Medical Center, University of California in San Francisco, 513 Parnassus Ave, CA 94143, San Francisco (United States); Rudelius, Martina; Piontek, Guido; Schlegel, Juergen [Institute of Pathology, Technical University, Munich (Germany); Metz, Stephan; Settles, Marcus; Rummeny, Ernst J. [Department of Radiology, Technical University, Munich (Germany); Pichler, Bernd [Department of Biomedical Engineering, University of California Davis, Davis (United States); Heinzmann, Ulrich [National Research Center for Environment and Health, Technical University, Munich (Germany); Oostendorp, Robert A.J. [3. Clinic of Internal Medicine, Laboratory of Stem Cell Physiology, Technical University, Munich (Germany)

    2004-09-01

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10{sup 6}-3 x 10{sup 8} labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10{sup 6} cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells

  10. The possibilities of improvement in the sensitivity of cancer fluorescence diagnostics by computer image processing

    Science.gov (United States)

    Ledwon, Aleksandra; Bieda, Robert; Kawczyk-Krupka, Aleksandra; Polanski, Andrzej; Wojciechowski, Konrad; Latos, Wojciech; Sieron-Stoltny, Karolina; Sieron, Aleksander

    2008-02-01

    Background: Fluorescence diagnostics uses the ability of tissues to fluoresce after exposition to a specific wavelength of light. The change in fluorescence between normal and progression to cancer allows to see early cancer and precancerous lesions often missed by white light. Aim: To improve by computer image processing the sensitivity of fluorescence images obtained during examination of skin, oral cavity, vulva and cervix lesions, during endoscopy, cystoscopy and bronchoscopy using Xillix ONCOLIFE. Methods: Function of image f(x,y):R2 --> R 3 was transformed from original color space RGB to space in which vector of 46 values refers to every point labeled by defined xy-coordinates- f(x,y):R2 --> R 46. By means of Fisher discriminator vector of attributes of concrete point analalyzed in the image was reduced according to two defined classes defined as pathologic areas (foreground) and healthy areas (background). As a result the highest four fisher's coefficients allowing the greatest separation between points of pathologic (foreground) and healthy (background) areas were chosen. In this way new function f(x,y):R2 --> R 4 was created in which point x,y corresponds with vector Y, H, a*, c II. In the second step using Gaussian Mixtures and Expectation-Maximisation appropriate classificator was constructed. This classificator enables determination of probability that the selected pixel of analyzed image is a pathologically changed point (foreground) or healthy one (background). Obtained map of probability distribution was presented by means of pseudocolors. Results: Image processing techniques improve the sensitivity, quality and sharpness of original fluorescence images. Conclusion: Computer image processing enables better visualization of suspected areas examined by means of fluorescence diagnostics.

  11. Pinhole X-ray Fluorescence Imaging of Gadolinium Nanoparticles: A Preliminary Monte Carlo Study

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Seong Moon; Sung, Won Mo; Ye, Sung Joon [Seoul National University, Seoul (Korea, Republic of)

    2014-10-15

    X-ray fluorescence imaging is a modality for the element-specific imaging of a subject through analysis of characteristic x-rays produced by exploiting the interaction of high atomic number elements and incoming x-rays. Previous studies have utilized a polychromatic x-ray source to investigate the production of in vivo x-ray fluorescence images for the assessment of concentrations and locations of gold nanoparticles. However, previous efforts have so far been unable to detect low concentrations, such as 0.001% gold by weight, which is an expected concentration accumulated in tumors. We examined the feasibility of a monochromatic synchrotron x-rays implementation of pinhole x-ray fluorescence imaging by Monte Carlo simulations using MCNP5. In the current study, gadolinium (Gd) nanoparticles, which have been widely used as a contrast agent in magnetic resonance imaging and also as a dose enhancer in radiation therapy, were chosen for tumor targeting. Since a monochromatic x-ray source is used, the increased x-ray fluorescence signals allow the detection of low concentrations of Gd. Two different monochromatic x-ray beam energies, 50.5 keV near the Kedge energy (i.e., 50.207 keV) of Gd and 55 keV, were compared by their respective imaging results. Using Monte Carlo simulations the feasibility of imaging low concentrations of Gd nanoparticles (e.g., 0.001 wt%) with x-ray fluorescence using monochromatic synchrotron x-rays of two different energies was shown. In the case of imaging a single Gd column inserted in the center of a water phantom, the fluorescence signals from 0.05 wt% and 0.1 wt% Gd columns irradiated with a 50.5 keV photon beam were higher than those irradiated with 55 keV. Below 0.05 wt% region no significant differences were found.

  12. Pinhole X-ray Fluorescence Imaging of Gadolinium Nanoparticles: A Preliminary Monte Carlo Study

    International Nuclear Information System (INIS)

    X-ray fluorescence imaging is a modality for the element-specific imaging of a subject through analysis of characteristic x-rays produced by exploiting the interaction of high atomic number elements and incoming x-rays. Previous studies have utilized a polychromatic x-ray source to investigate the production of in vivo x-ray fluorescence images for the assessment of concentrations and locations of gold nanoparticles. However, previous efforts have so far been unable to detect low concentrations, such as 0.001% gold by weight, which is an expected concentration accumulated in tumors. We examined the feasibility of a monochromatic synchrotron x-rays implementation of pinhole x-ray fluorescence imaging by Monte Carlo simulations using MCNP5. In the current study, gadolinium (Gd) nanoparticles, which have been widely used as a contrast agent in magnetic resonance imaging and also as a dose enhancer in radiation therapy, were chosen for tumor targeting. Since a monochromatic x-ray source is used, the increased x-ray fluorescence signals allow the detection of low concentrations of Gd. Two different monochromatic x-ray beam energies, 50.5 keV near the Kedge energy (i.e., 50.207 keV) of Gd and 55 keV, were compared by their respective imaging results. Using Monte Carlo simulations the feasibility of imaging low concentrations of Gd nanoparticles (e.g., 0.001 wt%) with x-ray fluorescence using monochromatic synchrotron x-rays of two different energies was shown. In the case of imaging a single Gd column inserted in the center of a water phantom, the fluorescence signals from 0.05 wt% and 0.1 wt% Gd columns irradiated with a 50.5 keV photon beam were higher than those irradiated with 55 keV. Below 0.05 wt% region no significant differences were found

  13. Fluorescence background subtraction technique for hybrid fluorescence molecular tomography/x-ray computed tomography imaging of a mouse model of early stage lung cancer

    Science.gov (United States)

    Ale, Angelique; Ermolayev, Vladimir; Deliolanis, Nikolaos C.; Ntziachristos, Vasilis

    2013-05-01

    The ability to visualize early stage lung cancer is important in the study of biomarkers and targeting agents that could lead to earlier diagnosis. The recent development of hybrid free-space 360-deg fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) imaging yields a superior optical imaging modality for three-dimensional small animal fluorescence imaging over stand-alone optical systems. Imaging accuracy was improved by using XCT information in the fluorescence reconstruction method. Despite this progress, the detection sensitivity of targeted fluorescence agents remains limited by nonspecific background accumulation of the fluorochrome employed, which complicates early detection of murine cancers. Therefore we examine whether x-ray CT information and bulk fluorescence detection can be combined to increase detection sensitivity. Correspondingly, we research the performance of a data-driven fluorescence background estimator employed for subtraction of background fluorescence from acquisition data. Using mice containing known fluorochromes ex vivo, we demonstrate the reduction of background signals from reconstructed images and sensitivity improvements. Finally, by applying the method to in vivo data from K-ras transgenic mice developing lung cancer, we find small tumors at an early stage compared with reconstructions performed using raw data. We conclude with the benefits of employing fluorescence subtraction in hybrid FMT-XCT for early detection studies.

  14. Functioning Nanomachines Seen in Real-Time in Living Bacteria Using Single-Molecule and Super-Resolution Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Sheng-Wen Chiu

    2011-04-01

    Full Text Available Molecular machines are examples of “pre-established” nanotechnology, driving the basic biochemistry of living cells. They encompass an enormous range of function, including fuel generation for chemical processes, transport of molecular components within the cell, cellular mobility, signal transduction and the replication of the genetic code, amongst many others. Much of our understanding of such nanometer length scale machines has come from in vitro studies performed in isolated, artificial conditions. Researchers are now tackling the challenges of studying nanomachines in their native environments. In this review, we outline recent in vivo investigations on nanomachines in model bacterial systems using state-of-the-art genetics technology combined with cutting-edge single-molecule and super-resolution fluorescence microscopy. We conclude that single-molecule and super-resolution fluorescence imaging provide powerful tools for the biochemical, structural and functional characterization of biological nanomachines. The integrative spatial, temporal, and single-molecule data obtained simultaneously from fluorescence imaging open an avenue for systems-level single-molecule cellular biophysics and in vivo biochemistry.

