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Sample records for multiparameter fluorescence imaging

  1. Monitoring dynamic systems with multiparameter fluorescence imaging.

    PubMed

    Kudryavtsev, Volodymyr; Felekyan, Suren; Woźniak, Anna K; König, Marcelle; Sandhagen, Carl; Kühnemuth, Ralf; Seidel, Claus A M; Oesterhelt, Filipp

    2007-01-01

    A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules. PMID:17160654

  2. FluoQ: a tool for rapid analysis of multiparameter fluorescence imaging data applied to oscillatory events.

    PubMed

    Stein, Frank; Kress, Manuel; Reither, Sabine; Piljić, Alen; Schultz, Carsten

    2013-09-20

    The number of fluorescent sensors and their use in living cells has significantly increased in the past years. Yet, the analysis of data from single cells or cell populations usually remains a very time-consuming enterprise. Here, we introduce FluoQ, a new macro for the image analysis software ImageJ, which enables fast analysis of multiparameter time-lapse fluorescence microscopy data with minimal manual input. FluoQ provides statistical analysis of all measured parameters and delivers the results in multiple graphic and numeric displays. We demonstrate the power of FluoQ by applying the macro to data analysis in the development and optimization of novel FRET reporters for monitoring the performance of calcium/calmodulin-binding inositol trisphosphate kinases A and B (ITPKA and ITPKB) in HeLa cells. We find that conformational changes in the ITPKA-based sensor follow receptor-mediated calcium oscillations. This indicates that ITPKA contributes to the regulation of intracellular calcium transients by limiting inositol trisphosphate levels. PMID:23882997

  3. Multiparameter fluorescence imaging for quantification of TH-1 and TH-2 cytokines at the single-cell level

    NASA Astrophysics Data System (ADS)

    Fekkar, Hakim; Benbernou, N.; Esnault, S.; Shin, H. C.; Guenounou, Moncef

    1998-04-01

    Immune responses are strongly influenced by the cytokines following antigenic stimulation. Distinct cytokine-producing T cell subsets are well known to play a major role in immune responses and to be differentially regulated during immunological disorders, although the characterization and quantification of the TH-1/TH-2 cytokine pattern in T cells remained not clearly defined. Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signaling pathways. The aim of this study was (1) to quantify the cytokine expression in T cells at the single cell level using optical imaging, (2) and to analyze the influence of cyclic AMP- dependent signal transduction pathway in the balance between the TH-1 and TH-2 cytokine profile. We attempted to study several cytokines (IL-2, IFN-(gamma) , IL-4, IL-10 and IL-13) in peripheral blood mononuclear cells. Cells were prestimulated in vitro using phytohemagglutinin and phorbol ester for 36h, and then further cultured for 8h in the presence of monensin. Cells were permeabilized and then simple-, double- or triple-labeled with the corresponding specific fluorescent monoclonal antibodies. The cell phenotype was also determined by analyzing the expression of each of CD4, CD8, CD45RO and CD45RA with the cytokine expression. Conventional images of cells were recorded with a Peltier- cooled CCD camera (B/W C5985, Hamamatsu photonics) through an inverted microscope equipped with epi-fluorescence (Diaphot 300, Nikon). Images were digitalized using an acquisition video interface (Oculus TCX Coreco) in 762 by 570 pixels coded in 8 bits (256 gray levels), and analyzed thereafter in an IBM PC computer based on an intel pentium processor with an adequate software (Visilog 4, Noesis). The first image processing step is the extraction of cell areas using an edge detection and a binary thresholding method. In order to reduce the background noise of fluorescence, we performed an opening

  4. Accurate single-molecule FRET studies using multiparameter fluorescence detection.

    PubMed

    Sisamakis, Evangelos; Valeri, Alessandro; Kalinin, Stanislav; Rothwell, Paul J; Seidel, Claus A M

    2010-01-01

    In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Förster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in a population of molecules. After a short introduction, the data analysis procedure is described in detail together with some experimental considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure determination based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when determining FRET-related distances with high accuracy. Furthermore, the broad time resolution (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomolecule of interest explores its

  5. Multiparameter image visualization with self-organizing maps

    NASA Astrophysics Data System (ADS)

    Manduca, Armando

    1994-05-01

    The effective display of multiparameter medical image data sets is assuming increasing importance as more distinct imaging modalities are becoming available. For medical purposes, one desirable goal is to fuse such data sets into a single most informative gray-scale image without making rigid classification decisions. A visualization technique based on a non-linear projection onto a 1D self-organizing map is described and examples are shown. The SOM visualization technique is fast, theoretically attractive, a useful complement to projection- pursuit or other linear techniques, and may be of particular value in calling attention to specific regions in a multiparameter image where the component images should be examined in detail.

  6. Multiparameter image visualization with self-organizing maps

    NASA Astrophysics Data System (ADS)

    Manduca, Armando

    1994-09-01

    The effective display of multi-parameter medical image data sets is assuming increasing importance as more distinct imaging modalities are becoming available. For medical pruposes, one desirable goal is to fuse such data sets into a single most informative gray-scale image without making rigid classification decisions. A visualization technique based on a non-linear projection onto a 1D self-organizing map is described and examples are shown. The SOM visualization technique is fast, theoretically attractive, and has properties which compliment those of projection-pursuit or other linear techniques. It may be of particular value in calling attention to specific regions in a multi-parameter image where the component images should be examined in detail.

  7. Multiparameter single-molecule fluorescence measurements of DNA intercalating fluorophores

    NASA Astrophysics Data System (ADS)

    Bowen, Benjamin P.; Enderlein, Jorg; Woodbury, Neal W. T.

    2003-06-01

    Experiments using single-molecules of TOTO-1 intercalated into dsDNA were performed to investigate the DNA sequence dependence on the fluorescence detectable with single-molecule fluorescence spectroscopy. Previous work has shown that there is a difference in the fluorescence lifetime when TOTO-1 is intercalated in poly-AT DNA or in poly-GC DNA. The fluorescence detected from single-molecules in this work for poly-GC and poly-AT DNA showed fluorescence lifetimes of 2.1 and 1.8 nsec, respectively. Analysis of the fluorescence intensity detected from single-molecules of TOTO-1 was performed by fluorescence cross-correlation spectroscopy. TOTO-1 is shown to spend large amounts of time in dark states. These dark states reduce the detectable fluorescence intensity to approximately 10 photons per millisecond on average.

  8. Quantitative Assessment of Retinopathy Using Multi-parameter Image Analysis.

    PubMed

    Ghanian, Zahra; Staniszewski, Kevin; Jamali, Nasim; Sepehr, Reyhaneh; Wang, Shoujian; Sorenson, Christine M; Sheibani, Nader; Ranji, Mahsa

    2016-01-01

    A multi-parameter quantification method was implemented to quantify retinal vascular injuries in microscopic images of clinically relevant eye diseases. This method was applied to wholemount retinal trypsin digest images of diabetic Akita/+, and bcl-2 knocked out mice models. Five unique features of retinal vasculature were extracted to monitor early structural changes and retinopathy, as well as quantifying the disease progression. Our approach was validated through simulations of retinal images. Results showed fewer number of cells (P = 5.1205e-05), greater population ratios of endothelial cells to pericytes (PCs) (P = 5.1772e-04; an indicator of PC loss), higher fractal dimension (P = 8.2202e-05), smaller vessel coverage (P = 1.4214e-05), and greater number of acellular capillaries (P = 7.0414e-04) for diabetic retina as compared to normal retina. Quantification using the present method would be helpful in evaluating physiological and pathological retinopathy in a high-throughput and reproducible manner. PMID:27186534

  9. Quantitative Assessment of Retinopathy Using Multi-parameter Image Analysis

    PubMed Central

    Ghanian, Zahra; Staniszewski, Kevin; Jamali, Nasim; Sepehr, Reyhaneh; Wang, Shoujian; Sorenson, Christine M.; Sheibani, Nader; Ranji, Mahsa

    2016-01-01

    A multi-parameter quantification method was implemented to quantify retinal vascular injuries in microscopic images of clinically relevant eye diseases. This method was applied to wholemount retinal trypsin digest images of diabetic Akita/+, and bcl-2 knocked out mice models. Five unique features of retinal vasculature were extracted to monitor early structural changes and retinopathy, as well as quantifying the disease progression. Our approach was validated through simulations of retinal images. Results showed fewer number of cells (P = 5.1205e-05), greater population ratios of endothelial cells to pericytes (PCs) (P = 5.1772e-04; an indicator of PC loss), higher fractal dimension (P = 8.2202e-05), smaller vessel coverage (P = 1.4214e-05), and greater number of acellular capillaries (P = 7.0414e-04) for diabetic retina as compared to normal retina. Quantification using the present method would be helpful in evaluating physiological and pathological retinopathy in a high-throughput and reproducible manner. PMID:27186534

  10. Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry.

    PubMed

    Nicholas, Katherine J; Greenplate, Allison R; Flaherty, David K; Matlock, Brittany K; Juan, Juan San; Smith, Rita M; Irish, Jonathan M; Kalams, Spyros A

    2016-03-01

    Mass and fluorescence cytometry are quantitative single cell flow cytometry approaches that are powerful tools for characterizing diverse tissues and cellular systems. Here mass cytometry was directly compared with fluorescence cytometry by studying phenotypes of healthy human peripheral blood mononuclear cells (PBMC) in the context of superantigen stimulation. One mass cytometry panel and five fluorescence cytometry panels were used to measure 20 well-established lymphocyte markers of memory and activation. Comparable frequencies of both common and rare cell subpopulations were observed with fluorescence and mass cytometry using biaxial gating. The unsupervised high-dimensional analysis tool viSNE was then used to analyze data sets generated from both mass and fluorescence cytometry. viSNE analysis effectively characterized PBMC using eight features per cell and identified similar frequencies of activated CD4+ T cells with both technologies. These results suggest combinations of unsupervised analysis programs and extended multiparameter cytometry will be indispensable tools for detecting perturbations in protein expression in both health and disease. PMID:26599989

  11. A Novel Staining Protocol for Multiparameter Assessment of Cell Heterogeneity in Phormidium Populations (Cyanobacteria) Employing Fluorescent Dyes

    PubMed Central

    Tashyreva, Daria; Elster, Josef; Billi, Daniela

    2013-01-01

    Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. We demonstrate a novel staining protocol for multiparameter assessment of individual cells in physiologically heterogeneous populations of cyanobacteria. The protocol employs fluorescent probes, i.e., redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, ‘dead cell’ nucleic acid stain SYTOX Green, and DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole, combined with microscopy image analysis. Our method allows simultaneous estimates of cellular respiration activity, membrane and nucleoid integrity, and allows the detection of photosynthetic pigments fluorescence along with morphological observations. The staining protocol has been adjusted for, both, laboratory and natural populations of the genus Phormidium (Oscillatoriales), and tested on 4 field-collected samples and 12 laboratory strains of cyanobacteria. Based on the mentioned cellular functions we suggest classification of cells in cyanobacterial populations into four categories: (i) active and intact; (ii) injured but active; (iii) metabolically inactive but intact; (iv) inactive and injured, or dead. PMID:23437052

  12. Multi-parameter optical image interpretations based on self-organizing mapping

    NASA Astrophysics Data System (ADS)

    Klose, Christian D.; Klose, A. K.; Netz, U.; Scheel, A.; Beuthan, J.; Hielscher, Andreas H.

    2008-02-01

    We found that using more than one parameter derived from optical tomographic images can lead to better image classification results compared to cases when only one parameter is used.. In particular we present a multi-parameter classification approach, called self-organizing mapping (SOM), for detecting synovitis in arthritic finger joints based on sagittal laser optical tomography (SLOT). This imaging modality can be used to determine various physical parameters such as minimal absorption and scattering coefficients in an image of the proximal interphalengeal joint. Results were compared to different gold standards: magnet resonance imaging, ultra-sonography and clinical evaluation. When compared to classifications based on single-parameters, e.g., absorption minimum only, the study reveals that multi-parameter classifications lead to higher classification sensitivities and specificities and statistical significances with p-values <5 per cent. Finally, the data suggest that image analyses are more reliable and avoid ambiguous interpretations when using more than one parameter.

  13. Associative image analysis: a method for automated quantification of 3D multi-parameter images of brain tissue

    PubMed Central

    Bjornsson, Christopher S; Lin, Gang; Al-Kofahi, Yousef; Narayanaswamy, Arunachalam; Smith, Karen L; Shain, William; Roysam, Badrinath

    2009-01-01

    Brain structural complexity has confounded prior efforts to extract quantitative image-based measurements. We present a systematic ‘divide and conquer’ methodology for analyzing three-dimensional (3D) multi-parameter images of brain tissue to delineate and classify key structures, and compute quantitative associations among them. To demonstrate the method, thick (~100 μm) slices of rat brain tissue were labeled using 3 – 5 fluorescent signals, and imaged using spectral confocal microscopy and unmixing algorithms. Automated 3D segmentation and tracing algorithms were used to delineate cell nuclei, vasculature, and cell processes. From these segmentations, a set of 23 intrinsic and 8 associative image-based measurements was computed for each cell. These features were used to classify astrocytes, microglia, neurons, and endothelial cells. Associations among cells and between cells and vasculature were computed and represented as graphical networks to enable further analysis. The automated results were validated using a graphical interface that permits investigator inspection and corrective editing of each cell in 3D. Nuclear counting accuracy was >89%, and cell classification accuracy ranged from 81–92% depending on cell type. We present a software system named FARSIGHT implementing our methodology. Its output is a detailed XML file containing measurements that may be used for diverse quantitative hypothesis-driven and exploratory studies of the central nervous system. PMID:18294697

  14. Fluorescent image tracking velocimeter

    DOEpatents

    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.

  15. Fluorescence Live Cell Imaging

    PubMed Central

    Ettinger, Andreas

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

  16. Towards a cellular multi-parameter analysis platform: fluorescence in situ hybridization (FISH) on microhole-array chips.

    PubMed

    Kurz, Christian M; Moosdijk, Stefan V D; Thielecke, Hagen; Velten, Thomas

    2011-01-01

    Highly-sensitive analysis systems based on cellular multi-parameter are needed in the diagnostics. Therefore we improved our previously developed chip platform for another additional analysis method, the fluorescence in situ hybridization. Fluorescence in situ hybridization (FISH) is a technique used in the diagnostics to determine the localization and the presence or absence of specific DNA sequence. To improve this labor- and cost-intensive method, we reduced the assay consumption by a factor of 5 compared to the standard protocol. Microhole chips were used for making the cells well addressable. The chips were fabricated by semiconductor technology on the basis of a Silicon wafer with a thin deposited silicon nitride layer (Si(3)N(4)). Human retina pigment epithelia (ARPE-19) cells were arrayed on 5-μm holes of a 35 × 35 microhole-array by a gently negative differential pressure of around 5 mbar. After 3 hours of incubation the cells were attached to the chip and the FISH protocol was applied to the positioned cells. A LabView software was developed to simplify the analysis. The software automatically counts the number of dots (positive labeled chromosome regions) as well as the distance between adjacent dots. Our developed platform reduces the assay consumption and the labor time. Furthermore, during the 3 hours of incubation non-invasive or minimal-invasive methods like Raman- and impedance-spectroscopy can be applied. PMID:22256298

  17. Fluorescence Imaging in Surgery

    PubMed Central

    Orosco, Ryan K.; Tsien, Roger Y.; Nguyen, Quyen T.

    2013-01-01

    Although the modern surgical era is highlighted by multiple technological advances and innovations, one area that has remained constant is the dependence of the surgeon's vision on white-light reflectance. This renders different body tissues in a limited palette of various shades of pink and red, thereby limiting the visual contrast available to the operating surgeon. Healthy tissue, anatomic variations, and diseased states are seen as slight discolorations relative to each other and differences are inherently limited in dynamic range. In the upcoming years, surgery will undergo a paradigm shift with the use of targeted fluorescence imaging probes aimed at augmenting the surgical armamentarium by expanding the “visible” spectrum available to surgeons. Such fluorescent “smart probes” will provide real-time, intraoperative, pseudo-color, high-contrast delineation of both normal and pathologic tissues. Fluorescent surgical molecular guidance promises another major leap forward to improve patient safety and clinical outcomes, and to reduce overall healthcare costs. This review provides an overview of current and future surgical applications of fluorescence imaging in diseased and nondiseased tissues and focus on the innovative fields of image processing and instrumentation. PMID:23335674

  18. High-Speed Multiparameter Photophysical Analyses of Fluorophore Libraries

    PubMed Central

    Dean, Kevin M.; Davis, Lloyd M.; Lubbeck, Jennifer L.; Manna, Premashis; Friis, Pia; Palmer, Amy E.; Jimenez, Ralph

    2015-01-01

    There is a critical need for high-speed multi-parameter photophysical measurements of large libraries of fluorescent probe variants for imaging and biosensor development. We present a microfluidic flow cytometer that rapidly assays 104–105 member cell-based fluorophore libraries, simultaneously measuring fluorescence lifetime and photo-bleaching. Together, these photophysical characteristics determine imaging performance. We demonstrate the ability to resolve the diverse photophysical characteristics of different library types and the ability to identify rare populations. PMID:25898152

  19. Fluorescent microthermographic imaging

    SciTech Connect

    Barton, D.L.

    1993-09-01

    In the early days of microelectronics, design rules and feature sizes were large enough that sub-micron spatial resolution was not needed. Infrared or IR thermal techniques were available that calculated the object`s temperature from infrared emission. There is a fundamental spatial resolution limitation dependent on the wavelengths of light being used in the image formation process. As the integrated circuit feature sizes began to shrink toward the one micron level, the limitations imposed on IR thermal systems became more pronounced. Something else was needed to overcome this limitation. Liquid crystals have been used with great success, but they lack the temperature measurement capabilities of other techniques. The fluorescent microthermographic imaging technique (FMI) was developed to meet this need. This technique offers better than 0.01{degrees}C temperature resolution and is diffraction limited to 0.3 {mu}m spatial resolution. While the temperature resolution is comparable to that available on IR systems, the spatial resolution is much better. The FMI technique provides better spatial resolution by using a temperature dependent fluorescent film that emits light at 612 nm instead of the 1.5 {mu}m to 12 {mu}m range used by IR techniques. This tutorial starts with a review of blackbody radiation physics, the process by which all heated objects emit radiation to their surroundings, in order to understand the sources of information that are available to characterize an object`s surface temperature. The processes used in infrared thermal imaging are then detailed to point out the limitations of the technique but also to contrast it with the FMI process. The FMI technique is then described in detail, starting with the fluorescent film physics and ending with a series of examples of past applications of FMI.

  20. Spectrally resolved multidepth fluorescence imaging

    PubMed Central

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

    2011-01-01

    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. PMID:21950929

  1. Spectrally resolved multidepth fluorescence imaging

    NASA Astrophysics Data System (ADS)

    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.

  2. Fiber-optic fluorescence imaging

    PubMed Central

    Flusberg, Benjamin A; Cocker, Eric D; Piyawattanametha, Wibool; Jung, Juergen C; Cheung, Eunice L M; Schnitzer, Mark J

    2010-01-01

    Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components. PMID:16299479

  3. Fluorescence lifetime imaging of coral fluorescent proteins.

    PubMed

    Cox, Guy; Matz, Mikhail; Salih, Anya

    2007-03-01

    Corals, like many other coelenterates, contain fluorescent pigments that show considerable homology with the well known green fluorescent protein of the jellyfish Aequoria. In corals, unlike jellyfish, multiple proteins are present and the range of excitations and emissions suggest the possibility of energy transfer. The occurrence of Förster resonant energy transfer (FRET) between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504, and amilFP593 (names indicate emission peaks) were 2.8, 2.9, and 2.9 ns, respectively. In the coral sample, imaging the entire emission spectrum from 420 nm, the mean lifetime was reduced to 1.5 ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3 ns, supporting this interpretation. In contrast, no reduction in lifetime is seen in the coral Euphyllia ancora, where the pigment distribution also suggests that the pigments are unlikely to be involved in photoprotection. This study set out to determine the extent of FRET between pigments in two corals, Acropora millepora and Euphyllia, ancora which differ in the arrangement of their pigments and hence possibly in pigment function. PMID:17279514

  4. Assessing Photosynthesis by Fluorescence Imaging

    ERIC Educational Resources Information Center

    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…

  5. Iterative Multiparameter Elastic Waveform Inversion Using Prestack Time Imaging and Kirchhoff approximation

    NASA Astrophysics Data System (ADS)

    Khaniani, Hassan

    This thesis proposes a "standard strategy" for iterative inversion of elastic properties from the seismic reflection data. The term "standard" refers to the current hands-on commercial techniques that are used for the seismic imaging and inverse problem. The method is established to reduce the computation time associated with elastic Full Waveform Inversion (FWI) methods. It makes use of AVO analysis, prestack time migration and corresponding forward modeling in an iterative scheme. The main objective is to describe the iterative inversion procedure used in seismic reflection data using simplified mathematical expression and their numerical applications. The frame work of the inversion is similar to (FWI) method but with less computational costs. The reduction of computational costs depends on the data conditioning (with or without multiple data), the level of the complexity of geological model and acquisition condition such as Signal to Noise Ratio (SNR). Many processing methods consider multiple events as noise and remove it from the data. This is the motivation for reducing the computational cost associated with Finite Difference Time Domain (FDTD) forward modeling and Reverse Time Migration (RTM)-based techniques. Therefore, a one-way solution of the wave equation for inversion is implemented. While less computationally intensive depth imaging methods are available by iterative coupling of ray theory and the Born approximation, it is shown that we can further reduce the cost of inversion by dropping the cost of ray tracing for traveltime estimation in a way similar to standard Prestack Time Migration (PSTM) and the corresponding forward modeling. This requires the model to have smooth lateral variations in elastic properties, so that the traveltime of the scatterpoints can be approximated by a Double Square Root (DSR) equation. To represent a more realistic and stable solution of the inverse problem, while considering the phase of supercritical angles, the

  6. Fluorescent eye test (image)

    MedlinePlus

    The fluorescent eye test is useful in determining if there is a scratch or other problem with the surface ... has thoroughly covered the eye a cobalt blue light is then directed on the eye. The light ...

  7. Voxel-based clustered imaging by multiparameter diffusion tensor images for glioma grading.

    PubMed

    Inano, Rika; Oishi, Naoya; Kunieda, Takeharu; Arakawa, Yoshiki; Yamao, Yukihiro; Shibata, Sumiya; Kikuchi, Takayuki; Fukuyama, Hidenao; Miyamoto, Susumu

    2014-01-01

    Gliomas are the most common intra-axial primary brain tumour; therefore, predicting glioma grade would influence therapeutic strategies. Although several methods based on single or multiple parameters from diagnostic images exist, a definitive method for pre-operatively determining glioma grade remains unknown. We aimed to develop an unsupervised method using multiple parameters from pre-operative diffusion tensor images for obtaining a clustered image that could enable visual grading of gliomas. Fourteen patients with low-grade gliomas and 19 with high-grade gliomas underwent diffusion tensor imaging and three-dimensional T1-weighted magnetic resonance imaging before tumour resection. Seven features including diffusion-weighted imaging, fractional anisotropy, first eigenvalue, second eigenvalue, third eigenvalue, mean diffusivity and raw T2 signal with no diffusion weighting, were extracted as multiple parameters from diffusion tensor imaging. We developed a two-level clustering approach for a self-organizing map followed by the K-means algorithm to enable unsupervised clustering of a large number of input vectors with the seven features for the whole brain. The vectors were grouped by the self-organizing map as protoclusters, which were classified into the smaller number of clusters by K-means to make a voxel-based diffusion tensor-based clustered image. Furthermore, we also determined if the diffusion tensor-based clustered image was really helpful for predicting pre-operative glioma grade in a supervised manner. The ratio of each class in the diffusion tensor-based clustered images was calculated from the regions of interest manually traced on the diffusion tensor imaging space, and the common logarithmic ratio scales were calculated. We then applied support vector machine as a classifier for distinguishing between low- and high-grade gliomas. Consequently, the sensitivity, specificity, accuracy and area under the curve of receiver operating characteristic

  8. Voxel-based clustered imaging by multiparameter diffusion tensor images for glioma grading

    PubMed Central

    Inano, Rika; Oishi, Naoya; Kunieda, Takeharu; Arakawa, Yoshiki; Yamao, Yukihiro; Shibata, Sumiya; Kikuchi, Takayuki; Fukuyama, Hidenao; Miyamoto, Susumu

    2014-01-01

    Gliomas are the most common intra-axial primary brain tumour; therefore, predicting glioma grade would influence therapeutic strategies. Although several methods based on single or multiple parameters from diagnostic images exist, a definitive method for pre-operatively determining glioma grade remains unknown. We aimed to develop an unsupervised method using multiple parameters from pre-operative diffusion tensor images for obtaining a clustered image that could enable visual grading of gliomas. Fourteen patients with low-grade gliomas and 19 with high-grade gliomas underwent diffusion tensor imaging and three-dimensional T1-weighted magnetic resonance imaging before tumour resection. Seven features including diffusion-weighted imaging, fractional anisotropy, first eigenvalue, second eigenvalue, third eigenvalue, mean diffusivity and raw T2 signal with no diffusion weighting, were extracted as multiple parameters from diffusion tensor imaging. We developed a two-level clustering approach for a self-organizing map followed by the K-means algorithm to enable unsupervised clustering of a large number of input vectors with the seven features for the whole brain. The vectors were grouped by the self-organizing map as protoclusters, which were classified into the smaller number of clusters by K-means to make a voxel-based diffusion tensor-based clustered image. Furthermore, we also determined if the diffusion tensor-based clustered image was really helpful for predicting pre-operative glioma grade in a supervised manner. The ratio of each class in the diffusion tensor-based clustered images was calculated from the regions of interest manually traced on the diffusion tensor imaging space, and the common logarithmic ratio scales were calculated. We then applied support vector machine as a classifier for distinguishing between low- and high-grade gliomas. Consequently, the sensitivity, specificity, accuracy and area under the curve of receiver operating characteristic

  9. Iterative Multiparameter Elastic Waveform Inversion Using Prestack Time Imaging and Kirchhoff approximation

    NASA Astrophysics Data System (ADS)

    Khaniani, Hassan

    This thesis proposes a "standard strategy" for iterative inversion of elastic properties from the seismic reflection data. The term "standard" refers to the current hands-on commercial techniques that are used for the seismic imaging and inverse problem. The method is established to reduce the computation time associated with elastic Full Waveform Inversion (FWI) methods. It makes use of AVO analysis, prestack time migration and corresponding forward modeling in an iterative scheme. The main objective is to describe the iterative inversion procedure used in seismic reflection data using simplified mathematical expression and their numerical applications. The frame work of the inversion is similar to (FWI) method but with less computational costs. The reduction of computational costs depends on the data conditioning (with or without multiple data), the level of the complexity of geological model and acquisition condition such as Signal to Noise Ratio (SNR). Many processing methods consider multiple events as noise and remove it from the data. This is the motivation for reducing the computational cost associated with Finite Difference Time Domain (FDTD) forward modeling and Reverse Time Migration (RTM)-based techniques. Therefore, a one-way solution of the wave equation for inversion is implemented. While less computationally intensive depth imaging methods are available by iterative coupling of ray theory and the Born approximation, it is shown that we can further reduce the cost of inversion by dropping the cost of ray tracing for traveltime estimation in a way similar to standard Prestack Time Migration (PSTM) and the corresponding forward modeling. This requires the model to have smooth lateral variations in elastic properties, so that the traveltime of the scatterpoints can be approximated by a Double Square Root (DSR) equation. To represent a more realistic and stable solution of the inverse problem, while considering the phase of supercritical angles, the

  10. Computer-controlled multi-parameter mapping of 3D compressible flowfields using planar laser-induced iodine fluorescence

    NASA Technical Reports Server (NTRS)

    Donohue, James M.; Victor, Kenneth G.; Mcdaniel, James C., Jr.

    1993-01-01

    A computer-controlled technique, using planar laser-induced iodine fluorescence, for measuring complex compressible flowfields is presented. A new laser permits the use of a planar two-line temperature technique so that all parameters can be measured with the laser operated narrowband. Pressure and temperature measurements in a step flowfield show agreement within 10 percent of a CFD model except in regions close to walls. Deviation of near wall temperature measurements from the model was decreased from 21 percent to 12 percent compared to broadband planar temperature measurements. Computer-control of the experiment has been implemented, except for the frequency tuning of the laser. Image data storage and processing has been improved by integrating a workstation into the experimental setup reducing the data reduction time by a factor of 50.

  11. Multiparameter double hole contrast detail phantom: Ability to detect image displacement due to off position anode stem

    NASA Astrophysics Data System (ADS)

    Pauzi, Nur Farahana; Majid, Zafri Azran Abdul; Sapuan, Abdul Halim; Azemin, Mohd Zulfaezal Che; Junet, Laila Kalidah

    2015-04-01

    Contrast Detail phantom is a quality control tool to analyze the performance of imaging devices. Currently, its function is solely to evaluate the contrast detail characteristic of imaging system. It consists of drilled hole which gives effect to the penetration of x-ray beam divergence to pass through the base of each hole. This effect will lead to false appearance of image from its original location but it does not being visualized in the radiograph. In this study, a new design of Contrast Detail phantom's hole which consists of double hole construction has been developed. It can detect the image displacement which is due to off position of anode stem from its original location. The double hole differs from previous milled hole, whereby it consists of combination of different hole diameters. Small hole diameter (3 mm) is positioned on top of larger hole diameter (10 mm). The thickness of double hole acrylic blocks is 13 mm. Result revealed` that, Multiparameter Double Hole Contrast Detail phantom can visualize the shifted flaw image quality produced by x-ray machine due to improper position of the anode stem which is attached to rotor and stator. The effective focal spot of x-ray beam also has been shifted from the center of collimator as a result of off-position anode stem. As a conclusion, the new design of double hole Contrast Detail phantom able to measure those parameters in a well manner.

  12. Multiparameter double hole contrast detail phantom: Ability to detect image displacement due to off position anode stem

    SciTech Connect

    Pauzi, Nur Farahana; Majid, Zafri Azran Abdul; Sapuan, Abdul Halim; Junet, Laila Kalidah; Azemin, Mohd Zulfaezal Che

    2015-04-24

    Contrast Detail phantom is a quality control tool to analyze the performance of imaging devices. Currently, its function is solely to evaluate the contrast detail characteristic of imaging system. It consists of drilled hole which gives effect to the penetration of x-ray beam divergence to pass through the base of each hole. This effect will lead to false appearance of image from its original location but it does not being visualized in the radiograph. In this study, a new design of Contrast Detail phantom’s hole which consists of double hole construction has been developed. It can detect the image displacement which is due to off position of anode stem from its original location. The double hole differs from previous milled hole, whereby it consists of combination of different hole diameters. Small hole diameter (3 mm) is positioned on top of larger hole diameter (10 mm). The thickness of double hole acrylic blocks is 13 mm. Result revealed that Multiparameter Double Hole Contrast Detail phantom can visualize the shifted flaw image quality produced by x-ray machine due to improper position of the anode stem which is attached to rotor and stator. The effective focal spot of x-ray beam also has been shifted from the center of collimator as a result of off-position anode stem. As a conclusion, the new design of double hole Contrast Detail phantom able to measure those parameters in a well manner.

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

    PubMed

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    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. PMID:26390855

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    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. PMID:26390855

  16. Fluorescence Lifetime Imaging of Apoptosis

    PubMed Central

    Xiao, Annie; Gibbons, Anne E.; Luker, Kathryn E.; Luker, Gary D.

    2015-01-01

    Genetically-encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools to analyze cell signaling and function at single cell resolution in standard two-dimensional cell cultures, but these reporters rarely have been applied to three-dimensional environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in absorption of light complicate this analysis method in three-dimensional settings. Here we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time, to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from two-dimensional cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity. PMID:26771007

  17. Development of an Immunologically Tolerated Combination of Fluorescent Proteins for In vivo Two-photon Imaging

    PubMed Central

    Gossa, Selamawit; Nayak, Debasis; Zinselmeyer, Bernd H.; McGavern, Dorian B.

    2014-01-01

    Combinations of fluorescent proteins (FPs) are routinely used for multi-parameter in vivo imaging experiments to visualize tagged proteins or cell populations of interest. Studies involving FPs are often limited by spectral overlap, toxicity, relative quantum efficiency, and the potential for immunological rejection upon transfer into a non-tolerant recipient. Here we evaluate the immunologic visibility of several commonly used FPs by the murine immune system and identify a spectrally compatible, immunologically tolerated combination of FPs well suited for in vivo two-photon imaging. PMID:25322934

  18. Local region statistics combining multi-parameter intensity fitting module for medical image segmentation with intensity inhomogeneity and complex composition

    NASA Astrophysics Data System (ADS)

    Zhao, Fan; Zhao, Jian; Zhao, Wenda; Qu, Feng; Sui, Long

    2016-08-01

    It is difficult to segment medical image with intensity inhomogeneity and complex composition, because most region-based modules relay on the intensity distributions. In this paper, we propose a novel method which uses local region statistics and multi-parameter intensity fitting as well. By replacing the original local region statistics with the novel local region statistics after bias field correction, the effect of intensity inhomogeneity can be eliminated. Then we devise a maximum likelihood energy function based on the distribution of each local region. Segmentation and bias field estimation can be jointly obtained by minimizing the proposed energy function. Furthermore, in order to characterize the features of each local region effectively, two parameters are used to fit the average intensity inside and outside of the counter, respectively. This can well handle the medical images with complex composition, such as larger gray difference even in the same region. Comparisons with several representative methods on synthetic and medical images demonstrate the superiority of the proposed method over other representative algorithms.