  15. Easy synthesis and imaging applications of cross-linked green fluorescent hollow carbon nanoparticles.

    Science.gov (United States)

    Fang, Youxing; Guo, Shaojun; Li, Dan; Zhu, Chengzhou; Ren, Wen; Dong, Shaojun; Wang, Erkang

    2012-01-24

    We propose an ingenious method for synthesizing cross-linked hollow fluorescent carbon nanoparticles (HFCNs) with green emission by simply mixing acetic acid, water, and diphosphorus pentoxide. This is an automatic method without external heat treatment to rapidly produce large quantities of HFCNs, in contrast to other syntheses of fluorescent carbon nanoparticles that required high temperature, complicated operations, or long reaction times. Characterizations of HFCNs through high-resolution transmission electron microscopy, infrared/Raman spectroscopy, and X-ray diffraction indicate that abundant small oxygenous graphite domains existed and endowed the HFCNs with fluorescent properties. After simple post-treatments, the cross-linked HFCNs can be used for cell-imaging applications. Compared with traditional dyes and CdTe quantum dots, HFCNs are the superior fluorescent bioimaging agent according to their low toxicity, stability, and resistance to photobleaching. The HFCNs were also applied to watermark ink and fluorescent powder, showing their promising potentials for further wide usage. PMID:22188541

  16. An exciton-polariton laser based on biologically produced fluorescent protein

    Science.gov (United States)

    Dietrich, Christof P.; Steude, Anja; Tropf, Laura; Schubert, Marcel; Kronenberg, Nils M.; Ostermann, Kai; Höfling, Sven; Gather, Malte C.

    2016-01-01

    Under adequate conditions, cavity polaritons form a macroscopic coherent quantum state, known as polariton condensate. Compared to Wannier-Mott excitons in inorganic semiconductors, the localized Frenkel excitons in organic emitter materials show weaker interaction with each other but stronger coupling to light, which recently enabled the first realization of a polariton condensate at room temperature. However, this required ultrafast optical pumping, which limits the applications of organic polariton condensates. We demonstrate room temperature polariton condensates of cavity polaritons in simple laminated microcavities filled with biologically produced enhanced green fluorescent protein (eGFP). The unique molecular structure of eGFP prevents exciton annihilation even at high excitation densities, thus facilitating polariton condensation under conventional nanosecond pumping. Condensation is clearly evidenced by a distinct threshold, an interaction-induced blueshift of the condensate, long-range coherence, and the presence of a second threshold at higher excitation density that is associated with the onset of photon lasing. PMID:27551686

  17. An exciton-polariton laser based on biologically produced fluorescent protein.

    Science.gov (United States)

    Dietrich, Christof P; Steude, Anja; Tropf, Laura; Schubert, Marcel; Kronenberg, Nils M; Ostermann, Kai; Höfling, Sven; Gather, Malte C

    2016-08-01

    Under adequate conditions, cavity polaritons form a macroscopic coherent quantum state, known as polariton condensate. Compared to Wannier-Mott excitons in inorganic semiconductors, the localized Frenkel excitons in organic emitter materials show weaker interaction with each other but stronger coupling to light, which recently enabled the first realization of a polariton condensate at room temperature. However, this required ultrafast optical pumping, which limits the applications of organic polariton condensates. We demonstrate room temperature polariton condensates of cavity polaritons in simple laminated microcavities filled with biologically produced enhanced green fluorescent protein (eGFP). The unique molecular structure of eGFP prevents exciton annihilation even at high excitation densities, thus facilitating polariton condensation under conventional nanosecond pumping. Condensation is clearly evidenced by a distinct threshold, an interaction-induced blueshift of the condensate, long-range coherence, and the presence of a second threshold at higher excitation density that is associated with the onset of photon lasing. PMID:27551686

  18. Development of fluorescence based handheld imaging devices for food safety inspection

    Science.gov (United States)

    Lee, Hoyoung; Kim, Moon S.; Chao, Kuanglin; Lefcourt, Alan M.; Chan, Diane E.

    2013-05-01

    For sanitation inspection in food processing environment, fluorescence imaging can be a very useful method because many organic materials reveal unique fluorescence emissions when excited by UV or violet radiation. Although some fluorescence-based automated inspection instrumentation has been developed for food products, there remains a need for devices that can assist on-site inspectors performing visual sanitation inspection of the surfaces of food processing/handling equipment. This paper reports the development of an inexpensive handheld imaging device designed to visualize fluorescence emissions and intended to help detect the presence of fecal contaminants, organic residues, and bacterial biofilms at multispectral fluorescence emission bands. The device consists of a miniature camera, multispectral (interference) filters, and high power LED illumination. With WiFi communication, live inspection images from the device can be displayed on smartphone or tablet devices. This imaging device could be a useful tool for assessing the effectiveness of sanitation procedures and for helping processors to minimize food safety risks or determine potential problem areas. This paper presents the design and development including evaluation and optimization of the hardware components of the imaging devices.

  19. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    International Nuclear Information System (INIS)

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases

  20. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dokyoung; Jun, Yong Woong; Ahn, Kyo Han [POSTECH, Pohang (Korea, Republic of)

    2014-05-15

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

  1. Temperature-modulated fluorescence tomography in a turbid media

    OpenAIRE

    Lin, Yuting; Bolisay, Linden; Ghijsen, Michael; Kwong, Tiffany C.; Gulsen, Gultekin

    2012-01-01

    High scattering in biological tissues makes fluorescence tomography inverse problem very challenging in thick medium. We describe an approach termed “temperature-modulated fluorescence tomography” that can acquire fluorescence images at focused ultrasound resolution. By utilizing recently emerged temperature sensitive fluorescence contrast agents, this technique provides fluorescence images with high resolution prior to any reconstruction process. We demonstrate that this technique is well su...

  2. Magnetic resonance-coupled fluorescence tomography scanner for molecular imaging of tissue

    Science.gov (United States)

    Davis, Scott C.; Pogue, Brian W.; Springett, Roger; Leussler, Christoph; Mazurkewitz, Peter; Tuttle, Stephen B.; Gibbs-Strauss, Summer L.; Jiang, Shudong S.; Dehghani, Hamid; Paulsen, Keith D.

    2008-06-01

    A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1nM in a 70mm diameter homogeneous phantom, and detection is feasible to near 10pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.

  3. Fluorescence lifetime imaging of endogenous biomarker of oxidative stress.

    OpenAIRE

    Rupsa Datta; Alba Alfonso-García; Rachel Cinco; Enrico Gratton

    2015-01-01

    Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be a...

  4. New image colocalization coefficient for fluorescence microscopy to quantify (bio-)molecular interactions

    OpenAIRE

    Herce, HD; Casas-Delucchi, CS; Cardoso, MC

    2013-01-01

    The spatial relationship, or degree of colocalization, between two or more types of molecules in live cells is commonly detected using fluorescence microscopy. This spatial distribution can be used to estimate the interaction between fluorescently labelled molecules. These interactions are usually quantified by analysing the correlation and/or the overlap between images, using the Pearson's and Manders’ coefficients, respectively. However, the correlation and overlap coefficients are paramete...

  5. Glucosamine-Bound Near-Infrared Fluorescent Probes with Lysosomal Specificity for Breast Tumor Imaging1

    OpenAIRE

    Li, Cong; Greenwood, Tiffany R; Glunde, Kristine

    2008-01-01

    Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of thes...

  6. Glucosamine-Bound Near-Infrared Fluorescent Probes with Lysosomal Specificity for Breast Tumor Imaging

    OpenAIRE

    Cong Li; Greenwood, Tiffany R; Kristine Glunde

    2008-01-01

    Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of thes...

  7. Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy

    OpenAIRE

    Day, Richard N

    2013-01-01

    The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster Resonance Energy Transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained fro...

  8. CMOS Time-Resolved, Contact, and Multispectral Fluorescence Imaging for DNA Molecular Diagnostics

    Directory of Open Access Journals (Sweden)

    Nan Guo

    2014-10-01

    Full Text Available Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art.

  9. Method and apparatus to image biological interactions in plants

    Science.gov (United States)

    Weisenberger, Andrew; Bonito, Gregory M.; Reid, Chantal D.; Smith, Mark Frederick

    2015-12-22

    A method to dynamically image the actual translocation of molecular compounds of interest in a plant root, root system, and rhizosphere without disturbing the root or the soil. The technique makes use of radioactive isotopes as tracers to label molecules of interest and to image their distribution in the plant and/or soil. The method allows for the study and imaging of various biological and biochemical interactions in the rhizosphere of a plant, including, but not limited to, mycorrhizal associations in such regions.

  10. Synthetic strategies for controlling inter- and intramolecular interactions: Applications in single-molecule fluorescence imaging, bioluminescence imaging, and palladium catalysis

    Science.gov (United States)

    Conley, Nicholas R.

    The field of synthetic organic chemistry has reached such maturity that, with sufficient effort and resources, the synthesis of virtually any small molecule which exhibits reasonable stability at room temperature can be realized. While representing a monumental achievement for the field, the ability to exert precise control over molecular structure is just a means to an end, and it is frequently the responsibility of the synthetic chemist to determine which molecules should actually be synthesized. For better or worse, there exists no competitive free market in academia for new molecules, and as a result, the decision of which compounds should be synthesized is seldom driven by the forces of supply and demand; rather, it is guided by the synthetic chemist's interest in an anticipated structure-function relationship or in the properties of a previously unstudied class of molecules. As a consequence, there exists a pervasive need for chemists with synthetic expertise in fields (e.g., molecular imaging) and subdisciplines of chemistry (e.g., physical chemistry) in which the identification of promising synthetic targets dramatically outpaces the synthetic output in that field or subdiscipline, and ample opportunities are available for synthetic chemists who choose to pursue such cross-disciplinary research. This thesis describes synthetic efforts that leverage these opportunities to realize applications in biological imaging and in palladium catalysis. In Part I, the synthesis and characterization of three novel luminophores and their imaging applications are discussed. The first is a molecular beacon that utilizes a fluorophorefluorophore pair which exhibits H-dimer quenching in the closed conformation. This probe offers several advantages over conventional fluorophore-quencher molecular beacons in the detection of oligonucleotides, both in bulk and at the single-molecule level. Secondly, a fluorescent, Cy3-Cy5 covalent heterodimer is reported, which on account of the

  11. A two-photon fluorescent turn-on probe for imaging of SO2 derivatives in living cells and tissues.

    Science.gov (United States)

    Zhu, Xiaoyan; Zhu, Longming; Liu, Hong-Wen; Hu, Xiaoxiao; Peng, Rui-Zi; Zhang, Jing; Zhang, Xiao-Bing; Tan, Weihong

    2016-09-21

    SO2 and its derivatives (bisulfite/sulfite) play crucial roles in several physiological processes. Therefore, development of reliable analytical methods for monitoring SO2 and its derivatives in biological systems is very significant. In this paper, a FRET-based two-photon fluorescent turn-on probe, A-HCy, was proposed for specific detection of SO2 derivatives through the bisulfite/sulfite-promoted Michael addition reaction. In this FRET system, an acedan (2-acetyl-6-dialkylaminonaphthalene) moiety was selected as a two-photon donor and a hemicyanine derivative served as both the quencher and the recognition unit for bisulfite/sulfite. A-HCy exhibited excellent selectivity and rapid response to HSO3(-) with a detection limit of 0.24 μM. More importantly, probe A-HCy was first successfully applied in two-photon fluorescence imaging of biological SO2 derivatives in living cells and tissues, suggesting its great potential for practical application in biological systems. PMID:27590555

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

    OpenAIRE

    Hino, Haruaki; Kamiya, Mako; Kitano, Kentaro; Mizuno, Kazue; Tanaka, Sayaka; Nishiyama, Nobuhiro; Kataoka, Kazunori; Urano, Yasuteru; Nakajima, Jun

    2016-01-01

    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 mod...

  13. Simulation of air shower image in fluorescence light based on energy deposits derived from CORSIKA

    OpenAIRE

    Gora, D.; Heck, D.; Homola, P.; Klages, H.; Pekala, J.; Risse, M.; Wilczynska, B.; Wilczynski, H.

    2004-01-01

    Spatial distributions of energy deposited by an extensive air shower in the atmosphere through ionization, as obtained from the CORSIKA simulation program, are used to find the fluorescence light distribution in the optical image of the shower. The shower image derived in this way is somewhat smaller than that obtained from the NKG lateral distribution of particles in the shower. The size of the image shows a small dependence on the primary particle type.

  14. Image-guided tumor surgery: will there be a role for fluorescent nanoparticles?

    Science.gov (United States)

    Hill, Tanner K; Mohs, Aaron M

    2016-07-01

    Image-guided surgery (IGS) using fluorescent nanoparticles (NPs) has the potential to substantially impact patient treatment. The use of fluorescence imaging provides surgeons with real-time feedback on the location of diseased tissue using safe, low-cost imaging agents and instrumentation. Fluorescent NPs are likely to play a role as they are capable of taking advantage of the enhanced permeability and retention (EPR) effect and can be modified to avoid clearance, increase circulation time, and specifically target tumors. Clinical trials of IGS using the FDA-approved fluorophores indocyanine green and methylene blue have already shown preliminary successes, and incorporation of fluorescent NPs will likely improve detection by providing higher signal to background ratio and reducing false-positive rates through active targeting. Preclinical development of fluorescent NP formulations is advancing rapidly, with strategies ranging from passive targeting to active targeting of cell surface receptors, creating pH-responsive NPs, and increasing cell uptake through cleavable proteins. This collective effort could lead to clinical trials using fluorescent NPs in the near future. WIREs Nanomed Nanobiotechnol 2016, 8:498-511. doi: 10.1002/wnan.1381 For further resources related to this article, please visit the WIREs website. PMID:26585556

  15. Highly confined, enhanced surface fluorescence imaging with two-dimensional silver nanoparticle sheets

    International Nuclear Information System (INIS)

    A method of obtaining highly confined, enhanced surface fluorescence imaging is proposed using two-dimensional (2D) silver nanoparticle (AgMy) sheets. This technique is based on the localized surface plasmon resonance excited homogeneously on a 2D silver nanoparticle sheet. The AgMy sheets are fabricated at the air–water interface by self-assembly and transferred onto hydrophobic glass substrates. These sheets can enhance the fluorescence only when the excitation wavelength overlaps with the plasmon resonance wavelength. To confirm the validity of this technique, two separate test experiments are performed. One is the epifluorescence microscope imaging of a quantum dot 2D sheet on the AgMy 2D sheet with a SiO2 spacer layer, where the fluorescence is maximized with the 20 nm SiO2 layer, determined by the Förster resonance energy transfer distances. The second experiment is the imaging of a single fluorescence bead with a total internal reflection fluorescent microscope. We confirmed that the AgMy sheet provides a 4-fold increase in fluorescence with a 160-nm spatial resolution at 30 ms/frame snapshot. The AgMy sheet will be a powerful tool for high sensitivity and high-resolution real time bioimaging at nanointerfaces

  16. Cisplatin Prodrug-Conjugated Gold Nanocluster for Fluorescence Imaging and Targeted Therapy of the Breast Cancer

    Science.gov (United States)

    Zhou, Fangyuan; Feng, Bing; Yu, Haijun; Wang, Dangge; Wang, Tingting; Liu, Jianping; Meng, Qingshuo; Wang, Siling; Zhang, Pengcheng; Zhang, Zhiwen; Li, Yaping

    2016-01-01

    Theranostic nanomedicine has emerged as a promising modality for cancer diagnosis and treatment. In this study, we report the fabrication of fluorescence gold nanoclusters (GNC) conjugated with a cisplatin prodrug and folic acid (FA) (FA-GNC-Pt) for fluorescence imaging and targeted chemotherapy of breast cancer. The physio-chemical properties of FA-GNC-Pt nanoparticles are thoroughly characterized by fluorescence/UV-Vis spectroscopic measurement, particle size and zeta-potential examination. We find that FA-modification significantly accelerated the cellular uptake and increased the cytotoxicity of GNC-Pt nanoparticles in murine 4T1 breast cancer cells. Fluorescence imaging in vivo using 4T1 tumor bearing nude mouse model shows that FA-GNC-Pt nanoparticles selectively accumulate in the orthotopic 4T1 tumor and generate strong fluorescence signal due to the tumor targeting effect of FA. Moreover, we demonstrate that FA-GNC-Pt nanoparticles significantly inhibit the growth and lung metastasis of the orthotopically implanted 4T1 breast tumors. All these data imply a good potential of the GNC-based theranostic nanoplatform for fluorescence tumor imaging and cancer therapy. PMID:27022415

  17. Improved tumor identification using dual tracer molecular imaging in fluorescence guided brain surgery

    Science.gov (United States)

    Xu, Xiaochun; Torres, Veronica; Straus, David; Brey, Eric M.; Byrne, Richard W.; Tichauer, Kenneth M.

    2015-03-01

    Brain tumors represent a leading cause of cancer death for people under the age of 40 and the probability complete surgical resection of brain tumors remains low owing to the invasive nature of these tumors and the consequences of damaging healthy brain tissue. Molecular imaging is an emerging approach that has the potential to improve the ability for surgeons to correctly discriminate between healthy and cancerous tissue; however, conventional molecular imaging approaches in brain suffer from significant background signal in healthy tissue or an inability target more invasive sections of the tumor. This work presents initial studies investigating the ability of novel dual-tracer molecular imaging strategies to be used to overcome the major limitations of conventional "single-tracer" molecular imaging. The approach is evaluated in simulations and in an in vivo mice study with animals inoculated orthotopically using fluorescent human glioma cells. An epidermal growth factor receptor (EGFR) targeted Affibody-fluorescent marker was employed as a targeted imaging agent, and the suitability of various FDA approved untargeted fluorescent tracers (e.g. fluorescein & indocyanine green) were evaluated in terms of their ability to account for nonspecific uptake and retention of the targeted imaging agent. Signal-to-background ratio was used to measure and compare the amount of reporter in the tissue between targeted and untargeted tracer. The initial findings suggest that FDA-approved fluorescent imaging agents are ill-suited to act as untargeted imaging agents for dual-tracer fluorescent guided brain surgery as they suffer from poor delivery to the healthy brain tissue and therefore cannot be used to identify nonspecific vs. specific uptake of the targeted imaging agent where current surgery is most limited.

  18. Fluorescence Imaging of Interscapular Brown Adipose Tissue in Living Mice†

    OpenAIRE

    Rice, Douglas R.; White, Alexander G.; Leevy, W. Matthew; Smith, Bradley D.

    2015-01-01

    Brown adipose tissue (BAT) plays a key role in energy expenditure and heat generation and is a promising target for diagnosing and treating obesity, diabetes and related metabolism disorders. While several nuclear and magnetic resonance imaging methods are established for detecting human BAT, there are no convenient protocols for high throughput imaging of BAT in small animal models. Here we disclose a simple but effective method for non-invasive optical imaging of interscapular BAT in mice u...