  19. Multi Spectral Fluorescence Imager (MSFI)

    NASA Technical Reports Server (NTRS)

    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.

  20. Fluorescence imaging spectrometer optical design

    NASA Astrophysics Data System (ADS)

    Taiti, A.; Coppo, P.; Battistelli, E.

    2015-09-01

    The optical design of the FLuORescence Imaging Spectrometer (FLORIS) studied for the Fluorescence Explorer (FLEX) mission is discussed. FLEX is a candidate for the ESA's 8th Earth Explorer opportunity mission. FLORIS is a pushbroom hyperspectral imager foreseen to be embarked on board of a medium size satellite, flying in tandem with Sentinel-3 in a Sun synchronous orbit at a height of about 815 km. FLORIS will observe the vegetation fluorescence and reflectance within a spectral range between 500 and 780 nm. Multi-frames acquisitions on matrix detectors during the satellite movement will allow the production of 2D Earth scene images in two different spectral channels, called HR and LR with spectral resolution of 0.3 and 2 nm respectively. A common fore optics is foreseen to enhance by design the spatial co-registration between the two spectral channels, which have the same ground spatial sampling (300 m) and swath (150 km). An overlapped spectral range between the two channels is also introduced to simplify the spectral coregistration. A compact opto-mechanical solution with all spherical and plane optical elements is proposed, and the most significant design rationales are described. The instrument optical architecture foresees a dual Babinet scrambler, a dioptric telescope and two grating spectrometers (HR and LR), each consisting of a modified Offner configuration. The developed design is robust, stable vs temperature, easy to align, showing very high optical quality along the whole field of view. The system gives also excellent correction for transverse chromatic aberration and distortions (keystone and smile).

  1. Fluorescence lifetime imaging of skin cancer

    NASA Astrophysics Data System (ADS)

    Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

    2011-03-01

    Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

  2. Fluorescence optical imaging in anticancer drug delivery.

    PubMed

    Etrych, Tomáš; Lucas, Henrike; Janoušková, Olga; Chytil, Petr; Mueller, Thomas; Mäder, Karsten

    2016-03-28

    In the past several decades, nanosized drug delivery systems with various targeting functions and controlled drug release capabilities inside targeted tissues or cells have been intensively studied. Understanding their pharmacokinetic properties is crucial for the successful transition of this research into clinical practice. Among others, fluorescence imaging has become one of the most commonly used imaging tools in pre-clinical research. The development of increasing numbers of suitable fluorescent dyes excitable in the visible to near-infrared wavelengths of the spectrum has significantly expanded the applicability of fluorescence imaging. This paper focuses on the potential applications and limitations of non-invasive imaging techniques in the field of drug delivery, especially in anticancer therapy. Fluorescent imaging at both the cellular and systemic levels is discussed in detail. Additionally, we explore the possibility for simultaneous treatment and imaging using theranostics and combinations of different imaging techniques, e.g., fluorescence imaging with computed tomography. PMID:26892751

  3. Combined fluorescence and phosphorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Shcheslavskiy, V. I.; Neubauer, A.; Bukowiecki, R.; Dinter, F.; Becker, W.

    2016-02-01

    We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.

  4. Multi-parameter high-resolution lithospheric imaging by source-independent full-waveform inversion of teleseismic data

    NASA Astrophysics Data System (ADS)

    Beller, S.; Monteiller, V.; Operto, S.; Nolet, G.

    2015-12-01

    Building broadband multi-parameter lithospheric models is one of the quest of earthquake seismology. Nowadays, deployment of dense arrays of broadband stations and advances in high-performance computing open new perspectives to achieve this goal by full waveform inversion (FWI) of teleseismic data. Compared to traveltime tomography, broadband images can be obtained by FWI when wavefields that are forward-scattered (i.e., transmission regime) and backward-scattered (reflection regime) by lithospheric heterogeneties to be imaged are involved in the inversion. In teleseismic setting, incident wavefields impinge the boundaries of the lithospheric target and propagate up to the surface where they are recorded by the stations, giving rise to the transmitted part of the recorded wavefield. The incident wavefield is also reflected back into the lithospheric target by the free surface acting as P- and S-waves secondary sources. The resulting wavefield is reflected by the lithospheric reflectors before being recorded by the stations, giving rise to the second-order reflection part of the recorded wavefield. While the transmitted part of the wavefield allows one to achieve a resolution close to that obtained by traveltime tomography, involving the reflected part of the wavefield in the FWI is amenable to the short-wavelength updates, hence broadaning the wavenumber spectrum of the lithospheric models toward high wavenumbers. Another benefit to involve the reflection regime in FWI is to increase the sensitivity of the FWI to the density parameter. In this study, we first discuss the feasibility of the density reconstruction in addition to that of the P- and S-waves velocities by FWI of teleseismic wavefields with a realistic synthetic study representative of the western Alps. The density reconstruction implies the extraction of information given by small amplitude secondary wavefields from the data that may be drastically affected by noise and trade-off between model parameter

  5. Hadamard-transform fluorescence-lifetime imaging.

    PubMed

    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

  6. Recent Progress in Fluorescent Imaging Probes.

    PubMed

    Pak, Yen Leng; Swamy, K M K; Yoon, Juyoung

    2015-01-01

    Due to the simplicity and low detection limit, especially the bioimaging ability for cells, fluorescence probes serve as unique detection methods. With the aid of molecular recognition and specific organic reactions, research on fluorescent imaging probes has blossomed during the last decade. Especially, reaction based fluorescent probes have been proven to be highly selective for specific analytes. This review highlights our recent progress on fluorescent imaging probes for biologically important species, such as biothiols, reactive oxygen species, reactive nitrogen species, metal ions including Zn(2+), Hg(2+), Cu(2+) and Au(3+), and anions including cyanide and adenosine triphosphate (ATP). PMID:26402684

  7. Recent Progress in Fluorescent Imaging Probes

    PubMed Central

    Pak, Yen Leng; Swamy, K. M. K.; Yoon, Juyoung

    2015-01-01

    Due to the simplicity and low detection limit, especially the bioimaging ability for cells, fluorescence probes serve as unique detection methods. With the aid of molecular recognition and specific organic reactions, research on fluorescent imaging probes has blossomed during the last decade. Especially, reaction based fluorescent probes have been proven to be highly selective for specific analytes. This review highlights our recent progress on fluorescent imaging probes for biologically important species, such as biothiols, reactive oxygen species, reactive nitrogen species, metal ions including Zn2+, Hg2+, Cu2+ and Au3+, and anions including cyanide and adenosine triphosphate (ATP). PMID:26402684

  8. Comprehensive phantom for interventional fluorescence molecular imaging.

    PubMed

    Anastasopoulou, Maria; Koch, Maximilian; Gorpas, Dimitris; Karlas, Angelos; Klemm, Uwe; Garcia-Allende, Pilar Beatriz; Ntziachristos, Vasilis

    2016-09-01

    Fluorescence imaging has been considered for over a half-century as a modality that could assist surgical guidance and visualization. The administration of fluorescent molecules with sensitivity to disease biomarkers and their imaging using a fluorescence camera can outline pathophysiological parameters of tissue invisible to the human eye during operation. The advent of fluorescent agents that target specific cellular responses and molecular pathways of disease has facilitated the intraoperative identification of cancer with improved sensitivity and specificity over nonspecific fluorescent dyes that only outline the vascular system and enhanced permeability effects. With these new abilities come unique requirements for developing phantoms to calibrate imaging systems and algorithms. We briefly review herein progress with fluorescence phantoms employed to validate fluorescence imaging systems and results. We identify current limitations and discuss the level of phantom complexity that may be required for developing a universal strategy for fluorescence imaging calibration. Finally, we present a phantom design that could be used as a tool for interlaboratory system performance evaluation. PMID:27304578

  9. Fluorescence goggle for intraoperative breast cancer imaging

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Bauer, Adam Q.; Akers, Walter; Sudlow, Gail; Liang, Kexian; Charanya, Tauseef; Mondal, Suman; Culver, Joseph P.; Achilefu, Samuel

    2012-03-01

    We have developed a fluorescence goggle device for intraoperative oncologic imaging. With our system design, the surgeon can directly visualize the fluorescence information from the eyepieces in real time without any additional monitor, which can improve one's coordination and surgical accuracy. In conjunction with targeting fluorescent dyes, the goggle device can successfully detect tumor margins and small nodules that are not obvious to naked eye. This can potentially decrease the incidence of incomplete resection.

  10. Scanning fluorescent microthermal imaging apparatus and method

    DOEpatents

    Barton, Daniel L.; Tangyunyong, Paiboon

    1998-01-01

    A scanning fluorescent microthermal imaging (FMI) apparatus and method is disclosed, useful for integrated circuit (IC) failure analysis, that uses a scanned and focused beam from a laser to excite a thin fluorescent film disposed over the surface of the IC. By collecting fluorescent radiation from the film, and performing point-by-point data collection with a single-point photodetector, a thermal map of the IC is formed to measure any localized heating associated with defects in the IC.

  11. Scanning fluorescent microthermal imaging apparatus and method

    DOEpatents

    Barton, D.L.; Tangyunyong, P.

    1998-01-06

    A scanning fluorescent microthermal imaging (FMI) apparatus and method is disclosed, useful for integrated circuit (IC) failure analysis, that uses a scanned and focused beam from a laser to excite a thin fluorescent film disposed over the surface of the IC. By collecting fluorescent radiation from the film, and performing point-by-point data collection with a single-point photodetector, a thermal map of the IC is formed to measure any localized heating associated with defects in the IC. 1 fig.

  12. Exogenous specific fluorescence marker location reconstruction using surface fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Avital, Garashi; Gannot, Israel; Chernomordik, Victor V.; Gannot, Gallya; Gandjbakhche, Amir H.

    2003-07-01

    Diseased tissue may be specifically marked by an exogenous fluorescent marker and then, following laser activation of the marker, optically and non-invasively detected through fluorescence imaging. Interaction of a fluorophore, conjugated to an appropriate antibody, with the antigen expressed by the diseased tissue, can indicate the presence of a specific disease. Using an optical detection system and a reconstruction algorithm, we were able to determine the fluorophore"s position in the tissue. We present 3D reconstructions of the location of a fluorescent marker, FITC, in the tongues of mice. One group of BALB/c mice was injected with squamous cell carcinoma (SqCC) cell line to the tongue, while another group served as the control. After tumor development, the mice"s tongues were injected with FITC conjugated to anti-CD3 and anti-CD 19 antibodies. An Argon laser excited the marker at 488 nm while a high precision fluorescent camera collected the emitted fluorescence. Measurements were performed with the fluorescent marker embedded at various simulated depths. The simulation was performed using agarose-based gel slabs applied to the tongue as tissue-like phantoms. A biopsy was taken from every mouse after the procedure and the excised tissue was histologically evaluated. We reconstruct the fluorescent marker"s location in 3D using an algorithm based on the random walk theory.

  13. High frame rate fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Agronskaia, A. V.; Tertoolen, L.; Gerritsen, H. C.

    2003-07-01

    A fast time-domain based fluorescence lifetime imaging (FLIM) microscope is presented that can operate at frame rates of hundreds of frames per second. A beam splitter in the detection path of a wide-field fluorescence microscope divides the fluorescence in two parts. One part is optically delayed with respect to the other. Both parts are viewed with a single time-gated intensified CCD camera with a gate width of 5 ns. The fluorescence lifetime image is obtained from the ratio of these two images. The fluorescence lifetime resolution of the FLIM microscope is verified both with dye solutions and fluorescent latex beads. The fluorescence lifetimes obtained from the reference specimens are in good agreement with values obtained from time correlated single photon counting measurements on the same specimens. The acquisition speed of the FLIM system is evaluated with a measurement of the calcium fluxes in neonatal rat myocytes stained with the calcium probe Oregon Green 488-Bapta. Fluorescence lifetime images of the calcium fluxes related to the beating of the myocytes are acquired with frame rates of up to 100 Hz.

  14. Ion-induced fluorescence imaging of endosomes

    NASA Astrophysics Data System (ADS)

    Norarat, R.; Marjomäki, V.; Chen, X.; Zhaohong, M.; Minqin, R.; Chen, C.-B.; Bettiol, A. A.; Whitlow, H. J.; Watt, F.

    2013-07-01

    Imaging laboratories at Jyväskylä and Singapore are collaborating on the development of fluorescence imaging of cytoplasmic endosomes using a combination of proton induced fluorescence (PIF) with direct Scanning Transmission Ion Microscopy (direct-STIM) for sub-cellular structural imaging. A549 lung carcinoma cells were cultivated and stained for epidermal growth factor receptor (EGFR) and receptor α2β1 integrin. In this paper, we demonstrate that cells can be imaged at sub-150 nm resolution using the PIF technique. In addition, the same target cell was imaged at 50 and 25 nm resolution by using proton and He-STIM, respectively. The combination of both techniques offer a powerful tool to improve fluorescence imaging beyond optical diffraction limits.

  15. 2D multi-parameter elastic seismic imaging by frequency-domain L1-norm full waveform inversion

    NASA Astrophysics Data System (ADS)

    Brossier, Romain; Operto, Stéphane; Virieux, Jean

    2010-05-01

    Full waveform inversion (FWI) is becoming a powerful and efficient tool to derive high-resolution quantitative models of the subsurface. In the frequency-domain, computationally efficient FWI algorithms can be designed for wide-aperture acquisition geometries by limiting inversion to few discrete frequencies. However, FWI remains an ill-posed and highly non-linear data-fitting procedure that is sensitive to noise, inaccuracies of the starting model and definition of multiparameter classes. The footprint of the noise in seismic imaging is conventionally mitigated by stacking highly redundant multifold data. However, when the data redundancy is decimated in the framework of efficient frequency-domain FWI, it is essential to assess the sensitivity of the inversion to noise. The impact of the noise in FWI, when applied to decimated data sets, has been marginally illustrated in the past and least-squares minimisation has remained the most popular approach. We investigate in this study the sensitivity of frequency-domain elastic FWI to noise for realistic onshore and offshore synthetic data sets contaminated by ambient random white noise. Four minimisation functionals are assessed in the framework of frequency domain FWI of decimated data: the classical least-square norm (L2), the least-absolute-values norm (L1), and some combinations of both (the Huber and the so-called Hybrid criteria). These functionals are implemented in a massively-parallel, 2D elastic frequency-domain FWI algorithm. A two-level hierarchical algorithm is implemented to mitigate the non-linearity of the inversion in complex environments. The first outer level consists of successive inversions of frequency groups of increasing high-frequency content. This level defines a multi-scale approach while preserving some data redundancy by means of simultaneous inversion of multiple frequencies. The second inner level used complex-valued frequencies for data preconditioning. This preconditioning controls the

  16. Photocontrollable Fluorescent Proteins for Superresolution Imaging

    PubMed Central

    Shcherbakova, Daria M.; Sengupta, Prabuddha; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V.

    2014-01-01

    Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization–based and nonlinear ensemble–based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine. PMID:24895855

  17. Reflectance and fluorescence hyperspectral elastic image registration

    NASA Astrophysics Data System (ADS)

    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.

  18. Fluorescent Cell Imaging in Regenerative Medicine

    PubMed Central

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

    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 intra-vital microscopy and Förster resonance energy transfer, are providing new insights into cellular behavior, including cell migration, morphology, and phenotypic changes in a dynamic environment. Such applications, combined with multimodal imaging, significantly expand the utility of fluorescent protein imaging in research and clinical applications of regenerative medicine. PMID:27158228

  19. Hyperspectral Fluorescence and Reflectance Imaging Instrument

    NASA Technical Reports Server (NTRS)

    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

  20. Fluorescence imaging using synthetic GFP chromophores.

    PubMed

    Walker, Christopher L; Lukyanov, Konstantin A; Yampolsky, Ilia V; Mishin, Alexander S; Bommarius, Andreas S; Duraj-Thatte, Anna M; Azizi, Bahareh; Tolbert, Laren M; Solntsev, Kyril M

    2015-08-01

    Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility. PMID:26117808

  1. Fluorescence Microscopy Imaging in Biomedical Sciences

    NASA Astrophysics Data System (ADS)

    Sun, Yuansheng; Periasamy, Ammasi

    Fluorescence microscopy is an important tool in biological sciences which provides excellent sensitivity for detecting very low concentrations of molecules over broad spatial and temporal dimensions. With fast developments of new fluorescent probes, advanced electronic and optical devices, and sophisticated data acquisition and analysis software, fluorescence microscopy resides on the central stage of life-sciences research. This chapter covers several commonly used and advanced fluorescence microscopy techniques and focuses on fluorescence lifetime imaging microscopy (FLIM). A number of FLIM systems and their applications are reviewed. As an example, we describe how we built and calibrated a two-photon excitation time-correlated single-photon counting (TPE-TCSPC) FLIM system and employed the system to investigate protein-protein interactions in living cells.