  19. Concentrations, size distributions and temporal variations of fluorescent biological aerosol particles in southern tropical India

    Science.gov (United States)

    Valsan, Aswathy; Krishna R, Ravi; CV, Biju; Huffman, Alex; Poschl, Ulrich; Gunthe, Sachin

    2015-04-01

    Biological aerosols constitute a wide range of dead and alive biological materials and structures that are suspended in the atmosphere. They play an important role in the atmospheric physical, chemical and biological processes and health of living being by spread of diseases among humans, plants, and, animals. The atmospheric abundance, sources, physical properties of PBAPs as compared to non-biological aerosols, however, is poorly characterized. The Indian tropical region, where large fraction of the world's total population is residing, experiences a distinctive meteorological phenomenon by means of Indian Summer Monsoon (IMS). Thus, the properties and characteristics of biological aerosols are also expected to be very diverse over the Indian subcontinent depending upon the seasons. Here we characterize the number concentration and size distribution of Fluorescent Biological Aerosol Particles (FBAP) at a high altitude continental site, Munnar (10.09 N, 77.06 E; 1605 m asl) in South India during the South-West monsoon, which constitute around 80 percent of the annual rainfall in Munnar. Continuous three months measurements (from 01 June 2014 to 21 Aug 2104) FBAPs were carried out at Munnar using Ultra Violet Aerodynamic Particle Sizer (UVAPS) during IMS. The mean number and mass concentration of coarse FBAP averaged over the entire campaign was 1.7 x 10-2 cm-3 and 0.24 µg m-3 respectively, which corresponds to 2 percent and 6 percent of total aerosol particle number and mass concentration. In agreement to other previous measurements the number size distribution of FBAP also peaks at 3.2 micron indicating the strong presence of fungal spores. This was also supported by the Scanning Electron Microscopic analysis of bioaerosols on filter paper. They also displayed a strong diurnal cycle with maximum concentration occurring at early morning hours. During periods of heavy and continuous rain where the wind is consistently blowing from South-West direction it was

  20. Potential of biological images for radiation therapy of cancer

    International Nuclear Information System (INIS)

    Full text: Recent technical advances in 3D conformal and intensity modulated radiotherapy (3DCRT and IMRT) based, on patient-specific CT and MRI images, have the potential of delivering exquisitely conformal dose distributions to the target volume while avoiding critical structures. Emerging clinical results in terms of reducing treatment-related morbidity and increasing local control appear promising. Recent developments in imaging have suggested that biological images may further positively impact cancer diagnosis, characterization and therapy. While in the past radiological images are largely anatomical, the new types of images can provide metabolic, biochemical, physiological, functional and molecular (genotypic and phenotypic) information. For radiation therapy, images that give information about factors (e.g. tumor hypoxia, Tpot) that influence radiosensitivity and treatment outcome can be regarded as radiobiological images. The ability of IMRT to 'paint' (in 2D) or 'sculpt' (in 3D) the dose, and produce exquisitely conformal dose distributions begs the '64 million dollar question' as to how to paint or sculpt, and whether biological imaging may provide the pertinent information. Can this new approach provide 'radiobiological phenotypes' non-invasively, and incrementally improve upon the predictive assays of radiobiological characteristics such as proliferative activity (Tpot - the potential doubling time), radiosensitivity (SF2 - the surviving fraction at a dose of 2 Gy), energy status (relative to sublethal damage repair), pH (a possible surrogate of hypoxia), tumor hypoxia, etc. as prognosticator(s) of radiation treatment outcome. Important for IMRT, the spatial (geometrical) distribution of the radiobiological phenotypes provide the basis for dose distribution design to conform to both the physical (geometrical) and the biological attributes. Copyright (2001) Australasian College of Physical Scientists and Engineers in Medicine

  1. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques

    International Nuclear Information System (INIS)

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied

  2. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques.

    Science.gov (United States)

    Wagner, D; Börgardts, M; Grünzweig, C; Lehmann, E; Müller, T J J; Egelhaaf, S U; Hermes, H E

    2015-09-01

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied. PMID:26429447

  3. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques

    Science.gov (United States)

    Wagner, D.; Börgardts, M.; Grünzweig, C.; Lehmann, E.; Müller, T. J. J.; Egelhaaf, S. U.; Hermes, H. E.

    2015-09-01

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied.

  4. Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

    Science.gov (United States)

    Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael, Jr.; Anton, Rein; Romanowski, Marek

    2015-10-01

    Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

  5. Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

    Science.gov (United States)

    Watson, Jeffrey R; Gainer, Christian F; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G Michael; Anton, Rein; Romanowski, Marek

    2015-10-01

    Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures. PMID:26440760

  6. Full-angle optical imaging of near-infrared fluorescent probes implanted in small animals

    Institute of Scientific and Technical Information of China (English)

    Gang Hu; Junjie Yao; Jing Bai

    2008-01-01

    To provide a valuable experimental platform for in vivo biomedical research of small animal model with fluorescence mediated approach, we developed a whole-body near-infrared fluorescence molecular imaging system as described in this paper. This system is based on a sensitive CCD camera and has the ability to achieve 360° full-angle source illuminations and projections capture of the targets to obtain the dense sampling by performing rotational scan. The measurement accuracy is validated from cylinder phantom experiments by the comparison between the experimental data and theoretical predictions. Finally, we also present typical in vivo images of fluorescent tube implanted into the mouse body. The results are promising and have proved the system imaging performance for macroscopic optical biomedical research.

  7. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, D.; Egelhaaf, S. U.; Hermes, H. E. [Condensed Matter Physics Laboratory, Heinrich Heine University, 40225 Düsseldorf (Germany); Börgardts, M.; Müller, T. J. J. [Institute for Organic and Macromolecular Chemistry, Heinrich Heine University, 40225 Düsseldorf (Germany); Grünzweig, C.; Lehmann, E. [Neutron Imaging and Activation Group, Paul Scherrer Institute, 5232 Villigen (Switzerland)

    2015-09-15

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied.

  8. Wide-field flexible endoscope for simultaneous color and NIR fluorescence image acquisition during surveillance colonoscopy

    Science.gov (United States)

    García-Allende, P. Beatriz; Nagengast, Wouter B.; Glatz, Jürgen; Ntziachristos, Vasilis

    2013-03-01

    Colorectal cancer (CRC) is the third most common form of cancer and, despite recent declines in both incidence and mortality, it still remains the second leading cause of cancer-related deaths in the western world. Colonoscopy is the standard for detection and removal of premalignant lesions to prevent CRC. The major challenges that physicians face during surveillance colonoscopy are the high adenoma miss-rates and the lack of functional information to facilitate decision-making concerning which lesions to remove. Targeted imaging with NIR fluorescence would address these limitations. Tissue penetration is increased in the NIR range while the combination with targeted NIR fluorescent agents provides molecularly specific detection of cancer cells, i.e. a red-flag detection strategy that allows tumor imaging with optimal sensitivity and specificity. The development of a flexible endoscopic fluorescence imaging method that can be integrated with standard medical endoscopes and facilitates the clinical use of this potential is described in this work. A semi-disposable coherent fiber optic imaging bundle that is traditionally employed in the exploration of biliary and pancreatic ducts is proposed, since it is long and thin enough to be guided through the working channel of a traditional video colonoscope allowing visualization of proximal lesions in the colon. A custom developed zoom system magnifies the image of the proximal end of the imaging bundle to fill the dimensions of two cameras operating in parallel providing the simultaneous color and fluorescence video acquisition.

  9. Novel fluorescence-based integrated sensor for chemical and biological agent detection

    Science.gov (United States)

    Frye-Mason, Greg; Leuschen, Martin; Wald, Lara; Paul, Kateri; Hancock, Lawrence F.; Fagan, Steve; Krouse, Justin; Hutchinson, Kira D.

    2004-12-01

    There is a renewed interest in the development of chemical and biological agent sensors due to the increased threat of weapons deployment by terrorist organizations and rogue states. Optically based sensors address the needs of military and homeland security forces in that they are reliable, rapidly deployed, and can provide continuous monitoring with little to no operator involvement. Nomadics has developed optically based chemical weapons sensors that utilize reactive fluorescent chromophores initially developed by Professor Tim Swager at MIT. The chromophores provide unprecedented sensitivity and selectivity toward toxic industrial chemicals and certain chemical weapon agents. The selectivity is based upon the reactivity of the G-class nerve agents (phosphorylation of acetylcholinesterase enzyme) that makes them toxic. Because the sensor recognizes the reactivity of strong electrophiles and not molecular weight, chemical affinity or ionizability, our system detects a specific class of reactive agents and will be able to detect newly developed or modified agents that are not currently known. We have recently extended this work to pursue a combined chemical/biological agent sensor system incorporating technologies based upon novel deep ultraviolet (UV) light emitting diodes (LEDs) developed out of the DARPA Semiconductor UV Optical Sources (SUVOS) program.

  10. Application of blue-green and ultraviolet micro-LEDs to biological imaging and detection

    International Nuclear Information System (INIS)

    This paper reviews authors' laboratory's work on the development of nitride-based blue-green and ultraviolet microscale LED devices with particular classes of imaging and spectroscopic applications in cellular level biology. Starting from neuroscience, we illustrate the utility of blue-green micro-LEDs for voltage-sensitive dye imaging of individual neural cells, as well as their ultraviolet counterparts for photostimulation of neurons. Arrays of micro-LEDs are also shown to be useful in projecting spatiotemporal patterns of photoexcitation to study the visual system development in living animals. As another illustration of the utility of the emerging nitride microdevice technology, we demonstrate the application of UV micro-LED arrays in bio-sensing technology as the core of a real-time fluorescence spectroscopy biowarning system. (invited paper)

  11. In vivo stepwise multi-photon activation fluorescence imaging of melanin in human skin

    Science.gov (United States)

    Lai, Zhenhua; Gu, Zetong; Abbas, Saleh; Lowe, Jared; Sierra, Heidy; Rajadhyaksha, Milind; DiMarzio, Charles

    2014-03-01

    The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

  12. Fluorescent Properties of Au Nanoclusters and Their Biological Applications%金团簇的荧光性质及其生物应用

    Institute of Scientific and Technical Information of China (English)

    任娟; 代雯乐; 付欣; 苏梦; 蔡晶晶; 马占芳

    2011-01-01

    金纳米团簇作为一类新型纳米材料具有独特的光学特性。当金纳米团簇颗粒的尺寸小到与电子的费米波长(〈1nm)相当时,由于量子尺寸效应,金颗粒会受激发射出荧光。作为一种新型荧光材料,金纳米团簇具有发光颜色随团簇尺寸可调、荧光不易猝灭等许多优势。本文主要综述了金纳米团簇的荧光性质及其在生物标记、生物成像以及生物检测等方面的应用。%Au nanoclusters have emerged as a class of materials with unique optical properties.When the size of Au nanocluster decrease to comparable to the Fermi wavelength(1nm) of an electron,it exhibits fluorescent characteristics while they are excited,which resulted from its quantum effect.As a novel fluorescent material,Au nanoclusters show size-dependent emission,a slower photobleaching rate and very good stability.The fluorescent properties of Au nanoclusters and their applications in the fields of biological labeling,biological imaging,and biodetection were mainly reviewed.

  13. Iodinated oil-loaded, fluorescent mesoporous silica-coated iron oxide nanoparticles for magnetic resonance imaging/computed tomography/fluorescence trimodal imaging

    Directory of Open Access Journals (Sweden)

    Xue S

    2014-05-01

    Full Text Available Sihan Xue,1 Yao Wang,1 Mengxing Wang,2 Lu Zhang,1 Xiaoxia Du,2 Hongchen Gu,1 Chunfu Zhang1,31School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, 2Shanghai Key Laboratory of Magnetic Resonance, Department of Physics, East China Normal University, 3State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaAbstract: In this study, a novel magnetic resonance imaging (MRI/computed tomography (CT/fluorescence trifunctional probe was prepared by loading iodinated oil into fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (i-fmSiO4@SPIONs. Fluorescent mesoporous silica-coated superparamagnetic iron oxide nanoparticles (fmSiO4@SPIONs were prepared by growing fluorescent dye-doped silica onto superparamagnetic iron oxide nanoparticles (SPIONs directed by a cetyltrimethylammonium bromide template. As prepared, fmSiO4@SPIONs had a uniform size, a large surface area, and a large pore volume, which demonstrated high efficiency for iodinated oil loading. Iodinated oil loading did not change the sizes of fmSiO4@SPIONs, but they reduced the MRI T2 relaxivity (r2 markedly. I-fmSiO4@SPIONs were stable in their physical condition and did not demonstrate cytotoxic effects under the conditions investigated. In vitro studies indicated that the contrast enhancement of MRI and CT, and the fluorescence signal intensity of i-fmSiO4@SPION aqueous suspensions and macrophages, were intensified with increased i-fmSiO4@SPION concentrations in suspension and cell culture media. Moreover, for the in vivo study, the accumulation of i-fmSiO4@SPIONs in the liver could also be detected by MRI, CT, and fluorescence imaging. Our study demonstrated that i-fmSiO4@SPIONs had great potential for MRI/C/fluorescence trimodal imaging.Keywords: multifunctional probe, SPIONs, mesoporous silica

  14. In vivo macroscopic HPD fluorescence reflectance imaging on small animals bearing surface ARO/NPA tumor

    Science.gov (United States)

    Autiero, Maddalena; Celentano, Luigi; Laccetti, Paolo; Marotta, Marcello; Mettivier, Giovanni; Montesi, Maria C.; Riccio, Patrizia; Russo, Paolo; Roberti, Giuseppe

    2005-08-01

    Recently multimodal imaging systems have been devised because the combination of different imaging modalities results in the complementarity and integration of the techniques and in a consequent improvement of the diagnostic capabilities of the multimodal system with respect to each separate imaging modality. We developed a simple and reliable HematoPorphyrin (HP) mediated Fluorescence Reflectance Imaging (FRI) system that allows for in vivo real time imaging of surface tumors with a large field of view. The tumor cells are anaplastic human thyroid carcinoma-derived ARO cells, or human papillary thyroid carcinoma-derived NPA cells. Our measurements show that the optical contrast of the tumor region image is increased by a simple digital subtraction of the background fluorescence and that HP fluorescence emissivity of ARO tumors is about 2 times greater than that of NPA tumors, and about 4 times greater than that of healthy tissues. This is also confirmed by spectroscopic measurements on histological sections of tumor and healthy tissues. It was shown also the capability of this system to distinguish the tumor type on the basis of the different intensity of the fluorescence emission, probably related to the malignancy degree. The features of this system are complementary with those ones of a pixel radionuclide detection system, which allows for relatively time expensive, narrow field of view measurements, and applicability to tumors also deeply imbedded in tissues. The fluorescence detection could be used as a large scale and quick analysis tool and could be followed by narrow field, higher resolution radionuclide measurements on previously determined highly fluorescent regions.

  15. Infrared spectroscopic imaging microscopy: Applications to biological systems

    International Nuclear Information System (INIS)

    The coupling of imaging modalities with spectroscopic techniques adds additional dimensions to sample analysis in both the spectroscopic and spatial domains. The particular ability of infrared (IR) imaging to explore the spatial distribution of chemically distinct species on length scales ranging from microns to kilometers demonstrates the versatility and diversity of spectroscopic imaging. In this paper, we focus on the further development of our Fourier-transform (FT) based mid-IR spectroscopic imaging technique which combines the analytical capabilities of mid-IR spectroscopy with the morphological information obtained from optical imaging. The seamless combination of spectroscopy for molecular analysis with the power of visualization represents the future of infrared microscopy. Our spectroscopic imaging instrument integrates several infrared focal-plane arrays with a Michelson step-scan interferometer, generating high-fidelity and high spectral resolution mid-infrared spectroscopic images. The instrumentation produces multidimensional, chemically specific images, while simultaneously obtaining high resolution spectra for each detector pixel. The spatial resolution of the images approaches the diffraction limit for mid-infrared wavelengths, while the spectral resolution is determined by the interferometer, and can be 4 cm-1 or higher. Data derived from a variety of materials, particularly biological samples, illustrate the capabilities of the technique for readily visualizing chemical complexity and for providing statistical data on sample heterogeneity

  16. Compact fluorescence and white-light imaging system for intraoperative visualization of nerves

    Science.gov (United States)

    Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; tan Hehir, Cristina

    2012-02-01

    Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

  17. A compact fluorescence and white light imaging system for intraoperative visualization of nerves

    Science.gov (United States)

    Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; Tan Hehir, Cristina

    2012-03-01

    Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

  18. Development of a noncontact 3-D fluorescence tomography system for small animal in vivo imaging

    Science.gov (United States)

    Zhang, Xiaofeng; Badea, Cristian; Jacob, Mathews; Johnson, G. Allan

    2009-02-01

    Fluorescence imaging is an important tool for tracking molecular-targeting probes in preclinical studies. It offers high sensitivity, but nonetheless low spatial resolution compared to other leading imaging methods such CT and MRI. We demonstrate our methodological development in small animal in vivo whole-body imaging using fluorescence tomography. We have implemented a noncontact fluid-free fluorescence diffuse optical tomography system that uses a raster-scanned continuous-wave diode laser as the light source and an intensified CCD camera as the photodetector. The specimen is positioned on a motorized rotation stage. Laser scanning, data acquisition, and stage rotation are controlled via LabVIEW applications. The forward problem in the heterogeneous medium is based on a normalized Born method, and the sensitivity function is determined using a Monte Carlo method. The inverse problem (image reconstruction) is performed using a regularized iterative algorithm, in which the cost function is defined as a weighted sum of the L-2 norms of the solution image, the residual error, and the image gradient. The relative weights are adjusted by two independent regularization parameters. Our initial tests of this imaging system were performed with an imaging phantom that consists of a translucent plastic cylinder filled with tissue-simulating liquid and two thin-wall glass tubes containing indocyanine green. The reconstruction is compared to the output of a finite element method-based software package NIRFAST and has produced promising results.

  19. Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method

    OpenAIRE

    Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J.; Saldi, Stephanie; Qiu, Jean

    2014-01-01

    The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometr...

  20. Large area synchrotron X-ray fluorescence mapping of biological samples

    International Nuclear Information System (INIS)

    Large area mapping of inorganic material in biological samples has suffered severely from prohibitively long acquisition times. With the advent of new detector technology we can now generate statistically relevant information for studying cell populations, inter-variability and bioinorganic chemistry in large specimen. We have been implementing ultrafast synchrotron-based XRF mapping afforded by the MAIA detector for large area mapping of biological material. For example, a 2.5 million pixel map can be acquired in 3 hours, compared to a typical synchrotron XRF set-up needing over 1 month of uninterrupted beamtime. Of particular focus to us is the fate of metals and nanoparticles in cells, 3D tissue models and animal tissues. The large area scanning has for the first time provided statistically significant information on sufficiently large numbers of cells to provide data on intercellular variability in uptake of nanoparticles. Techniques such as flow cytometry generally require analysis of thousands of cells for statistically meaningful comparison, due to the large degree of variability. Large area XRF now gives comparable information in a quantifiable manner. Furthermore, we can now image localised deposition of nanoparticles in tissues that would be highly improbable to 'find' by typical XRF imaging. In addition, the ultra fast nature also makes it viable to conduct 3D XRF tomography over large dimensions. This technology avails new opportunities in biomonitoring and understanding metal and nanoparticle fate ex-vivo. Following from this is extension to molecular imaging through specific anti-body targeted nanoparticles to label specific tissues and monitor cellular process or biological consequence

  1. Sentinel lymph nodes detection with an imaging system using Patent Blue V dye as fluorescent tracer

    Science.gov (United States)

    Tellier, F.; Steibel, J.; Chabrier, R.; Rodier, J. F.; Pourroy, G.; Poulet, P.