  2. Multi-parameter seismic imaging by Full Waveform Inversion : an application to the Valhall oil field in North Sea

    NASA Astrophysics Data System (ADS)

    Brossier, R.; Gholami, Y.; Prieux, V.; Operto, S.; Ribodetti, A.; Virieux, J.

    2012-12-01

    Full Waveform Inversion (FWI) is becoming an appealing technique to delineate high resolution models of the Earth at various scales. At the exploration scale, FWI is mainly applied in the acoustic approximation for velocity model building before migration. The P-wave velocity has generally the strongest influence on data but seismic waveforms also contain useful information on density, attenuation, S-wave velocity, and seismic anisotropy. These parameters affect either traveltimes, amplitudes, or the full waveform. This complex information requires to be carefully extracted by multi-parameter inversion, as some trade-off exist between parameters. When multiple classes of parameters are reconstructed, FWI becomes highly non-linear that can require to design hierarchical approaches both over the data components and the model parameter classes. In this study, we present an application of 2D multi-parameter FWI to multicomponent ocean-bottom cable data from the shallow-water Valhall field, North Sea. We follow a data-driven hierarchical workflow to retrieve model of P-wave velocity, anisotropy, density, P-wave quality factor and the shear-wave velocity. The shallow-water (70 m) and the soft sediment of the target zone (shear-wave around 300 m/s at the sea-floor) prevent to generate significant P-to-S conversion on the sea bed. Therefore, a limited footprint of shear waves is present in the data, and FWI in the acoustic approximation can be used in a first step of reconstruction. The target contains shale layers in the shallow part (above the 1.5 km depth) that generates a significant VTI anisotropy: anisotropy is a key parameter for FWI reconstruction in this zone. In a first part of this study, we show the strong impact of the VTI anisotropy on anisotropic and isotropic acoustic FWI : horizontal velocities, up to 15% faster than the vertical velocities, are reconstructed by isotropic FWI in the upper part of the target, where the FWI is mainly driven by the wide

  3. Laser-induced fluorescence imaging of bacteria

    NASA Astrophysics Data System (ADS)

    Hilton, Peter J.

    1998-12-01

    This paper outlines a method for optically detecting bacteria on various backgrounds, such as meat, by imaging their laser induced auto-fluorescence response. This method can potentially operate in real-time, which is many times faster than current bacterial detection methods, which require culturing of bacterial samples. This paper describes the imaging technique employed whereby a laser spot is scanned across an object while capturing, filtering, and digitizing the returned light. Preliminary results of the bacterial auto-fluorescence are reported and plans for future research are discussed. The results to date are encouraging with six of the eight bacterial strains investigated exhibiting auto-fluorescence when excited at 488 nm. Discrimination of these bacterial strains against red meat is shown and techniques for reducing background fluorescence discussed.

  4. Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

    PubMed Central

    Marriott, G; Clegg, R M; Arndt-Jovin, D J; Jovin, T M

    1991-01-01

    An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:1723311

  5. Fluorescence imaging of early lung cancer

    NASA Astrophysics Data System (ADS)

    Lam, Stephen; MacAulay, Calum E.; Le Riche, Jean C.; Ikeda, Norihiko; Palcic, Branko

    1995-01-01

    The performance of a fluorescence imaging device was compared with conventional white-light bronchoscopy in 100 patients with lung cancer, 46 patients with resected State I nonsmall cell lung cancer, 10 patients with head and neck cancer, and 67 volunteers who had smoked at least one pack of cigarettes per day for twenty-five years or more. Using differences in tissue autofluorescence between premalignant, malignant and normal tissues, fluorescence bronchoscopy was found to detect more than twice as many moderate-severe dysplasia and carcinoma in situ sites than conventional white-light bronchoscopy. The use of fluorescence imaging to detect small peripheral lung nodules was investigated in a micro metastatic lung model of mice implanted with Lewis lung tumor cells. Fluorescence imaging was found to be able to detect small malignant lung lesions. The use of (delta) -aminolevulinic acid (ALA) to enhance fluorescence detection of CIS was investigated in a patient after oral administration of 60 mg/kg of ALA four hours prior to bronchoscopy, although ALA enhanced the tumor's visibility, multiple sites of false positive fluorescence were observed in areas of inflammation or metaplasia.

  6. Fluorescence imaging in the last two decades

    PubMed Central

    Miyawaki, Atsushi

    2013-01-01

    In commemoration of the 20th anniversary of the molecular cloning of the gene for the green fluorescent protein from the jellyfish Aequorea victoria, I would like to reflect on the development of new fluorescence imaging technology in the last two decades. As this technology has become increasingly diversified, it has become more and more of a challenge to come up with a comprehensive and exhaustive review of it. Here I will focus on optogenetics and large-scale, three-dimensional reconstruction. Those two technological innovations have been achieved in the neuroscience community owing to the combined efforts of molecular biologists and light microscopists. In addition, modern fluorescence imaging has indeed improved our understanding of the spatiotemporal regulation of fundamental biological functions at cellular level. As an example, I will introduce some findings we made regarding the movement of biomolecules across the nuclear membrane. The above-mentioned imaging approaches are possible today but were impossible two decades ago. PMID:23393311

  7. ICG fluorescence imaging and its medical applications

    NASA Astrophysics Data System (ADS)

    Miwa, Mitsuharu; Shikayama, Takahiro

    2008-12-01

    This paper presents a novel optical angiography system, and introduces its medical applications. We developed the optical enhanced imaging system which can observe the blood and lymphatic vessels as the Indocyanine green (ICG) fluorescence image. The imaging system consists of 760nm light emitted diode (LED) as excite light, CCD camera as a detector, a high-pass optical filter in front of the CCD and video processing system. The advantage of ICG fluorescence method is safe (radiation free), high sensitive, real time monitoring of blood and/or lymphatic flow, small size, easy to operate and cost effective compared to conventional X-ray angiography or scintigraphy. We have applied this method to several clinical applications such as breast cancer sentinel lymph node (SLN) navigation, lymph edema diagnostic and identification of liver segmentation. In each application, ICG fluorescence method shows useful result. It's indicated that this method is promising technique as optical angiography.

  8. Two-photon fluorescence anisotropy imaging

    NASA Astrophysics Data System (ADS)

    Li, Wei; Wang, Yi; Shao, Hanrong; He, Yonghong; Ma, Hui

    2006-09-01

    We have developed a novel method for imaging the fluorescence intensity and anisotropy by two-photon fluorescence microscopy and tested its capability in biological application. This method is applied to model sample including FITC and FITC-CD44 antibody solution and also FITC-CD44 stained cells. The fluorescence anisotropy (FA) of FITC-CD44ab solution is higher than the FITC solution with the same concentration. The fluorescence in cell sample has even higher FA than in solution because the rotation diffusion is restrained in membrane. The method is employed to study the effect of berberine a kind of Chinese medicine, on tumor metastasis. The results indicated that tumor cell membrane fluidity is decreasing with increasing the concentration of berberine in culture medium.

  9. Toward Fourier interferometry fluorescence excitation/emission imaging of malignant cells combined with photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Kohen, Elli; Hirschberg, Joseph G.; Berry, John P.; Ozkutuk, Nuri; Ornek, Ceren; Monti, Marco; Leblanc, Roger M.; Schachtschabel, Dietrich O.; Haroon, Sumaira

    2003-10-01

    Dual excitation fluorescence imaging has been used as a first step towards multi-wavelength excitation/emission fluorescence spectral imaging. Target cells are transformed keratinocytes, and other osteosarcoma, human breast and color cancer cells. Mitochondrial membrane potential probes, e.g. TMRM (tetramethylrhodamine methyl ester), Mitotracker Green (Molecular Probes, Inc., Eugene OR,USA; a recently synthesized mitochondrial oxygen probe, [PRE,P1"- pyrene butyl)-2-rhodamine ester] allow dual excitation in the UV plus in teh blue-green spectral regions. Also, using the natural endogenous probe NAD(P)H, preliminary results indicate mitochondrial responses to metabolic challenges (e.g. glucose addition), plus changes in mitochonrial distribution and morphology. In terms of application to biomedicine (for diagnostiscs, prognostsics and drug trials) three parameters have been selected in addition to the natural probe NAD(P)H, i.e. vital fluorescence probing of mitochondria, lysosomes and Golgi apparatus. It is hoped that such a multiparameter approach will allow malignant cell characterization and grading. A new area being introduced is the use of similar methodology for biotechnical applications such as the study of the hydrogen-producing alga Chlamydomonas Reinhardtii, and possible agricultural applications, such as Saccharomyces yeast for oenology. Complementation by Photoacoustic Microscopy is also contemplated, to study the internal conversion component which follows the excitation by photons.

  10. Intraoperative imaging and fluorescence image guidance in oncologic surgery using a wearable fluorescence goggle system

    NASA Astrophysics Data System (ADS)

    Mondal, Suman B.; Gao, Shengkui; Zhu, Nan; Liu, Yang; Sudlow, Gail P.; Akers, Walter J.; Liang, Rongguang; Gruev, Viktor; Achilefu, Samuel

    2014-03-01

    We have developed a wearable, fluorescence goggle based system for intraoperative imaging of tumors and image guidance in oncologic surgery. Our system can detect fluorescence from cancer selective near infra-red (NIR) contrast agent, facilitating intraoperative visualization of surgical margins and tumors otherwise not apparent to the surgeon. The fluorescence information is displayed directly to the head mounted display (HMD) of the surgeon in real time, allowing unhindered surgical procedure under image guidance. This system has the potential of improving surgical outcomes in oncologic surgery and reduce the chances of cancer recurrence.

  11. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

    NASA Astrophysics Data System (ADS)

    Betzig, Eric; Patterson, George H.; Sougrat, Rachid; Lindwasser, O. Wolf; Olenych, Scott; Bonifacino, Juan S.; Davidson, Michael W.; Lippincott-Schwartz, Jennifer; Hess, Harald F.

    2006-09-01

    We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to ~2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method-termed photoactivated localization microscopy-to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

  12. Molecular Probes for Fluorescence Lifetime Imaging

    PubMed Central

    Sarder, Pinaki; Maji, Dolonchampa; Achilefu, Samuel

    2015-01-01

    Visualization of biological processes and pathologic conditions at the cellular and tissue levels largely rely on the use of fluorescence intensity signals from fluorophores or their bioconjugates. To overcome the concentration dependency of intensity measurements, evaluate subtle molecular interactions, and determine biochemical status of intracellular or extracellular microenvironments, fluorescence lifetime (FLT) imaging has emerged as a reliable imaging method complementary to intensity measurements. Driven by a wide variety of dyes exhibiting stable or environment-responsive FLTs, information multiplexing can be readily accomplished without the need for ratiometric spectral imaging. With knowledge of the fluorescent states of the molecules, it is entirely possible to predict the functional status of biomolecules or microevironment of cells. Whereas the use of FLT spectroscopy and microscopy in biological studies is now well established, in vivo imaging of biological processes based on FLT imaging techniques is still evolving. This review summarizes recent advances in the application of the FLT of molecular probes for imaging cells and small animal models of human diseases. It also highlights some challenges that continue to limit the full realization of the potential of using FLT molecular probes to address diverse biological problems, and outlines areas of potential high impact in the future. PMID:25961514

  13. Fluorescent nanoparticle probes for imaging of cancer.

    PubMed

    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

  14. Hyperspectral confocal fluorescence imaging of cells

    NASA Astrophysics Data System (ADS)

    Haaland, David M.; Jones, Howland D. T.; Sinclair, Michael B.; Carson, Bryan; Branda, Catherine; Poschet, Jens F.; Rebeil, Roberto; Tian, Bing; Liu, Ping; Brasier, Allan R.

    2007-09-01

    Confocal fluorescence imaging of biological systems is an important method by which researchers can investigate molecular processes occurring in live cells. We have developed a new 3D hyperspectral confocal fluorescence microscope that can further enhance the usefulness of fluorescence microscopy in studying biological systems. The new microscope can increase the information content obtained from the image since, at each voxel, the microscope records 512 wavelengths from the emission spectrum (490 to 800 nm) while providing optical sectioning of samples with diffraction-limited spatial resolution. When coupled with multivariate curve resolution (MCR) analyses, the microscope can resolve multiple spatially and spectrally overlapped emission components, thereby greatly increasing the number of fluorescent labels, relative to most commercial microscopes, that can be monitored simultaneously. The MCR algorithm allows the "discovery" of all emitting sources and estimation of their relative concentrations without cross talk, including those emission sources that might not have been expected in the imaged cells. In this work, we have used the new microscope to obtain time-resolved hyperspectral images of cellular processes. We have quantitatively monitored the translocation of the GFP-labeled RelA protein (without interference from autofluorescence) into and out of the nucleus of live HeLa cells in response to continuous stimulation by the cytokine, TNFα. These studies have been extended to imaging live mouse macrophage cells with YFP-labeled RelA and GFP-labeled IRF3 protein. Hyperspectral imaging coupled with MCR analysis makes possible, for the first time, quantitative analysis of GFP, YFP, and autofluorescence without concern for cross-talk between emission sources. The significant power and quantitative capabilities of the new hyperspectral imaging system are further demonstrated with the imaging of a simple fluorescence dye (SYTO 13) traditionally used to stain the

  15. Imaging efficacy of a targeted imaging agent for fluorescence endoscopy

    NASA Astrophysics Data System (ADS)

    Healey, A. J.; Bendiksen, R.; Attramadal, T.; Bjerke, R.; Waagene, S.; Hvoslef, A. M.; Johannesen, E.

    2008-02-01

    Colorectal cancer is a major cause of cancer death. A significant unmet clinical need exists in the area of screening for earlier and more accurate diagnosis and treatment. We have identified a fluorescence imaging agent targeted to an early stage molecular marker for colorectal cancer. The agent is administered intravenously and imaged in a far red imaging channel as an adjunct to white light endoscopy. There is experimental evidence of preclinical proof of mechanism for the agent. In order to assess potential clinical efficacy, imaging was performed with a prototype fluorescence endoscope system designed to produce clinically relevant images. A clinical laparoscope system was modified for fluorescence imaging. The system was optimised for sensitivity. Images were recorded at settings matching those expected with a clinical endoscope implementation (at video frame rate operation). The animal model was comprised of a HCT-15 xenograft tumour expressing the target at concentration levels expected in early stage colorectal cancer. Tumours were grown subcutaneously. The imaging agent was administered intravenously at a dose of 50nmol/kg body weight. The animals were killed 2 hours post administration and prepared for imaging. A 3-4mm diameter, 1.6mm thick slice of viable tumour was placed over the opened colon and imaged with the laparoscope system. A receiver operator characteristic analysis was applied to imaging results. An area under the curve of 0.98 and a sensitivity of 87% [73, 96] and specificity of 100% [93, 100] were obtained.

  16. Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy

    PubMed Central

    Min, Wei; Freudiger, Christian W.; Lu, Sijia; Xie, X. Sunney

    2012-01-01

    The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy, including stimulated Raman scattering and two photon absorption, and pump-probe microscopy, including stimulated emission, excited state absorption and ground state depletion, provide distinct and powerful image contrasts for non-fluorescent species. Thanks to high-frequency modulation transfer scheme, they exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles, excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques. PMID:21453061

  17. Fluorescence labeled microbubbles for multimodal imaging.

    PubMed

    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

  18. Temporal resolution in fluorescence imaging

    PubMed Central

    Mondal, Partha Pratim

    2014-01-01

    Temporal resolution is a key factor for imaging rapidly occurring events in biology. In this feature article, I investigate an approximate estimate for determining the temporal resolution limit. The condition that led to this limit is, the time taken by the ensemble (99.9%) of excited molecules to relax to ground state, assuming all the emitted photons are detected. In a simplistic three-level system, the temporal resolution is, ≈3τp, where τp = (loge10)/(kf + knr) and, kf and knr are respectively the radiative and non-radiative emission rates. This further assumes the ideal condition that, the quantum efficiency of the detector is unity and there are no other loses. We discuss few state-of-art microscopy techniques that are capable of high temporal resolution. This includes techniques such as multifocal multiphoton microscopy (MMM), multifocal plane microscopy, multiple excitation spot optical microscopy (MESO), multiplane microscopy and multiple light-sheet microscopy (MLSM). PMID:25988152

  19. Hyperspectral fluorescence lifetime imaging for optical biopsy.

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Fluorescence lifetime imaging in turbid media

    NASA Astrophysics Data System (ADS)

    O'Leary, M. A.; Boas, D. A.; Li, X. D.; Chance, B.; Yodh, A. G.