    2013-03-01

    Sentinel lymph node biopsy is the gold standard to detect metastatic invasion from primary breast cancer. This method can help patients avoid full axillary chain dissection, thereby decreasing the risk of morbidity. We propose an alternative to the traditional isotopic method, to detect and map the sentinel lymph nodes. Indeed, Patent Blue V is the most widely used dye in clinical routine for the visual detection of sentinel lymph nodes. A Recent study has shown the possibility of increasing the fluorescence quantum yield of Patent Blue V, when it is bound to human serum albumin. In this study we present a preclinical fluorescence imaging system to detect sentinel lymph nodes labeled with this fluorescent tracer. The setup is composed of a black and white CCD camera and two laser sources. One excitation source with a laser emitting at 635 nm and a second laser at 785 nm to illuminate the region of interest. The prototype is operated via a laptop. Preliminary experiments permitted to determine the device sensitivity in the μmol.L-1 range as regards the detection of PBV fluorescence signals. We also present a preclinical evaluation performed on Lewis rats, during which the fluorescence imaging setup detected the accumulation and fixation of the fluorescent dye on different nodes through the skin.

  2. Endoscopic fluorescence imaging for early assessment of anastomotic recurrence of Crohn's disease

    Science.gov (United States)

    Mordon, Serge R.; Maunoury, Vincent; Geboes, K.; Klein, Olivier; Desreumaux, P.; Debaert, A.; Colombel, Jean-Frederic

    1999-02-01

    Crohn's disease is an inflammatory bowel disease of unknown etiology. The mechanism of the initial mucosal alterations is still unclear: ulcerations overlying lymphoid follicles and/or vasculitis have been proposed as the early lesions. We have developed a new and original method combining endoscopy of fluorescence angiography for identifying the early pathological lesions, occurring in the neo-terminal ileum after right ileocolonic resection. The patient population consisted of 10 subjects enrolled in a prospective protocol of endoscopic follow-up at 3 and 12 months after surgery. Fluorescence imaging showed small spots giving a bright fluorescence distributed singly in mucosa which appeared normal in routine endoscopy. Histopathological examination demonstrated that the fluorescence of small spots originated from small, usually superficial, erosive lesions. In several cases, these erosive lesions occurred over lymphoid follicles. Endoscopic fluorescence imaging provides a suitable means of investigating the initial aspect of the Crohn's disease process in displaying some correlative findings between fluorescent aspects and early pathological mucosal alterations.

  3. Colocalization of fluorescence and Raman microscopic images for the identification of subcellular compartments: a validation study.

    Science.gov (United States)

    Krauß, Sascha D; Petersen, Dennis; Niedieker, Daniel; Fricke, Inka; Freier, Erik; El-Mashtoly, Samir F; Gerwert, Klaus; Mosig, Axel

    2015-04-01

    A major promise of Raman microscopy is the label-free detailed recognition of cellular and subcellular structures. To this end, identifying colocalization patterns between Raman spectral images and fluorescence microscopic images is a key step to annotate subcellular components in Raman spectroscopic images. While existing approaches to resolve subcellular structures are based on fluorescence labeling, we propose a combination of a colocalization scheme with subsequent training of a supervised classifier that allows label-free resolution of cellular compartments. Our colocalization scheme unveils statistically significant overlapping regions by identifying correlation between the fluorescence color channels and clusters from unsupervised machine learning methods like hierarchical cluster analysis. The colocalization scheme is used as a pre-selection to gather appropriate spectra as training data. These spectra are used in the second part as training data to establish a supervised random forest classifier to automatically identify lipid droplets and nucleus. We validate our approach by examining Raman spectral images overlaid with fluorescence labelings of different cellular compartments, indicating that specific components may indeed be identified label-free in the spectral image. A Matlab implementation of our colocalization software is available at . PMID:25679809

  4. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds.

    Science.gov (United States)

    Holt, David; Parthasarathy, Ashwin B; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16 . Ten animals showed no residual tumor cells in the wound bed (mean SBRP1-year follow-up. In five animals, the mean SBR of the wound was >15 , and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations. PMID:26160347

  5. Lysosome triggered near-infrared fluorescence imaging of cellular trafficking processes in real time.

    Science.gov (United States)

    Grossi, Marco; Morgunova, Marina; Cheung, Shane; Scholz, Dimitri; Conroy, Emer; Terrile, Marta; Panarella, Angela; Simpson, Jeremy C; Gallagher, William M; O'Shea, Donal F

    2016-01-01

    Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools. PMID:26927507

  6. Recent advances in near-infrared fluorescence-guided imaging surgery using indocyanine green.

    Science.gov (United States)

    Namikawa, Tsutomu; Sato, Takayuki; Hanazaki, Kazuhiro

    2015-12-01

    Near-infrared (NIR) fluorescence imaging has better tissue penetration, allowing for the effective rejection of excitation light and detection deep inside organs. Indocyanine green (ICG) generates NIR fluorescence after illumination by an NIR ray, enabling real-time intraoperative visualization of superficial lymphatic channels and vessels transcutaneously. The HyperEye Medical System (HEMS) can simultaneously detect NIR rays under room light to provide color imaging, which enables visualization under bright light. Thus, NIR fluorescence imaging using ICG can provide for excellent diagnostic accuracy in detecting sentinel lymph nodes in cancer and microvascular circulation in various ischemic diseases, to assist us with intraoperative decision making. Including HEMS in this system could further improve the sentinel lymph node mapping and intraoperative identification of blood supply in reconstructive organs and ischemic diseases, making it more attractive than conventional imaging. Moreover, the development of new laparoscopic imaging systems equipped with NIR will allow fluorescence-guided surgery in a minimally invasive setting. Future directions, including the conjugation of NIR fluorophores to target specific cancer markers might be realistic technology with diagnostic and therapeutic benefits. PMID:25820596

  7. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    Science.gov (United States)

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-01

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

  8. Lysosome triggered near-infrared fluorescence imaging of cellular trafficking processes in real time

    Science.gov (United States)

    Grossi, Marco; Morgunova, Marina; Cheung, Shane; Scholz, Dimitri; Conroy, Emer; Terrile, Marta; Panarella, Angela; Simpson, Jeremy C.; Gallagher, William M.; O'Shea, Donal F.

    2016-01-01

    Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools. PMID:26927507

  9. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    Science.gov (United States)

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  10. Three approaches for direct analysis of biological samples by Total reflection X-Ray Fluorescence analysis

    International Nuclear Information System (INIS)

    One of the main advantages of the Total reflection X-Ray Fluorescence technique is feasibility of the direct analysis of the sample. taking in account this fact, three methods for the direct analysis of biological samples were evaluated For the evaluation of the method of Compton peak standardization serum, brain tissue, urine and amniotic fluid matrices were analyzed using compton Peak and the elements Co, and V as internal standard. In the case the sample itself is used as calibration standard. The method is reliable for the analysis of serum and brain tissue samples. The analytical quality of the results was similar to that obtained by the conventional method. The results were in good agreements with those obtained by the atomic absorption technique. The second method was applied to the certified Bovine Liver Standard sample 1577a. Experimental conditions for the microwave acid digestion of the solid sample directly on the quartz reflector were found, The percent of recovery of the elements S, Ci, K, Ca, Mn, Fe, Cu, Zn, Se, Br, and Rb carried between 85% and 115% and The precision was below the 10% of relative standards deviations for ten independent replicates. In the third method the concept of chemical modifications is adapted to the Total reflection X-Ray Fluorescence technique. In these experiments the main objective was the elimination of Chlorine from the matrix following the in situ addition of the reactive ammonium nitrate and heating. The method was applied to high saline content samples, i.e. amniotic fluid samples, Special care was given to the volatile elements and to the quality of the thin layer. an enhancement of the sensitivity was found for those elements with signals near the chlorine k-Alfa signal after the modifications procedure

  11. Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

    Science.gov (United States)

    Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

    2016-01-19

    This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

  12. Intracellular Dual Fluorescent Lightup Bioprobes for Image-Guided Photodynamic Cancer Therapy.

    Science.gov (United States)

    Han, Haijie; Jin, Qiao; Wang, Haibo; Teng, Wenzhuo; Wu, Jina; Tong, Hongxin; Chen, Tingting; Ji, Jian

    2016-07-01

    An intracellular dual fluorescent light-up bioprobe with aggregation-induced emission features and endogenously producing photosensitizer protoporphyrin IX (PpIX) abilities is designed and synthesized. The bioprobe is nonemissive in physiological environment. However, the bioprobe can selectively light up cancer cells with blue fluorescence of tetraphenylene (TPE) and red fluorescence of PpIX, owing to the release of TPE and methyl aminolevulinate after targeted internalization by cancer cells. Moreover, upon endogenous generation and accumulation of PpIX in cancer cells, efficient photodynamic ablation of cancer cells after light irradiation is demonstrated with easy regulation for optimal therapeutic efficacy. The design of such dual fluorescent light-up bioprobes might provide a new opportunity for targeted and image-guided photodynamic cancer therapy. PMID:27322139

  13. Near-IR Two-Photon Fluorescent Sensor for K(+) Imaging in Live Cells.

    Science.gov (United States)

    Sui, Binglin; Yue, Xiling; Kim, Bosung; Belfield, Kevin D

    2015-08-19

    A new two-photon excited fluorescent K(+) sensor is reported. The sensor comprises three moieties, a highly selective K(+) chelator as the K(+) recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (>52-fold) in detecting K(+) over other physiological metal cations. Upon binding K(+), the sensor switches from nonfluorescent to highly fluorescent, emitting red to near-IR (NIR) fluorescence. The sensor exhibited a good two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K(+) sensing in living cells. PMID:26258885

  14. Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation

    Science.gov (United States)

    Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.