    1996-01-01

    The lifetime of a fluorophore generally varies in different environments, making the molecule a sensitive indicator of tissue oxygenation, pH, and glucose. However, lifetime measurements are complicated when the fluorophore is embedded in an optically thick, highly scattering medium such as human tissue. We formulate the inverse problem for fluorescence lifetime tomography using diffuse photon density waves, and we demonstrate the technique by deriving spatial images of heterogeneous fluorophore distribution and lifetime, using simulated measurements in heterogeneous turbid media.

  2. Multispectral fluorescence imaging device for malignancy detection

    NASA Astrophysics Data System (ADS)

    Bocher, Thomas; Luhmann, Till; Baier, S.; Dierolf, Marc; Naumann, M.; Beuthan, Juergen; Berlien, Hans-Peter; Mueller, Gerhard J.

    1997-12-01

    In medical diagnosis of superficial lesions at inner or outer surfaces of the human body fluorescence imaging techniques are able to deliver additional information on the metabolic and structural state of the observed tissue. To subtract background fluorescence and to achieve a differential diagnosis a multispectral analysis in several wavelength windows is needed. Additionally, special image algorithms have to be applied which depend on the examined malignancy. For this purpose a multispectral fluorescence imaging device was developed. It can be used both endoscopically and in combination with a standard operational microscope from Carl Zeiss, Germany. In this paper, the device and first clinical results are presented. The device was built to detect superficial lesions like tumors, inflammations, etc. Target chromophores are NADH, Protoporphyrin IX, collagen and other. The measured optical bands are (405 plus or minus 5) nm, (442 plus or minus 5) nm, (458 plus or minus 5) nm, (550 plus or minus 5) nm, (630 plus or minus 5) nm and (690 plus or minus 5) nm. A special UV-source with a liquid light guide is used as the illumination source in two excitation bands of (365 plus or minus 10) nm and (420 plus or minus 20) nm. First clinical investigations of superficial malignancies like squamous cell carcinoma and basalioma are presented.

  3. Fluorescence lifetime-based optical molecular imaging.

    PubMed

    Kumar, Anand T N

    2011-01-01

    Fluorescence lifetime is a powerful contrast mechanism for in vivo molecular imaging. In this chapter, we describe instrumentation and methods to optimally exploit lifetime contrast using a time domain fluorescence tomography system. The key features of the system are the use of point excitation in free-space using ultrashort laser pulses and non-contact detection using a gated, intensified CCD camera. The surface boundaries of the imaging volume are acquired using a photogrammetric camera integrated with the imaging system, and implemented in theoretical models of light propagation in biological tissue. The time domain data are optimally analyzed using a lifetime-based tomography approach, which is based on extracting a tomographic set of lifetimes and decay amplitudes from the long time decay portion of the time domain data. This approach improves the ability to locate in vivo targets with a resolution better than conventional optical methods. The application of time domain lifetime multiplexing and tomography are illustrated using phantoms and tumor bearing mouse model of breast adenocarcinoma. In the latter application, the time domain approach allows an improved detection of fluorescent protein signals from intact nude mice in the presence of background autofluorescence. This feature has potential applications for longitudinal pre-clinical evaluation of drug treatment response as well as to address fundamental questions related to tumor physiology and metastasis. PMID:21153381

  4. Mosaic-Detector-Based Fluorescence Spectral Imager

    NASA Technical Reports Server (NTRS)

    Son, Kyung-Ah; Moon, Jeong

    2007-01-01

    A battery-powered, pen-sized, portable instrument for measuring molecular fluorescence spectra of chemical and biological samples in the field has been proposed. Molecular fluorescence spectroscopy is among the techniques used most frequently in laboratories to analyze compositions of chemical and biological samples. Heretofore, it has been possible to measure fluorescence spectra of molecular species at relative concentrations as low as parts per billion (ppb), with a few nm spectral resolution. The proposed instrument would include a planar array (mosaic) of detectors, onto which a fluorescence spectrum would be spatially mapped. Unlike in the larger laboratory-type molecular fluorescence spectrometers, mapping of wavelengths to spatial positions would be accomplished without use of relatively bulky optical parts. The proposed instrument is expected to be sensitive enough to enable measurement of spectra of chemical species at relative concentrations <1 ppb, with spectral resolution that could be tailored by design to be comparable to a laboratory molecular fluorescence spectrometer. The proposed instrument (see figure) would include a button-cell battery and a laser diode, which would generate the monochromatic ultraviolet light needed to excite fluorescence in a sample. The sample would be held in a cell bounded by far-ultraviolet-transparent quartz or optical glass. The detector array would be, more specifically, a complementary metal oxide/ semiconductor or charge-coupled- device imaging photodetector array, the photodetectors of which would be tailored to respond to light in the wavelength range of the fluorescence spectrum to be measured. The light-input face of the photodetector array would be covered with a matching checkerboard array of multilayer thin film interference filters, such that each pixel in the array would be sensitive only to light in a spectral band narrow enough so as not to overlap significantly with the band of an adjacent pixel. The

  5. Coherent nonlinear optical imaging: beyond fluorescence microscopy.

    PubMed

    Min, Wei; Freudiger, Christian W; Lu, Sijia; Xie, X Sunney

    2011-01-01

    The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy (including stimulated Raman scattering and two-photon absorption) and pump-probe microscopy (including excited-state absorption, stimulated emission, and ground-state depletion), provides new image contrasts for nonfluorescent species. Thanks to the high-frequency modulation transfer scheme, these imaging techniques exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles and excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques. PMID:21453061

  6. Fluorescence confocal endomicroscopy in biological imaging

    NASA Astrophysics Data System (ADS)

    Delaney, Peter; Thomas, Steven; Allen, John; McLaren, Wendy; Murr, Elise; Harris, Martin

    2007-02-01

    In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices. There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo microscopy systems. A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system). Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204 sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of a high-density optical fibre bundle (~30,000 fibres) of <1mm diameter to transfer the confocal imaging plane to tissue in intact small animal organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and

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

    PubMed

    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

  8. Fluorescence imaging to quantify crop residue cover

    NASA Technical Reports Server (NTRS)

    Daughtry, C. S. T.; Mcmurtrey, J. E., III; Chappelle, E. W.

    1994-01-01

    Crop residues, the portion of the crop left in the field after harvest, can be an important management factor in controlling soil erosion. Methods to quantify residue cover are needed that are rapid, accurate, and objective. Scenes with known amounts of crop residue were illuminated with long wave ultraviolet (UV) radiation and fluorescence images were recorded with an intensified video camera fitted with a 453 to 488 nm band pass filter. A light colored soil and a dark colored soil were used as background for the weathered soybean stems. Residue cover was determined by counting the proportion of the pixels in the image with fluorescence values greater than a threshold. Soil pixels had the lowest gray levels in the images. The values of the soybean residue pixels spanned nearly the full range of the 8-bit video data. Classification accuracies typically were within 3(absolute units) of measured cover values. Video imaging can provide an intuitive understanding of the fraction of the soil covered by residue.

  9. Fluorescence Ratio Imaging Of Dynamic Intracellular Signals

    NASA Astrophysics Data System (ADS)

    Harootunian, Alec T.; Kao, J. P.; Tsien, Roger Y.

    1989-12-01

    Traditional biochemical assays of cellular messengers require grinding up thousands or millions of cells for each data point. Such destructive measurements use up large amounts of tissue, have poor time resolution, and cannot assess heterogeneity between individual cells or dynamic spatial localizations. Recent technical advances now enable important ionic signals to be continuously imaged inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+. Binding of these ions shifts the fluorescence excitation spectrum of the corresponding indicator. The ratio of excitation amplitudes at two wavelengths measures the free ion concentration while canceling out intensity variations due to nonuniform cell thickness or dye content. By rapidly alternating between the two ion-sensitive excitation wavelengths, a fluorescence microscope equipped with a low-light television camera and digital image processor can produce dynamic images of intracellular messenger levels. In many populations of cells traditionally assumed to be homogeneous, we find that neighboring individual cells can differ enormously in their cytosolic Ca2+ response to agonist stimulation, some ignoring the stimulus, others raising cytosolic Ca2+ transiently, others showing oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of graded inputs; we are investigating the mechanism of their generation using light flashes to generate pulses of intracellular messengers. Spatial gradients of cytosolic Ca t+ within single cells have been observed in embryos during fertilization and development, neurons exposed to electrical or drug stimulation and in cytotoxic T lymphocytes during killing of target

  10. A novel multiwavelength fluorescence image-guided surgery imaging system

    NASA Astrophysics Data System (ADS)

    Volpi, D.; Tullis, I. D. C.; Laios, A.; Pathiraja, P. N. J.; Haldar, K.; Ahmed, A. A.; Vojnovic, B.

    2014-02-01

    We describe the development and performance analysis of two clinical near-infrared fluorescence image-guided surgery (FIGS) devices that aim to overcome some of the limitations of current FIGS systems. The devices operate in a widefield-imaging mode and can work (1) in conjunction with a laparoscope, during minimally invasive surgery, and (2) as a hand-held, open surgery imaging system. In both cases, narrow-band excitation light, delivered at multiple wavelengths, is efficiently combined with white reflectance light. Light is delivered to ~100 cm2 surgical field at 1-2 mW/cm2 for white light and 3-7 mW/cm2 (depending on wavelength) of red - near infrared excitation, at a typical working distance of 350 mm for the hand-held device and 100 mm for the laparoscope. A single, sensitive, miniaturized color camera collects both fluorescence and white reflectance light. The use of a single imager eliminates image alignment and software overlay complexity. A novel filtering and illumination arrangement allows simultaneous detection of white reflectance and fluorescence emission from multiple dyes in real-time. We will present both fluorescence detection sensitivity modeling and practical performance data. We have demonstrated the efficiency and the advantages of the devices both pre-clinically and during live surgery on humans. Both the hand-held and the laparoscopic systems have proved to be reliable and beneficial in an ongoing clinical trial involving sentinel lymph node detection in gynecological cancers. We will show preliminary results using two clinically approved dyes, Methylene blue and indocyanine green. We anticipate that this technology can be integrated and routinely used in a larger variety of surgical procedures.

  11. Carbon Quantum Dots for Zebrafish Fluorescence Imaging.

    PubMed

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-01-01

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model. PMID:26135470

  12. Carbon Quantum Dots for Zebrafish Fluorescence Imaging

    PubMed Central

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-01-01

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model. PMID:26135470

  13. Carbon Quantum Dots for Zebrafish Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-07-01

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model.

  14. Multimodal light-sheet microscopy for fluorescence live imaging

    NASA Astrophysics Data System (ADS)

    Oshima, Y.; Kajiura-Kobayashi, H.; Nonaka, S.

    2012-03-01

    Light-sheet microscopy, it is known as single plane illumination microscope (SPIM), is a fluorescence imaging technique which can avoid phototoxic effects to living cells and gives high contrast and high spatial resolution by optical sectioning with light-sheet illumination in developmental biology. We have been developed a multifunctional light-sheet fluorescence microscopy system with a near infrared femto-second fiber laser, a high sensitive image sensor and a high throughput spectrometer. We performed that multiphoton fluorescence images of a transgenic fish and a mouse embryo were observed on the light-sheet microscope. As the results, two photon images with high contrast and high spatial resolution were successfully obtained in the microscopy system. The system has multimodality, not only mutiphoton fluorescence imaging, but also hyperspectral imaging, which can be applicable to fluorescence unmixing analysis and Raman imaging. It enables to obtain high specific and high throughput molecular imaging in vivo and in vitro.

  15. Multiparameter breast cancer cell image analysis for objective estimation of nuclear grade: comparison with light microscopic observational data

    NASA Astrophysics Data System (ADS)

    Berzins, Juris; Sneiders, Uldis; Plegere, Daina; Freivalds, Talivaldis; Grigalinovica, Romalda

    2000-04-01

    We performed a multi parameter image analysis assessment of breast cancer cell population nuclear grade (NG), which is regarded as one of the main prognostic factors for treatment efficacy and survival of the patients and compared it with light microscopic estimation of NG. Cytological imprint slides from 20 ductal carcinomas were stained according to Leischmann-AzureII-eosine method, and NG was estimated by light microscopic observation according to Black in Fisher's modification. Simultaneously, using specially elaborated software, in each patient 100 cancer cells were analyzed for nuclear perimeter, diameter, area, nucleolar area, and average intensity of staining. The chromatin structure was assessed using mean diameter of chromatin grains and relatively chromatic are within the nucleus. Light microscopic estimation revealed 4/15 grade 2 and 7/15 grade 3 tumors out of 15 filtrating ductal carcinomas, with 4/15 classified as intermediate between grade 2-3. Multifactoral linear correlation coefficient r equals 0.39, p < 0.001 for ductal cancer, higher NG comes with increasing nucleolar area, nuclear roundness factor, nuclear are, and chromatin area within the cell nucleus. Image analysis may yield precise information on NG as a prognostic factor in breast cancer patients.

  16. Temporal multiparameter airborne DLR E-SAR images for crop monitoring: summary of the CLEOPATRA campaign 1992

    NASA Astrophysics Data System (ADS)

    Schmullius, Christiane C.; Nithack, Juergen

    1997-01-01

    From May 11 to July 31, 1992 the Cloud Experiment OberPfaffenhofen And Transports took place as a field experimental contribution to the global energy and water cycle experiment. The DLR Institute of Radio Frequency Technology participated with its experimental SAR system E- SAR. Multitemporal X-, C- and L-band data from 8 dates and three ERS-1 images between May 20 and July 30, 1992 are analyzed in regard to the influence of changing plant backscatter constituents and to investigate the impact of increasing ground cover in the different wavelength on soil moisture mapping. Backscatter curves of four crops are shown, which indicate the possibility for crop monitoring and preferred times for crop classification. Detection of soil moisture changes is only possible with L-band and only under grain crops. Maximum likelihood and isocluster classifications were applied on several single- and multifrequency, mono- and multitemporal channel combinations. The overall classification accuracies were higher than with supervised methods. Maximum likelihood classification allowed identification of ten crop types with accuracies of up to 84 percent, when a temporal multifrequency data set was used.

  17. Multicolor Conjugated Polymer Dots for Biological Fluorescence Imaging

    PubMed Central

    Wu, Changfeng; Bull, Barbara; Szymanski, Craig; Christensen, Kenneth; McNeill, Jason

    2009-01-01

    Highly fluorescent conjugated polymer dots were developed for demanding applications such as fluorescence imaging in live cells. These nanoparticles exhibit small particle diameters, extraordinary fluorescence brightness, and excellent photostability. Single particle fluorescence imaging and kinetic studies indicate much higher emission rates (∼108 s-1) and little or no blinking of the nanoparticles as compared to typical results for single dye molecules and quantum dots. Analysis of single particle photobleaching trajectories reveals excellent photostability — as many as 109 or more photons emitted per nanoparticle prior to irreversible photobleaching. The superior figures of merit of these new fluorescent probes, together with the demonstration of cellular imaging, indicate their enormous potential for demanding fluorescence-based imaging and sensing applications such as high speed super-resolution single molecule/particle tracking and highly sensitive assays. PMID:19206410

  18. Wide-Field Multi-Parameter FLIM: Long-Term Minimal Invasive Observation of Proteins in Living Cells

    PubMed Central

    Vitali, Marco; Picazo, Fernando; Prokazov, Yury; Duci, Alessandro; Turbin, Evgeny; Götze, Christian; Llopis, Juan; Hartig, Roland; Visser, Antonie J. W. G.; Zuschratter, Werner

    2011-01-01

    Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes. PMID:21311595

  19. Multiparameter analysis of apoptosis in puromycin-treated Saccharomyces cerevisiae.

    PubMed

    Citterio, Barbara; Albertini, Maria Cristina; Ghibelli, Lina; Falcieri, Elisabetta; Battistelli, Michela; Canonico, Barbara; Rocchi, Marco B L; Teodori, Laura; Ciani, Maurizio; Piatti, Elena

    2015-08-01

    In Saccharomyces cerevisiae, a typical apoptotic phenotype is induced by some stress factors such as sugars, acetic acid, hydrogen peroxide, aspirin and age. Nevertheless, no data have been reported for apoptosis induced by puromycin, a damaging agent known to induce apoptosis in mammalian cells. We treated S. cerevisiae with puromycin to induce apoptosis and evaluated the percentage of dead cells by using Hoechst 33342 staining, transmission electron microscopy (TEM) and Annexin V flow cytometry (FC) analysis. Hoechst 33342 fluorescence images were processed to acquire parameters to use for multiparameter analysis [and perform a principal component analysis, (PCA)]. Cell viability was evaluated by Rhodamine 123 (Rh 123) and Acridine Orange microscope fluorescence staining. The results show puromycin-induced apoptosis in S. cerevisiae, and the PCA analysis indicated that the increasing percentage of apoptotic cells delineated a well-defined graph profile. The results were supported by TEM and FC. This study gives new insights into yeast apoptosis using puromycin as inducer agent, and PCA analysis may complement molecular analysis facilitating further studies to its detection. PMID:25868793

  20. Fluorescence Imaging Study of Impinging Underexpanded Jets

    NASA Technical Reports Server (NTRS)

    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.