    2016-03-01

    Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

  15. Biodistribution and imaging of fluorescently-tagged iron oxide nanoparticles in a breast cancer mouse model

    Science.gov (United States)

    Tate, Jennifer A.; Savellano, Mark D.; Hoopes, P. Jack

    2013-02-01

    Iron oxide nanoparticle (IONP) hyperthermia is an emerging treatment that shows great potential as a cancer therapy both alone and in synergy with conventional modalities. Pre-clinical studies are attempting to elucidate the mechanisms of action and distributions of IONP in various in vitro and in vivo models, however these studies would greatly benefit from real-time imaging of IONP locations both in cellular and in mammalian systems. To this end, fluorescently-tagged IONP (fIONP) have been employed for real time tracking and co-registration of IONP with iron content. Starch-coated IONP were fluorescently-tagged, purified and analyzed for fluorescent signal at various concentrations. fIONP were incubated with MTGB cells for varying times and cellular uptake analyzed using confocal microscopy, flow cytometry and inductively-coupled plasma mass spectrometry (ICP-MS). fIONP were also injected into a bilateral mouse tumor model for radiation modification of tumor tissue and enhanced fIONP deposition assessed using a Xenogen IVIS fluorescent imager. Results demonstrated that fIONP concentrations in vitro correlated with ICPMS iron readings. fIONP could be tracked in vitro as well as in tissue samples from an in vivo model. Future work will employ whole animal fluorescent imaging to track the biodistribution of fIONP over time.

  16. Evaluation of Copper Oxide Nanoparticles Toxicity Using Chlorophyll a Fluorescence Imaging in Lemna gibba

    Directory of Open Access Journals (Sweden)

    François Perreault

    2010-01-01

    Full Text Available Copper oxide nanoparticles (CuO NPs, used in antifouling paints of boats, are released in the environment and can induce toxicity to aquatic organisms. In this report, we used chlorophyll a fluorescence imaging to evaluate CuO NPs toxicity in Lemna gibba. This approach allowed to evaluate the differential effect of CuO NPs on photosynthesis of whole L. gibba plants. Exposure to 0.1 to 0.4 g/L CuO NPs during 48h induced strong inhibition of photosynthetic processes resulting in a decrease of plant growth. By using fluorescence imaging, different photosynthetic parameters were evaluated simultaneously in microplate conditions. Imaging of FO fluorescence yield showed the decrease of leaf photosynthetic active surface for whole plants exposed to CuO NPs. This method showed that CuO NPs inhibited photosystem II maximal, photosystem II operational quantum yields, and photochemical quenching of fluorescence associated with electron transport. Nonphotochemical fluorescence quenching as an indicator of energy dissipation not used in photosynthesis was shown to be increased by the effect of CuO NPs. Such approach in microplate conditions provides synchronous high repetition measurements for numerous plants. This study may give a reliable methodological approach to evaluate toxicity risk of NPs in aquatic ecosystems.

  17. Tracking graphene by fluorescence imaging: a tool for detecting multiple populations of graphene in solution

    Science.gov (United States)

    Guidetti, G.; Cantelli, A.; Mazzaro, R.; Ortolani, L.; Morandi, V.; Montalti, M.

    2016-04-01

    Most methods used for the characterization of graphene produced by liquid phase exfoliation require the deposition of the liquid sample on a substrate and subsequent drying. Because of this or other post-synthetic treatments, the reliability of the data in describing the actual features of the graphene particles in the pristine solution becomes questionable. Hence there is a need for new methods that permit the study of graphene directly in solution. Fluorescence imaging is at present the most convenient and sensitive method to visualize nanosized objects in solution. Here we report the development of a new method for visualizing and tracking exfoliated graphene directly in solution using a conventional set-up for fluorescence microscopy. We functionalized a fluorescent surfactant typically used for exfoliating graphite in aqueous phase (Pluronic P123) with two different fluorophores, in order to make graphene detectable by fluorescence microscopy. The photophysical interactions between the fluorescent surfactant and graphene were investigated at the bulk level. Finally, fluorescence microscopy allowed us to track the carbon particles produced and to identify two different populations of particles with sizes of 265 +/- 25 and 1100 +/- 200 nm respectively. The correlation of these results with TEM and DLS data is discussed.Most methods used for the characterization of graphene produced by liquid phase exfoliation require the deposition of the liquid sample on a substrate and subsequent drying. Because of this or other post-synthetic treatments, the reliability of the data in describing the actual features of the graphene particles in the pristine solution becomes questionable. Hence there is a need for new methods that permit the study of graphene directly in solution. Fluorescence imaging is at present the most convenient and sensitive method to visualize nanosized objects in solution. Here we report the development of a new method for visualizing and tracking

  18. Fluorescent non-conjugated polymer dots for targeted cell imaging

    Science.gov (United States)

    Sun, Bin; Zhao, Bin; Wang, Dandan; Wang, Yibo; Tang, Qi; Zhu, Shoujun; Yang, Bai; Sun, Hongchen

    2016-05-01

    Through the chemical crosslinking of the sub-fluorophore, linear non-conjugated polymers can possess strong photoluminescence (PL), which is a very important fluorescence behavior and the non-conjugated polymer dots (PDs) are efficient bio-fluorophores for bio-based applications. Herein, the new type of non-conjugated polyethyleneimine (PEI) PDs was further modified by targeting molecules (folic acid) for a new generation of bio-fluorophores. The free folic acid can quench the PL of PDs by energy transfer, while the conjugated folic acid@PDs (FA@PDs) can still maintain their PL properties to a certain degree. The FA@PDs also possess lower toxicity compared with free PDs, which is possibly due to blocking of the amino groups. Moreover, we investigated the targeted bioimaging applications of the FA@PDs, which gave a very important direction for application of these types of materials.Through the chemical crosslinking of the sub-fluorophore, linear non-conjugated polymers can possess strong photoluminescence (PL), which is a very important fluorescence behavior and the non-conjugated polymer dots (PDs) are efficient bio-fluorophores for bio-based applications. Herein, the new type of non-conjugated polyethyleneimine (PEI) PDs was further modified by targeting molecules (folic acid) for a new generation of bio-fluorophores. The free folic acid can quench the PL of PDs by energy transfer, while the conjugated folic acid@PDs (FA@PDs) can still maintain their PL properties to a certain degree. The FA@PDs also possess lower toxicity compared with free PDs, which is possibly due to blocking of the amino groups. Moreover, we investigated the targeted bioimaging applications of the FA@PDs, which gave a very important direction for application of these types of materials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01909a

  19. pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

    OpenAIRE

    Shen, Yi; Rosendale, Morgane; Robert E Campbell; Perrais, David

    2014-01-01

    Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultan...

  20. Multi-Dimensional Time-Correlated Single Photon Counting (TCSPC) Fluorescence Lifetime Imaging Microscopy (FLIM) to Detect FRET in Cells

    OpenAIRE

    Duncan, R. R.; Bergmann, A; Cousin, M. A.; Apps, D. K.; Shipston, M. J.

    2004-01-01

    We present a novel, multi-dimensional, time-correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser-scanning microscope operated at a pixel dwell-time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This tec...

  1. Development of a Regional Coral Observation Method by a Fluorescence Imaging LIDAR Installed in a Towable Buoy

    OpenAIRE

    Masahiko Sasano; Motonobu Imasato; Hiroya Yamano; Hiroyuki Oguma

    2016-01-01

    Coral bleaching and mortality is predicted to increase under global climate change. A new observation technique is required to monitor regional coral conditions. To this end, we developed a light detection and ranging (LIDAR) system installed in a towable buoy for boat observations, which acquires continuous fluorescent images of the seabed during day-time. Most corals have innate fluorescent proteins in their tissue, and they emit fluorescence by ultraviolet excitation. This fluorescence dis...