  1. 3D fluorescence anisotropy imaging using selective plane illumination microscopy.

    PubMed

    Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

    2015-08-24

    Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein. PMID:26368202

  2. 3D fluorescence anisotropy imaging using selective plane illumination microscopy

    PubMed Central

    Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

    2015-01-01

    Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein. PMID:26368202

  3. Clinical application of indocyanine green-fluorescence imaging during hepatectomy.

    PubMed

    Ishizawa, Takeaki; Saiura, Akio; Kokudo, Norihiro

    2016-08-01

    In hepatobiliary surgery, the fluorescence and bile excretion of indocyanine green (ICG) can be used for real-time visualization of biological structure. Fluorescence cholangiography is used to obtain fluorescence images of the bile ducts following intrabiliary injection of 0.025-0.5 mg/mL ICG or intravenous injection of 2.5 mg ICG. Recently, the latter technique has been used in laparoscopic/robotic cholecystectomy. Intraoperative fluorescence imaging can be used to identify subcapsular hepatic tumors. Primary and secondary hepatic malignancy can be identified by intraoperative fluorescence imaging using preoperative intravenous injection of ICG through biliary excretion disorders that exist in cancerous tissues of hepatocellular carcinoma (HCC) and in non-cancerous hepatic parenchyma around adenocarcinoma foci. Intraoperative fluorescence imaging may help detect tumors to be removed, especially during laparoscopic hepatectomy, in which visual inspection and palpation are limited, compared with open surgery. Fluorescence imaging can also be used to identify hepatic segments. Boundaries of hepatic segments can be visualized following injection of 0.25-2.5 mg/mL ICG into the portal veins or by intravenous injection of 2.5 mg ICG following closure of the proximal portal pedicle toward hepatic regions to be removed. These techniques enable identification of hepatic segments before hepatectomy and during parenchymal transection for anatomic resection. Advances in imaging systems will increase the use of fluorescence imaging as an intraoperative navigation tool that can enhance the safety and accuracy of open and laparoscopic/robotic hepatobiliary surgery. PMID:27500144

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

    PubMed

    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

  5. Advances in fluorescence labeling strategies for dynamic cellular imaging

    PubMed Central

    Dean, Kevin M; Palmer, Amy E

    2014-01-01

    Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein– and bio-orthogonal–based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging. PMID:24937069

  6. Multispectral imaging fluorescence microscopy for lymphoid tissue analysis

    NASA Astrophysics Data System (ADS)

    Monici, Monica; Agati, Giovanni; Fusi, Franco; Mazzinghi, Piero; Romano, Salvatore; Pratesi, Riccardo; Alterini, Renato; Bernabei, Pietro A.; Rigacci, Luigi

    1999-01-01

    Multispectral imaging autofluorescence microscopy (MIAM) is used here for the analysis of lymphatic tissues. Lymph node biopsies, from patients with lympthoadenopathy of different origin have been examined. Natural fluorescence (NF) images of 3 micrometers sections were obtained using three filters peaked at 450, 550 and 680 nm with 50 nm bandpass. Monochrome images were combined together in a single RGB image. NF images of lymph node tissue sections show intense blue-green fluorescence of the connective stroma. Normal tissue shows follicles with faintly fluorescent lymphocytes, as expected fro the morphologic and functional characteristics of these cells. Other more fluorescent cells (e.g., plasma cells and macrophages) are evidenced. Intense green fluorescence if localized in the inner wall of the vessels. Tissues coming from patients affected by Hodgkin's lymphoma show spread fluorescence due to connective infiltration and no evidence of follicle organization. Brightly fluorescent large cells, presumably Hodgkin cells, are also observed. These results indicate that MIAM can discriminate between normal and pathological tissues on the basis of their natural fluorescence pattern, and, therefore, represent a potentially useful technique for diagnostic applications. Analysis of the fluorescence spectra of both normal and malignant lymphoid tissues resulted much less discriminatory than MIAM.

  7. Dual PET and Near-Infrared Fluorescence Imaging Probes as Tools for Imaging in Oncology

    PubMed Central

    An, Fei-Fei; Chan, Mark; Kommidi, Harikrishna; Ting, Richard

    2016-01-01

    OBJECTIVE The purpose of this article is to summarize advances in PET fluorescence resolution, agent design, and preclinical imaging that make a growing case for clinical PET fluorescence imaging. CONCLUSION Existing SPECT, PET, fluorescence, and MRI contrast imaging techniques are already deeply integrated into the management of cancer, from initial diagnosis to the observation and management of metastases. Combined positron-emitting fluorescent contrast agents can convey new or substantial benefits that improve on these proven clinical contrast agents. PMID:27223168

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

    PubMed

    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

  9. Multispectral fluorescence imaging techniques for nondestructive food safety inspection

    NASA Astrophysics Data System (ADS)

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

    2004-03-01

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

  10. Imaging Septum Formation by Fluorescence Microscopy.

    PubMed

    Ribas, Juan Carlos; Cortés, Juan Carlos G

    2016-01-01

    Fungal cleavage furrow formation during cytokinesis relays in the coordinated contraction of an actomyosin-based ring and the centripetal synthesis of both new plasma membrane and a special wall structure named division septum. Through transmission electron microscopy, the septum exhibits a three-layered structure with a central primary septum, flanked at both sides by the secondary septum. In contrast to the chitinous primary septum present in most of fungi, the fission yeast Schizosaccharomyces pombe does not contain chitin, instead it divides through the formation of a linear β(1,3)glucan-rich primary septum, which has been shown to be specifically stained by the fluorochrome Calcofluor white. Recent findings in S. pombe have revealed the importance of septum synthesis for the steady contraction of the ring during cytokinesis. Therefore, to study the molecular mechanisms that connect the extracellular septum wall with the other components of the cytokinetic machinery located in the plasma membrane and cytoplasm, new experimental approaches are needed. Here we describe the methods developed to image the septum structure by fluorescence microscopy, with a special focus in the analysis of septum progression by the use of time-lapse microscopy. PMID:26519306

  11. In vivo imaging with near-infrared fluorescence lifetime contrast

    NASA Astrophysics Data System (ADS)

    Akers, Walter J.; Berezin, Mikhail Y.; Lee, Hyeran; Achilefu, Samuel

    2009-02-01

    Fluorescence imaging is a mainstay of biomedical research, allowing detection of molecular events in both fixed and living cells, tissues and whole animals. Such high resolution fluorescence imaging is hampered by unwanted signal from intrinsic background fluorescence and scattered light. The signal to background ratio can be improved by using extrinsic contrast agents and greatly enhanced by multispectral imaging methods. Unfortunately, these methods are insufficient for deep tissue imaging where high contrast and speedy acquisition are necessary. Fluorescence lifetime (FLT) is an inherent characteristic of each fluorescent species that can be independent of intensity and spectral properties. Accordingly, FLT-based detection provides an additional contrast mechanism to optical measurements. This contrast is particularly important in the near-infrared (NIR) due to relative transparency of tissue as well as the broad absorption and emission spectra of dyes that are active in this region. Here we report comparative analysis of signal distribution of several NIR fluorescent polymethine dyes in living mice and their correlations with lifetimes obtained in vitro using solution models. The FLT data obtained from dyes dissolved in serum albumin solution correlated well with FLTs measured in vivo. Thus the albumin solution model could be used as a good predictive model for in vivo FLT behavior of newly developed fluorescent reporters. Subsequent experiments in vivo, including monitoring slow release kinetics and detecting proteinuria, demonstrate the complementary nature of FLT for fluorescence intensity imaging.

  12. Fluorescence imaging of historical buildings by lidar remote sensing

    NASA Astrophysics Data System (ADS)

    Raimondi, Valentina; Weibring, Petter K. A.; Cecchi, Giovanna; Edner, Hans; Johansson, Thomas; Pantani, Luca; Sundner, Barbro; Svanberg, Sune

    1998-10-01

    This paper reports on the first lidar imaging experiments carried out on a historical monument. The measurements were carried out by scanning the northern facade of the Lund cathedral from a distance of at least 60 m with a mobile fluorescence lidar. Two different arrangements were used for the receiver: an optical multi-spectral analyzer or two photomultipliers equipped with interference filters. Depending on the wavelength the fluorescence images allow the mapping of biodirection colonization or of the different stony materials. To our knowledge these were the first images of a historical building detected by a fluorescence lidar.

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

    PubMed

    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

  14. Quantification of tumor fluorescence during intraoperative optical cancer imaging

    PubMed Central

    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

  15. Fluorescent imaging of cancerous tissues for targeted surgery

    PubMed Central

    Bu, Lihong; Shen, Baozhong; Cheng, Zhen

    2014-01-01

    To maximize tumor excision and minimize collateral damage is the primary goal of cancer surgery. Emerging molecular imaging techniques have to “image-guided surgery” developing into “molecular imaging-guided surgery”, which is termed “targeted surgery” in this review. Consequently, the precision of surgery can be advanced from tissue-scale to molecule-scale, enabling “targeted surgery” to be a component of “targeted therapy”. Evidence from numerous experimental and clinical studies has demonstrated significant benefits of fluorescent imaging in targeted surgery with preoperative molecular diagnostic screening. Fluorescent imaging can help to improve intraoperative staging and enable more radical cytoreduction, detect obscure tumor lesions in special organs, highlight tumor margins, better map lymph node metastases, and identify important normal structures intraoperatively. Though limited tissue penetration of fluorescent imaging and tumor heterogeneity are two major hurdles for current targeted surgery, multimodality imaging and multiplex imaging may provide potential solutions to overcome these issues, respectively. Moreover, though many fluorescent imaging techniques and probes have been investigated, targeted surgery remains at a proof-of-principle stage. The impact of fluorescent imaging on cancer surgery will likely be realized through persistent interdisciplinary amalgamation of research in diverse fields. PMID:25064553

  16. Intraoperative Fluorescence Imaging and Multimodal Surgical Navigation Using Goggle System.

    PubMed

    Mela, Christopher A; Papay, Francis A; Liu, Yang

    2016-01-01

    Intraoperative imaging is an invaluable tool in many surgical procedures. We have developed a wearable stereoscopic imaging and display system entitled Integrated Imaging Goggle, which can provide real-time multimodal image guidance. With the Integrated Imaging Goggle, wide field-of-view fluorescence imaging is tracked and registered with intraoperative ultrasound imaging and preoperative tomography-based surgical navigation, to provide integrated multimodal imaging capabilities in real-time. Herein we describe the system instrumentation and the methods of using the Integrated Imaging Goggle to guide surgeries. PMID:27283420

  17. Detection of rheumatoid arthritis in humans by fluorescence imaging

    NASA Astrophysics Data System (ADS)

    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.

  18. FY08 Annual Report for Nuclear Resonance Fluorescence Imaging

    SciTech Connect

    Warren, Glen A.; Caggiano, Joseph A.

    2009-01-06

    FY08 annual report for project the "Nuclear Resonance Fluorescence Imaging" project. Reviews accomplishments of last 3 years, including U-235 signature search, comparison of different photon sources, and examination of NRF measurements using monochromatic photon source.

  19. Cell imaging by transient fluorescence detected infrared microscopy

    NASA Astrophysics Data System (ADS)

    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.

  20. Soft fluorescent nanomaterials for biological and biomedical imaging

    PubMed Central

    Peng, Hong-Shang; Chiu, Daniel T.

    2015-01-01

    Soft fluorescent nanomaterials have attracted recent attention as imaging agents for biological applications, because they provide the advantages of good biocompatibility, high brightness, and easy biofunctionalization. Here, we provide a survey of recent developments in fluorescent soft nano-sized biological imaging agents. Various soft fluorescent nanoparticles (NPs) (including dye-doped polymer NPs, semiconducting polymer NPs, small-molecule organic NPs, nanogels, micelles, vesicles, and biomaterial-based NPs) are summarized from the perspectives of preparation method, structure, optical property, and surface functionalization. Based on both optical and functional properties of the nano-sized imaging agents, their applications are then reviewed in terms of in vitro imaging, in vivo imaging, and cellular-process imaging, by means of specific or nonspecific targeting. PMID:25531691

  1. Intrinsic fluorescence of selenium nanoparticles for cellular imaging applications

    NASA Astrophysics Data System (ADS)

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Integrated ultrasonic particle positioning and low excitation light fluorescence imaging

    SciTech Connect

    Bernassau, A. L.; Al-Rawhani, M.; Beeley, J.; Cumming, D. R. S.

    2013-12-09

    A compact hybrid system has been developed to position and detect fluorescent micro-particles by combining a Single Photon Avalanche Diode (SPAD) imager with an acoustic manipulator. The detector comprises a SPAD array, light-emitting diode (LED), lenses, and optical filters. The acoustic device is formed of multiple transducers surrounding an octagonal cavity. By stimulating pairs of transducers simultaneously, an acoustic landscape is created causing fluorescent micro-particles to agglomerate into lines. The fluorescent pattern is excited by a low power LED and detected by the SPAD imager. Our technique combines particle manipulation and visualization in a compact, low power, portable setup.

  4. Integrated ultrasonic particle positioning and low excitation light fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Bernassau, A. L.; Al-Rawhani, M.; Beeley, J.; Cumming, D. R. S.

    2013-12-01

    A compact hybrid system has been developed to position and detect fluorescent micro-particles by combining a Single Photon Avalanche Diode (SPAD) imager with an acoustic manipulator. The detector comprises a SPAD array, light-emitting diode (LED), lenses, and optical filters. The acoustic device is formed of multiple transducers surrounding an octagonal cavity. By stimulating pairs of transducers simultaneously, an acoustic landscape is created causing fluorescent micro-particles to agglomerate into lines. The fluorescent pattern is excited by a low power LED and detected by the SPAD imager. Our technique combines particle manipulation and visualization in a compact, low power, portable setup.

  5. Note: Raman microspectroscopy integrated with fluorescence and dark field imaging

    NASA Astrophysics Data System (ADS)

    Li, Haibo; Wang, Hailong; Huang, Dianshuai; Liang, Lijia; Gu, Yuejiao; Liang, Chongyang; Xu, Shuping; Xu, Weiqing

    2014-05-01

    A Raman detection platform integrated with both fluorescence and dark field microscopes was built for in situ Raman detection with the assistance of fluorescence and dark field imaging to locate the target micro regions. Cells and organelles can be easily found via fluorescence imaging with labeling techniques. Besides, nano-sized particles could be observed and located by dark field microscopes. Therefore, comparing with the commercial Raman spectrometers, much more researches based on Raman spectroscopy could be carried out on this integrated Raman platform, especially in the fields of analyzing biological tissues and subwavelength samples.

  6. Clinical application of indocyanine green-fluorescence imaging during hepatectomy

    PubMed Central

    Ishizawa, Takeaki; Saiura, Akio

    2016-01-01

    In hepatobiliary surgery, the fluorescence and bile excretion of indocyanine green (ICG) can be used for real-time visualization of biological structure. Fluorescence cholangiography is used to obtain fluorescence images of the bile ducts following intrabiliary injection of 0.025−0.5 mg/mL ICG or intravenous injection of 2.5 mg ICG. Recently, the latter technique has been used in laparoscopic/robotic cholecystectomy. Intraoperative fluorescence imaging can be used to identify subcapsular hepatic tumors. Primary and secondary hepatic malignancy can be identified by intraoperative fluorescence imaging using preoperative intravenous injection of ICG through biliary excretion disorders that exist in cancerous tissues of hepatocellular carcinoma (HCC) and in non-cancerous hepatic parenchyma around adenocarcinoma foci. Intraoperative fluorescence imaging may help detect tumors to be removed, especially during laparoscopic hepatectomy, in which visual inspection and palpation are limited, compared with open surgery. Fluorescence imaging can also be used to identify hepatic segments. Boundaries of hepatic segments can be visualized following injection of 0.25−2.5 mg/mL ICG into the portal veins or by intravenous injection of 2.5 mg ICG following closure of the proximal portal pedicle toward hepatic regions to be removed. These techniques enable identification of hepatic segments before hepatectomy and during parenchymal transection for anatomic resection. Advances in imaging systems will increase the use of fluorescence imaging as an intraoperative navigation tool that can enhance the safety and accuracy of open and laparoscopic/robotic hepatobiliary surgery. PMID:27500144

  7. Ion beam induced fluorescence imaging in biological systems

    NASA Astrophysics Data System (ADS)

    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 (<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.

  8. Removal of subsurface fluorescence in cryo-imaging using deconvolution.

    PubMed

    Krishnamurthi, Ganapathy; Wang, Charlie Y; Steyer, Grant; Wilson, David L

    2010-10-11

    We compared image restoration methods [Richardson-Lucy (RL), Wiener, and Next-image] with measured "scatter" point-spread-functions, for removing subsurface fluorescence from section-and-image cryo-image volumes. All methods removed haze, delineated single cells from clusters, and improved visualization, but RL best represented structures. Contrast-to-noise and contrast-to-background improvement from RL and Wiener were comparable and 35% better than Next-image. Concerning detection of labeled cells, ROC analyses showed RL ≈Wiener > Next-image > no processing. Next-image was faster than other methods and less prone to image processing artifacts. RL is recommended for the best restoration of the shape and size of fluorescent structures. PMID:20941133

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

    PubMed

    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

  10. Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging

    PubMed Central

    Stoltzfus, Caleb R.; Rebane, Aleksander

    2016-01-01

    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 cm2. PMID:27231620

  11. Multiplexed Spectral Imaging of 120 Different Fluorescent Labels

    PubMed Central

    Valm, Alex M.; Oldenbourg, Rudolf; Borisy, Gary G.

    2016-01-01

    The number of fluorescent labels that can unambiguously be distinguished in a single image when acquired through band pass filters is severely limited by the spectral overlap of available fluorophores. The recent development of spectral microscopy and the application of linear unmixing algorithms to spectrally recorded image data have allowed simultaneous imaging of fluorophores with highly overlapping spectra. However, the number of distinguishable fluorophores is still limited by the unavoidable decrease in signal to noise ratio when fluorescence signals are fractionated over multiple wavelength bins. Here we present a spectral image analysis algorithm to greatly expand the number of distinguishable objects labeled with binary combinations of fluorophores. Our algorithm utilizes a priori knowledge about labeled specimens and imposes a binary label constraint on the unmixing solution. We have applied our labeling and analysis strategy to identify microbes labeled by fluorescence in situ hybridization and here demonstrate the ability to distinguish 120 differently labeled microbes in a single image. PMID:27391327

  12. Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2013-01-01

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water. PMID:23603893

  13. Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

    PubMed Central

    Zhu, Hongying; Ozcan, Aydogan

    2013-01-01

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 μm over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water. PMID:23603893

  14. Double-excitation fluorescence spectral imaging: eliminating tissue auto-fluorescence from in vivo PPIX measurements

    NASA Astrophysics Data System (ADS)

    Torosean, Sason; Flynn, Brendan; Samkoe, Kimberley S.; Davis, Scott C.; Gunn, Jason; Axelsson, Johan; Pogue, Brian W.

    2012-02-01

    An ultrasound coupled handheld-probe-based optical fluorescence molecular tomography (FMT) system has been in development for the purpose of quantifying the production of Protoporphyrin IX (PPIX) in aminolevulinic acid treated (ALA), Basal Cell Carcinoma (BCC) in vivo. The design couples fiber-based spectral sampling of PPIX fluorescence emission with a high frequency ultrasound imaging system, allowing regionally localized fluorescence intensities to be quantified [1]. The optical data are obtained by sequential excitation of the tissue with a 633nm laser, at four source locations and five parallel detections at each of the five interspersed detection locations. This method of acquisition permits fluorescence detection for both superficial and deep locations in ultrasound field. The optical boundary data, tissue layers segmented from ultrasound image and diffusion theory are used to estimate the fluorescence in tissue layers. To improve the recovery of the fluorescence signal of PPIX, eliminating tissue autofluorescence is of great importance. Here the approach was to utilize measurements which straddled the steep Qband excitation peak of PPIX, via the integration of an additional laser source, exciting at 637 nm; a wavelength with a 2 fold lower PPIX excitation value than 633nm.The auto-fluorescence spectrum acquired from the 637 nm laser is then used to spectrally decouple the fluorescence data and produce an accurate fluorescence emission signal, because the two wavelengths have very similar auto-fluorescence but substantially different PPIX excitation levels. The accuracy of this method, using a single source detector pair setup, is verified through animal tumor model experiments, and the result is compared to different methods of fluorescence signal recovery.

  15. Fluorescence Linear Dichroism Imaging for Quantifying Membrane Order

    PubMed Central

    Benninger, Richard K.P.

    2014-01-01

    The plasma membrane of a cell is an ordered environment, giving rise to anisotropic orientations and restricted motion of constituent lipids and proteins. The membrane environment is also dynamic and heterogeneous, which is important for the regulation of membrane-localized signaling. A number of fluorescent microscopy approaches enable the membrane order to be quantified with high spatial and temporal resolution. A polarization-resolved fluorescence method, termed fluorescent linear dichroism (fLD) imaging, can quantify the orientation of membrane bound fluorophores which allows spatially resolved measurement of membrane order and sub-resolution membrane topology (ruffling). Here we describe the detailed methods for performing fLD imaging in biological membrane environments such as the plasma membrane of living cells. This includes the preparation of the sample with appropriate fluorescent dyes, the requirements of the microscope system, the data collection protocol, and post-acquisition image processing, analysis, and interpretation. PMID:25331136

  16. Wide Field-of-View Fluorescence Imaging of Coral Reefs

    PubMed Central

    Treibitz, Tali; Neal, Benjamin P.; Kline, David I.; Beijbom, Oscar; Roberts, Paul L. D.; Mitchell, B. Greg; Kriegman, David

    2015-01-01

    Coral reefs globally are declining rapidly because of both local and global stressors. Improved monitoring tools are urgently needed to understand the changes that are occurring at appropriate temporal and spatial scales. Coral fluorescence imaging tools have the potential to improve both ecological and physiological assessments. Although fluorescence imaging is regularly used for laboratory studies of corals, it has not yet been used for large-scale in situ assessments. Current obstacles to effective underwater fluorescence surveying include limited field-of-view due to low camera sensitivity, the need for nighttime deployment because of ambient light contamination, and the need for custom multispectral narrow band imaging systems to separate the signal into meaningful fluorescence bands. Here we describe the Fluorescence Imaging System (FluorIS), based on a consumer camera modified for greatly increased sensitivity to chlorophyll-a fluorescence, and we show high spectral correlation between acquired images and in situ spectrometer measurements. This system greatly facilitates underwater wide field-of-view fluorophore surveying during both night and day, and potentially enables improvements in semi-automated segmentation of live corals in coral reef photographs and juvenile coral surveys. PMID:25582836

  17. Wide Field-of-View Fluorescence Imaging of Coral Reefs

    NASA Astrophysics Data System (ADS)

    Treibitz, Tali; Neal, Benjamin P.; Kline, David I.; Beijbom, Oscar; Roberts, Paul L. D.; Mitchell, B. Greg; Kriegman, David

    2015-01-01

    Coral reefs globally are declining rapidly because of both local and global stressors. Improved monitoring tools are urgently needed to understand the changes that are occurring at appropriate temporal and spatial scales. Coral fluorescence imaging tools have the potential to improve both ecological and physiological assessments. Although fluorescence imaging is regularly used for laboratory studies of corals, it has not yet been used for large-scale in situ assessments. Current obstacles to effective underwater fluorescence surveying include limited field-of-view due to low camera sensitivity, the need for nighttime deployment because of ambient light contamination, and the need for custom multispectral narrow band imaging systems to separate the signal into meaningful fluorescence bands. Here we describe the Fluorescence Imaging System (FluorIS), based on a consumer camera modified for greatly increased sensitivity to chlorophyll-a fluorescence, and we show high spectral correlation between acquired images and in situ spectrometer measurements. This system greatly facilitates underwater wide field-of-view fluorophore surveying during both night and day, and potentially enables improvements in semi-automated segmentation of live corals in coral reef photographs and juvenile coral surveys.

  18. In vivo simultaneous multispectral fluorescence imaging with spectral multiplexed volume holographic imaging system

    NASA Astrophysics Data System (ADS)

    Lv, Yanlu; Zhang, Jiulou; Zhang, Dong; Cai, Wenjuan; Chen, Nanguang; Luo, Jianwen

    2016-06-01

    A simultaneous multispectral fluorescence imaging system incorporating multiplexed volume holographic grating (VHG) is developed to acquire multispectral images of an object in one shot. With the multiplexed VHG, the imaging system can provide the distribution and spectral characteristics of multiple fluorophores in the scene. The implementation and performance of the simultaneous multispectral imaging system are presented. Further, the system's capability in simultaneously obtaining multispectral fluorescence measurements is demonstrated with in vivo experiments on a mouse. The demonstrated imaging system has the potential to obtain multispectral images fluorescence simultaneously.

  19. Hyperspectral fluorescence imaging with multi wavelength LED excitation

    NASA Astrophysics Data System (ADS)

    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.

  20. Biological applications of fluorescence lifetime imaging beyond microscopy

    NASA Astrophysics Data System (ADS)

    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.

  1. Quantitative Imaging in Laboratory: Fast Kinetics and Fluorescence Quenching

    ERIC Educational Resources Information Center

    Cumberbatch, Tanya; Hanley, Quentin S.

    2007-01-01

    The process of quantitative imaging, which is very commonly used in laboratory, is shown to be very useful for studying the fast kinetics and fluorescence quenching of many experiments. The imaging technique is extremely cheap and hence can be used in many absorption and luminescence experiments.

  2. Fluorescence lifetime imaging of oxygen in dental biofilm

    NASA Astrophysics Data System (ADS)

    Gerritsen, Hans C.; de Grauw, Cees J.

    2000-12-01

    Dental biofilm consists of micro-colonies of bacteria embedded in a matrix of polysaccharides and salivary proteins. pH and oxygen concentration are of great importance in dental biofilm. Both can be measured using fluorescence techniques. The imaging of dental biofilm is complicated by the thickness of the biofilms that can be up to several hundred micrometers thick. Here, we employed a combination of two-photon excitation microscopy with fluorescence lifetime imaging to quantify the oxygen concentration in dental biofilm. Collisional quenching of fluorescent probes by molecular oxygen leads to a reduction of the fluorescence lifetime of the probe. We employed this mechanism to measure the oxygen concentration distribution in dental biofilm by means of fluorescence lifetime imaging. Here, TRIS Ruthenium chloride hydrate was used as an oxygen probe. A calibration procedure on buffers was use to measure the lifetime response of this Ruthenium probe. The results are in agreement with the Stern-Volmer equation. A linear relation was found between the ratio of the unquenched and the quenched lifetime and the oxygen concentration. The biofilm fluorescence lifetime imaging results show a strong oxygen gradient at the buffer - biofilm interface and the average oxygen concentration in the biofilm amounted to 50 μM.

  3. Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

    PubMed Central

    Bixler, Joel N.; Kong, Ying; Cirillo, Jeffrey D.; Maitland, Kristen C.

    2016-01-01

    Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU. PMID:26901051

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Optical imaging-guided cancer therapy with fluorescent nanoparticles

    PubMed Central

    Jiang, Shan; Gnanasammandhan, Muthu Kumara; Zhang, Yong

    2010-01-01

    The diagnosis and treatment of cancer have been greatly improved with the recent developments in nanotechnology. One of the promising nanoscale tools for cancer diagnosis is fluorescent nanoparticles (NPs), such as organic dye-doped NPs, quantum dots and upconversion NPs that enable highly sensitive optical imaging of cancer at cellular and animal level. Furthermore, the emerging development of novel multi-functional NPs, which can be conjugated with several functional molecules simultaneously including targeting moieties, therapeutic agents and imaging probes, provides new potentials for clinical therapies and diagnostics and undoubtedly will play a critical role in cancer therapy. In this article, we review the types and characteristics of fluorescent NPs, in vitro and in vivo imaging of cancer using fluorescent NPs and multi-functional NPs for imaging-guided cancer therapy. PMID:19759055

  6. Microspectrofluorometry and fluorescence imaging in the study of human cytopathology.

    PubMed

    Kohen, E; Gatt, S; Schachtschabel, A; Schachtschabel, D O; Kohen, C; Agmon, V; Hirschberg, J G; Monti, M

    2000-12-01

    The study of energy pools and dynamics of specific pathways in living cells by microspectrofluorometry and fluorescence imaging produces spectral and topographic images characterizing structural and functional changes associated with cytopathology. Microspectro-fluorometry and fluorescence imaging have been applied, together with organelle morphometry to a number of cells mimicking certain cytopathologies, including melanoma cells, long-term malignant cells, and gene-defective cells. These investigations of cellular pathology indicate that there is a convergence of various physiopathological processes. Cellular states that have similarities include senescence, detoxification, and transformation. While the NAD(P)H metabolic transients have been studied before, our emphasis in this article is on very rapidly scanned fluorescence images related to organelle integration and photoinduced cellular senescence. PMID:11074618

  7. Green Fluorescent Protein with Anionic Tryptophan-Based Chromophore and Long Fluorescence Lifetime

    PubMed Central

    Sarkisyan, Karen S.; Goryashchenko, Alexander S.; Lidsky, Peter V.; Gorbachev, Dmitry A.; Bozhanova, Nina G.; Gorokhovatsky, Andrey Yu.; Pereverzeva, Alina R.; Ryumina, Alina P.; Zherdeva, Victoria V.; Savitsky, Alexander P.; Solntsev, Kyril M.; Bommarius, Andreas S.; Sharonov, George V.; Lindquist, Jake R.; Drobizhev, Mikhail; Hughes, Thomas E.; Rebane, Aleksander; Lukyanov, Konstantin A.; Mishin, Alexander S.

    2015-01-01

    Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP—the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins. PMID:26200874

  8. Biomedical imaging of colorectal cancer by near infrared fluorescent nanoparticles.

    PubMed

    Tivony, Ran; Larush, Liraz; Sela-Tavor, Osnat; Magdassi, Shlomo

    2014-06-01

    In this paper we describe the preparation of novel Near Infrared (NIR) fluorescent nanoparticles for application in medical imaging of colorectal tumors. The nanoparticles are prepared by using only non-covalent binding processes of molecules which are approved for clinical use. The preparation process is based on the precipitation of a polycation, Eudragit-RS, followed by sequential adsorption of a blocking protein, sodium caseinate, NIR fluorescent dye, Indocyanine Green (ICG) and optionally, a targeting molecule, anti-CEA antibody. Fluorescence measurements have shown that these nanoparticles have higher resistance to photobleaching and higher quantum yield relatively to free ICG. Imaging experiments in orthotopic colorectal cancer mice models have shown that these fluorescent nanoparticles are capable of binding to LS174T human colon tumors in vivo with high specificity, even without the targeting molecule. These nanoparticles, composed of all FDA approved materials, open the way to clinical bioimaging and diagnostics of colon cancer. PMID:24749398

  9. A Time Domain Fluorescence Tomography System for Small Animal Imaging

    PubMed Central

    Raymond, Scott B.; Dunn, Andrew K.; Bacskai, Brian J.; Boas, David A.

    2010-01-01

    We describe the application of a time domain diffuse fluorescence tomography system for whole body small animal imaging. The key features of the system are the use of point excitation in free space using ultrashort laser pulses and noncontact detection using a gated, intensified charge-coupled device (CCD) camera. Mouse shaped epoxy phantoms, with embedded fluorescent inclusions, were used to verify the performance of a recently developed asymptotic lifetime-based tomography algorithm. The asymptotic algorithm is based on a multiexponential analysis of the decay portion of the data. The multiexponential model is shown to enable the use of a global analysis approach for a robust recovery of the lifetime components present within the imaging medium. The surface boundaries of the imaging volume were acquired using a photogrammetric camera integrated with the imaging system, and implemented in a Monte-Carlo model of photon propagation in tissue. The tomography results show that the asymptotic approach is able to separate axially located fluorescent inclusions centered at depths of 4 and 10 mm from the surface of the mouse phantom. The fluorescent inclusions had distinct lifetimes of 0.5 and 0.95 ns. The inclusions were nearly overlapping along the measurement axis and shown to be not resolvable using continuous wave (CW) methods. These results suggest the practical feasibility and advantages of a time domain approach for whole body small animal fluorescence molecular imaging, particularly with the use of lifetime as a contrast mechanism. PMID:18672432

  10. Examining Adsorbed Polymer Conformations with Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    Parkes, Maria; Chennaoui, Mourad; Wong, Janet; Tribology Group, Dept. of Mechanical Engineering Team

    2011-03-01

    The conformation of adsorbed polymers can have significant impact on their properties such as dynamics and elasticity as well as their ability to take part in reactions with other molecules. Experimental research to determine adsorbed polymer conformation has relied mainly on atomic force microscopy (AFM) studies. During an AFM scan, the contact between the scanning probe and the polymer could affect the polymer conformation, particularly where parts of the polymer might have formed projected loops and tails. In this work, conformations of model polymers are examined with total internal reflection fluorescence microscopy (TIRFM). The advantage of TIRFM over AFM is that TIRFM is a non contact technique. Lambda DNA labelled along its length with fluorescent probes was adsorbed in a projected 2D -- 3D state. With TIRFM, the relationship between intensity and depth was used as a basis to determine how the conformation of the adsorbed polymers evolved with time using our custom algorithm.