  2. Multispectral fluorescence imaging of human ovarian and Fallopian tube tissue for early stage cancer detection

    Science.gov (United States)

    Tate, Tyler; Baggett, Brenda; Rice, Photini; Watson, Jennifer; Orsinger, Gabe; Nymeyer, Ariel C.; Welge, Weston A.; Keenan, Molly; Saboda, Kathylynn; Roe, Denise J.; Hatch, Kenneth; Chambers, Setsuko; Black, John; Utzinger, Urs; Barton, Jennifer

    2015-03-01

    With early detection, five year survival rates for ovarian cancer are over 90%, yet no effective early screening method exists. Emerging consensus suggests that perhaps over 50% of the most lethal form of the disease, high grade serous ovarian cancer, originates in the Fallopian tube. Cancer changes molecular concentrations of various endogenous fluorophores. Using specific excitation wavelengths and emissions bands on a Multispectral Fluorescence Imaging (MFI) system, spatial and spectral data over a wide field of view can be collected from endogenous fluorophores. Wavelength specific reflectance images provide additional information to normalize for tissue geometry and blood absorption. Ratiometric combination of the images may create high contrast between neighboring normal and abnormal tissue. Twenty-six women undergoing oophorectomy or debulking surgery consented the use of surgical discard tissue samples for MFI imaging. Forty-nine pieces of ovarian tissue and thirty-two pieces of Fallopian tube tissue were collected and imaged with excitation wavelengths between 280 nm and 550 nm. After imaging, each tissue sample was fixed, sectioned and HE stained for pathological evaluation. Comparison of mean intensity values between normal, benign, and cancerous tissue demonstrate a general trend of increased fluorescence of benign tissue and decreased fluorescence of cancerous tissue when compared to normal tissue. The predictive capabilities of the mean intensity measurements are tested using multinomial logistic regression and quadratic discriminant analysis. Adaption of the system for in vivo Fallopian tube and ovary endoscopic imaging is possible and is briefly described.

  3. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

    NARCIS (Netherlands)

    Shaner, Nathan C; Campbell, Robert E; Steinbach, Paul A; Giepmans, Ben N G; Palmer, Amy E; Tsien, Roger Y

    2004-01-01

    Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red

  4. Uranium speciation studies in biological medium by capillary electrophoresis and time-resolved laser-induced fluorescence

    International Nuclear Information System (INIS)

    Uranium speciation studies in biological medium by capillary electrophoresis and time-resolved laser-induced fluorescence. The knowledge of the chemical state of uranium in biological medium requires more detailed investigations to understand the behaviour of uranium in the organism. The characterization of the different complexes of uranium can be improved by combining two techniques: time-resolved laser-induced fluorescence (TRLIF) and capillary electrophoresis (CE). CE, indeed, using the isoelectric focusing mode (CIEF), allows for the separation of the different complexes as a function of their isoelectric points (pI) and TRLIF as a speciation method leads to the identification at very low level of different uranyl complexes by temporal resolution and spectral unfolding. Results obtained on various inorganic chemical systems (phosphate, bicarbonate) together with biological systems (citrate, transferrin) will be presented and discussed. The complexation between uranium and human transferrin has been pointed out through CIEF. (authors)

  5. 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

    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...

  6. Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil

    OpenAIRE

    Boschi, F.; Fontanella, M.; Calderan, L; Sbarbati, A

    2011-01-01

    Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical propert...

  7. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    Kecheng Li; Douglas W. Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. Withthe thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration, and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  8. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    KechengLi; DouglasW.Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. With the thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  9. In-vivo fluorescence and reflectance imaging of human cervical tissue

    Science.gov (United States)

    Gustafsson, Ulf P.; McLaughlin, Elisabeth; Jacobsen, Ellen; Hakansson, Johan; Troy, Paul; DeWeert, Michael J.; Svanberg, Katarina; Palsson, Sara; Soto Thompson, Marcelo; Svanberg, Sune; Vaitkuviene, Aurelija

    2003-05-01

    A hyperspectral imaging spectrograph has been used to measure the fluorescence and reflectance of cervical tissue in vivo. The instrument was employed in a clinical trial in Vilnius, Lithuania, where 111 patients were examined. The patients were initially screened by Pap smear, examined by colposcopy and a tissue sampling procedure was performed. Detailed histopathological assessments were performed on the biopsies, and these assessments were correlated with spectra and images. The results of the spectroscopic investigations show that different tissue types within one biopsy region exhibit different spectral signatures. A spectral analysis of the entire image localizes dysplastic regions in both fluorescence and reflectance, suggesting that the hyperspectral imaging technique is useful in the management of cervical malignancies.

  10. Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice.

    Science.gov (United States)

    Wahl, Daniel J; Jian, Yifan; Bonora, Stefano; Zawadzki, Robert J; Sarunic, Marinko V

    2016-01-01

    Cellular-resolution in vivo fluorescence imaging is a valuable tool for longitudinal studies of retinal function in vision research. Wavefront sensorless adaptive optics (WSAO) is a developing technology that enables high-resolution imaging of the mouse retina. In place of the conventional method of using a Shack-Hartmann wavefront sensor to measure the aberrations directly, WSAO uses an image quality metric and a search algorithm to drive the shape of the adaptive element (i.e. deformable mirror). WSAO is a robust approach to AO and it is compatible with a compact, low-cost lens-based system. In this report, we demonstrated a hill-climbing algorithm for WSAO with a variable focus lens and deformable mirror for non-invasive in vivo imaging of EGFP (enhanced green fluorescent protein) labelled ganglion cells and microglia cells in the mouse retina. PMID:26819812

  11. Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime.

    Science.gov (United States)

    Battisti, Antonella; Panettieri, Silvio; Abbandonato, Gerardo; Jacchetti, Emanuela; Cardarelli, Francesco; Signore, Giovanni; Beltram, Fabio; Bizzarri, Ranieri

    2013-07-01

    The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress. PMID:23780224

  12. Photoacoustic imaging of the near-infrared fluorescent protein iRFP in vivo

    Science.gov (United States)

    Krumholz, Arie; Filonov, Grigory S.; Xia, Jun; Yao, Junjie; Verkhusha, Vladislav V.; Wang, Lihong V.

    2012-02-01

    Genetically encoded probes powerfully and non-invasively target specific tissues, cells, and subcellular locations. iRFP, a novel near-infrared fluorescent protein with low quantum yield whose absorption and fluorescence maxima are located at wavelengths longer than the Q-band of hemoglobin absorption, is ideal for PAT. Here, we report on an in vitro comparison of iRFP with other far-red fluorescent proteins, and its use in imaging a mouse tumor xenograft model. In an in vivo experiment, we stably transfected iRFP into MTLn3 adenocarcinoma cells and injected them into the mammary fat pad of female SCID/NCr mice, then imaged the resulting tumors two and three weeks post injection. The contrast increase from the protein expression was high enough to clearly separate the tumor region from the rest of the animal.

  13. Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging

    Science.gov (United States)

    Yamanaka, Masahito; Saito, Kenta; Smith, Nicholas I.; Arai, Yoshiyuki; Uegaki, Kumiko; Yonemaru, Yasuo; Mochizuki, Kentaro; Kawata, Satoshi; Nagai, Takeharu; Fujita, Katsumasa

    2015-10-01

    The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.

  14. Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging.

    Science.gov (United States)

    Ortega Arroyo, Jaime; Cole, Daniel; Kukura, Philipp

    2016-04-01

    Interferometric scattering microscopy (iSCAT) is a light scattering-based imaging modality that offers a unique combination of imaging speed and precision for tracking nanoscopic labels and enables label-free optical sensing down to the single-molecule level. In contrast to fluorescence, iSCAT does not suffer from limitations associated with dye photochemistry and photophysics, or the requirement for fluorescent labeling. Here we present a protocol for constructing an iSCAT microscope from commercially available optical components and demonstrate its compatibility with simultaneously operating single-molecule, objective-type, total internal reflection fluorescence microscopy. Given an intermediate level of experience with optics and microscopy, for instance graduate-level familiarity with laser beam steering and optical components, this protocol can be completed in a time frame of 2 weeks. PMID:26938114

  15. Demonstration of x-ray fluorescence imaging of a high-energy-density plasma

    International Nuclear Information System (INIS)

    Experiments at the Trident Laser Facility have successfully demonstrated the use of x-ray fluorescence imaging (XRFI) to diagnose shocked carbonized resorcinol formaldehyde (CRF) foams doped with Ti. One laser beam created a shock wave in the doped foam. A second laser beam produced a flux of vanadium He-α x-rays, which in turn induced Ti K-shell fluorescence within the foam. Spectrally resolved 1D imaging of the x-ray fluorescence provided shock location and compression measurements. Additionally, experiments using a collimator demonstrated that one can probe specific regions within a target. These results show that XRFI is a capable alternative to path-integrated measurements for diagnosing hydrodynamic experiments at high energy density

  16. Detecting thermal phase transitions in corneal stroma by fluorescence micro-imaging analysis

    Science.gov (United States)

    Matteini, P.; Rossi, F.; Ratto, F.; Bruno, I.; Nesi, P.; Pini, R.

    2008-02-01

    Thermal modifications induced in corneal stroma were investigated by the use of fluorescence microscopy. Freshly extracted porcine corneas were immersed for 5 minutes in a water bath at temperatures in the 35-90°C range and stored in formalin. The samples were then sliced in 200-μm-thick transversal sections and analyzed under a stereomicroscope to assess corneal shrinkage. Fluorescence images of the thermally treated corneal samples were acquired using a slow-scan cooled CCD camera, after staining the slices with Indocyanine Green (ICG) fluorescent dye which allowed to detect fluorescence signal from the whole tissue. All measurements were performed using an inverted epifluorescence microscope equipped with a mercury lamp. The thermally-induced modifications to the corneal specimens were evaluated by studying the grey level distribution in the fluorescence images. For each acquired image, Discrete Fourier Transform (DFT) and entropy analyses were performed. The spatial distribution of DFT absolute value indicated the spatial orientation of the lamellar planes, while entropy was used to study the image texture, correlated to the stromal structural transitions. As a result, it was possible to indicate a temperature threshold value (62°C) for high thermal damage, resulting in a disorganization of the lamellar p