  11. Enhanced speed in fluorescence imaging using beat frequency multiplexing

    NASA Astrophysics Data System (ADS)

    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.

  12. A portable fluorescence microscopic imaging system for cholecystectomy

    NASA Astrophysics Data System (ADS)

    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.

  13. Exploiting Molecular Biology by Time-Resolved Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    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

  14. Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

    PubMed Central

    Warren, Sean C.; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J.; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda

    2013-01-01

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

  15. Metal–Dielectric Waveguides for High Efficiency Fluorescence Imaging

    PubMed Central

    Zhu, Liangfu; Zhang, Douguo; Wang, Ruxue; Wang, Pei; Ming, Hai; Badugu, Ramachandram; Du, Luping; Yuan, Xiaocong; Lakowicz, Joseph R.

    2015-01-01

    We demonstrate that Metal–Dielectric Waveguide structures (MDWs) with high efficiency of fluorescence coupling can be suitable as substrates for fluorescence imaging. This hybrid MDWs consists of a continuous metal film and a dielectric top layer. The optical modes sustaining inside this structure can be excited with a high numerical aperture (N.A) objective, and then focused into a virtual optical probe with high intensity, leading to efficient excitation of fluorophores deposited on top of the MDWs. The emitted fluorophores couple with the optical modes thus enabling the directional emission, which is verified by the back focal plane (BFP) imaging. These unique properties of MDWs have been adopted in a scanning laser confocal optical microscopy, and show the merit of high efficiency fluorescence imaging. MDWs can be easily fabricated by vapor deposition and/or spin coating, the silica surface of the MDWs is suitable for biomolecule tethering, and will offer new opportunities for cell biology and biophysics research. PMID:26525494

  16. Photoactivatable Synthetic Dyes for Fluorescence Imaging at the Nanoscale.

    PubMed

    Raymo, Françisco M

    2012-09-01

    The transition from conventional to photoactivatable fluorophores can bring the resolution of fluorescence images from the micrometer to the nanometer level. Indeed, fluorescence photoactivation can overcome the limitations that diffraction imposes on the resolution of optical microscopes. Specifically, distinct fluorophores positioned within the same subdiffraction volume can be resolved only if their emissions are activated independently at different intervals of time. Under these conditions, the sequential localization of multiple probes permits the reconstruction of images with a spatial resolution that is otherwise impossible to achieve with conventional fluorophores. The irreversible photolysis of protecting groups or the reversible transformations of photochromic compounds can be employed to control the emission of appropriate fluorescent chromophores and allow the implementation of these ingenious operating principles for superresolution imaging. Such molecular constructs enable the spatiotemporal control that is required to avoid diffraction and can become invaluable analytical tools for the optical visualization of biological specimens and nanostructured materials with unprecedented resolution. PMID:26292118

  17. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

    1995-01-01

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

  18. Mitigating fluorescence spectral overlap in wide-field endoscopic imaging

    PubMed Central

    Hou, Vivian; Nelson, Leonard Y.; Seibel, Eric J.

    2013-01-01

    Abstract. The number of molecular species suitable for multispectral fluorescence imaging is limited due to the overlap of the emission spectra of indicator fluorophores, e.g., dyes and nanoparticles. To remove fluorophore emission cross-talk in wide-field multispectral fluorescence molecular imaging, we evaluate three different solutions: (1) image stitching, (2) concurrent imaging with cross-talk ratio subtraction algorithm, and (3) frame-sequential imaging. A phantom with fluorophore emission cross-talk is fabricated, and a 1.2-mm ultrathin scanning fiber endoscope (SFE) is used to test and compare these approaches. Results show that fluorophore emission cross-talk could be successfully avoided or significantly reduced. Near term, the concurrent imaging method of wide-field multispectral fluorescence SFE is viable for early stage cancer detection and localization in vivo. Furthermore, a means to enhance exogenous fluorescence target-to-background ratio by the reduction of tissue autofluorescence background is demonstrated. PMID:23966226

  19. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

    1995-11-28

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

  20. Widely accessible method for superresolution fluorescence imaging of living systems.

    PubMed

    Dedecker, Peter; Mo, Gary C H; Dertinger, Thomas; Zhang, Jin

    2012-07-01

    Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging. PMID:22711840

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

    NASA Astrophysics Data System (ADS)

    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. Tumor-stem cells interactions by fluorescence imaging

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    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. TOPICAL REVIEW: Fluorescence lifetime imaging microscopy in life sciences

    NASA Astrophysics Data System (ADS)

    Willem Borst, Jan; Visser, Antonie J. W. G.

    2010-10-01

    Fluorescence lifetime imaging microscopy (FLIM) and fluorescence anisotropy imaging microscopy (FAIM) are versatile tools for the investigation of the molecular environment of fluorophores in living cells. Owing to nanometre-scale interactions via Förster resonance energy transfer (FRET), FLIM and FAIM are powerful microscopy methods for the detection of conformational changes and protein-protein interactions reflecting the biochemical status of live cells. This review provides an overview of recent advances in photonics techniques, quantitative data analysis methods and applications in the life sciences.

  5. Vertical nanopillars for highly localized fluorescence imaging

    PubMed Central

    Xie, Chong; Hanson, Lindsey; Cui, Yi; Cui, Bianxiao

    2011-01-01

    Observing individual molecules in a complex environment by fluorescence microscopy is becoming increasingly important in biological and medical research, for which critical reduction of observation volume is required. Here, we demonstrate the use of vertically aligned silicon dioxide nanopillars to achieve below-the-diffraction-limit observation volume in vitro and inside live cells. With a diameter much smaller than the wavelength of visible light, a transparent silicon dioxide nanopillar embedded in a nontransparent substrate restricts the propagation of light and affords evanescence wave excitation along its vertical surface. This effect creates highly confined illumination volume that selectively excites fluorescence molecules in the vicinity of the nanopillar. We show that this nanopillar illumination can be used for in vitro single-molecule detection at high fluorophore concentrations. In addition, we demonstrate that vertical nanopillars interface tightly with live cells and function as highly localized light sources inside the cell. Furthermore, specific chemical modification of the nanopillar surface makes it possible to locally recruit proteins of interest and simultaneously observe their behavior within the complex, crowded environment of the cell. PMID:21368157

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Photon budget analysis for fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Zhao, Qiaole; Young, Ian T.; de Jong, Jan Geert Sander

    2011-08-01

    We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon ``budget.'' These measures are relevant to many fluorescence microscope users and the results are not restricted to FLIM but applicable to widefield fluorescence microscopy in general. Limitations in photon numbers, however, are more of an issue with FLIM compared to other less quantitative types of imaging. By modeling a typical experimental configuration, examples are given for fluorophores whose absorption peaks span the visible spectrum from Fura-2 to Cy5. We have performed experiments to validate the assumptions and parameters used in our mathematical model. The influence of fluorophore concentration on the intensity of the fluorescence emission light and the Poisson distribution assumption of the detected fluorescence emission light have been validated. The experimental results agree well with the mathematical model. This photon budget is important in order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems.

  8. Multilayer fluorescence imaging on a single-pixel detector

    PubMed Central

    Guo, Kaikai; Jiang, Shaowei; Zheng, Guoan

    2016-01-01

    A critical challenge for fluorescence imaging is the loss of high frequency components in the detection path. Such a loss can be related to the limited numerical aperture of the detection optics, aberrations of the lens, and tissue turbidity. In this paper, we report an imaging scheme that integrates multilayer sample modeling, ptychography-inspired recovery procedures, and lensless single-pixel detection to tackle this challenge. In the reported scheme, we directly placed a 3D sample on top of a single-pixel detector. We then used a known mask to generate speckle patterns in 3D and scanned this known mask to different positions for sample illumination. The sample was then modeled as multiple layers and the captured 1D fluorescence signals were used to recover multiple sample images along the z axis. The reported scheme may find applications in 3D fluorescence sectioning, time-resolved and spectrum-resolved imaging. It may also find applications in deep-tissue fluorescence imaging using the memory effect.

  9. Multilayer fluorescence imaging on a single-pixel detector.

    PubMed

    Guo, Kaikai; Jiang, Shaowei; Zheng, Guoan

    2016-07-01

    A critical challenge for fluorescence imaging is the loss of high frequency components in the detection path. Such a loss can be related to the limited numerical aperture of the detection optics, aberrations of the lens, and tissue turbidity. In this paper, we report an imaging scheme that integrates multilayer sample modeling, ptychography-inspired recovery procedures, and lensless single-pixel detection to tackle this challenge. In the reported scheme, we directly placed a 3D sample on top of a single-pixel detector. We then used a known mask to generate speckle patterns in 3D and scanned this known mask to different positions for sample illumination. The sample was then modeled as multiple layers and the captured 1D fluorescence signals were used to recover multiple sample images along the z axis. The reported scheme may find applications in 3D fluorescence sectioning, time-resolved and spectrum-resolved imaging. It may also find applications in deep-tissue fluorescence imaging using the memory effect. PMID:27446679

  10. FluoSTIC: miniaturized fluorescence image-guided surgery system.

    PubMed

    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. PMID:23052561

  11. Discriminating enumeration of subseafloor life using automated fluorescent image analysis

    NASA Astrophysics Data System (ADS)

    Morono, Y.; Terada, T.; Masui, N.; Inagaki, F.

    2008-12-01

    Enumeration of microbial cells in marine subsurface sediments has provided fundamental information for understanding the extent of life and deep-biosphere on Earth. The microbial population has been manually evaluated by direct cell count under the microscopy because the recognition of cell-derived fluorescent signals has been extremely difficult. Here, we improved the conventional method by removing the non- specific fluorescent backgrounds and enumerated the cell population in sediments using a newly developed automated microscopic imaging system. Although SYBR Green I is known to specifically bind to the double strand DNA (Lunau et al., 2005), we still observed some SYBR-stainable particulate matters (SYBR-SPAMs) in the heat-sterilized control sediments (450°C, 6h), which assumed to be silicates or mineralized organic matters. Newly developed acid-wash treatments with hydrofluoric acid (HF) followed by image analysis successfully removed these background objects and yielded artifact-free microscopic images. To obtain statistically meaningful fluorescent images, we constructed a computer-assisted automated cell counting system. Given the comparative data set of cell abundance in acid-washed marine sediments evaluated by SYBR Green I- and acridine orange (AO)-stain with and without the image analysis, our protocol could provide the statistically meaningful absolute numbers of discriminating cell-derived fluorescent signals.

  12. FluoSTIC: miniaturized fluorescence image-guided surgery system

    NASA Astrophysics Data System (ADS)

    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.

  13. Optofluidic Fluorescent Imaging Cytometry on a Cell Phone

    PubMed Central

    Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F.; Yaglidere, Oguzhan; Ozcan, Aydogan

    2012-01-01

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in

  14. Towards Whole-Body Fluorescence Imaging in Humans

    PubMed Central

    Piper, Sophie K.; Habermehl, Christina; Schmitz, Christoph H.; Kuebler, Wolfgang M.; Obrig, Hellmuth; Steinbrink, Jens; Mehnert, Jan

    2013-01-01

    Dynamic near-infrared fluorescence (DNIF) whole-body imaging of small animals has become a popular tool in experimental biomedical research. In humans, however, the field of view has been limited to body parts, such as rheumatoid hands, diabetic feet or sentinel lymph nodes. Here we present a new whole-body DNIF-system suitable for adult subjects. We explored whether this system (i) allows dynamic whole-body fluorescence imaging and (ii) can detect modulations in skin perfusion. The non-specific fluorescent probe indocyanine green (ICG) was injected intravenously into two subjects, and fluorescence images were obtained at 5 Hz. The in- and out-flow kinetics of ICG have been shown to correlate with tissue perfusion. To validate the system, skin perfusion was modulated by warming and cooling distinct areas on the chest and the abdomen. Movies of fluorescence images show a bolus passage first in the face, then in the chest, abdomen and finally in the periphery (∼10, 15, 20 and 30 seconds, respectively). When skin perfusion is augmented by warming, bolus arrives about 5 seconds earlier than when the skin is cooled and perfusion decreased. Calculating bolus arrival times and spatial fitting of basis time courses extracted from different regions of interest allowed a mapping of local differences in subcutaneous skin perfusion. This experiment is the first to demonstrate the feasibility of whole-body dynamic fluorescence imaging in humans. Since the whole-body approach demonstrates sensitivity to circumscribed alterations in skinperfusion, it may be used to target autonomous changes in polyneuropathy and to screen for peripheral vascular diseases. PMID:24391820

  15. Design of Environmentally Responsive Fluorescent Polymer Probes for Cellular Imaging.

    PubMed

    Yamada, Arisa; Hiruta, Yuki; Wang, Jian; Ayano, Eri; Kanazawa, Hideko

    2015-08-10

    We report the development of environmentally responsive fluorescent polymers. The reversible temperature-induced phase transition of copolymers composed of N-isopropylacrylamide and a fluorescent monomer based on the fluorescein (FL), coumarin (CO), rhodamine (RH), or dansyl (DA) skeleton was used as a molecular switch to control the fluorescence intensity. The poly(N-isopropylacrylamide) (PNIPAAm) chain showed an expanded coil conformation below the lower critical solution temperature (LCST) due to hydration, but it changed to a globular form above the LCST due to dehydration. Through the combination of a polarity-sensitive fluorophore with PNIPAAm, the synthetic fluorescent polymer displayed a response to external temperature, with the fluorescence strength dramatically changing close to the LCST. Additionally, the P(NIPAAm-co-FL) and P(NIPAAm-co-CO) polymers, containing fluorescein and coumarin groups, respectively, exhibited pH responsiveness. The environmental responsiveness of the reported polymers is derived directly from the PNIPAAm and fluorophore structures, thus allowing for the cellular uptake of the fluorescence copolymer by RAW264.7 cells to be temperature-controlled. Cellular uptake was suppressed below the LCST but enhanced above the LCST. Furthermore, the cellular uptake of both P(NIPAAm-co-CO) and P(NIPAAm-co-RH) conjugated with a fusogenic lipid, namely, l-α-phosphatidylethanolamine, dioleoyl (DOPE), was enhanced. Such lipid-conjugated fluorescence probes are expected to be useful as physiological indicators for intracellular imaging. PMID:26121103

  16. Dual-function fluorescent probe for cancer imaging and therapy.

    PubMed

    Cui, Hongjing; Wang, Ran; Zhou, Ying; Shu, Chang; Song, Fengjuan; Zhong, Wenying

    2016-05-01

    To date, several fluorescent probes modified by a single targeting agent have been explored. However, studies on the preparation of dual-function quantum dot (QD) fluorescent probes with dual-targeting action and a therapeutic effect are rare. Here, a dual-targeting CdTe/CdS QD fluorescent probe with a bovine serum albumin-glycyrrhetinic acid conjugate and arginine-glycine-aspartic acid was successfully prepared that could induce the apoptosis of liver cancer cells and showed enhanced targeting in in vitro cell imaging. Therefore, the as-prepared fluorescent probe in this work is an efficient diagnostic tool for the simultaneous detection of liver cancer and breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26387677

  17. Structure of mouse spleen investigated by 7-color fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Tsurui, Hiromichi; Niwa, Shinichirou; Hirose, Sachiko; Okumura, Ko; Shirai, Toshikazu

    2001-07-01

    Multi-color fluorescence imaging of tissue samples has been an urgent requirement in current biology. As far as fluorescence signals should be isolated by optical bandpass filter-sets, rareness of the combination of chromophores with little spectral overlap has hampered to satisfy this demand. Additivity of signals in a fluorescence image accepts applying linear unmixing of superposed spectra based on singular value decomposition, hence complete separation of the fluorescence signals fairly overlapping each other. We have developed 7-color fluorescence imaging based on this principle and applied the method to the investigation of mouse spleen. Not only rough structural features in a spleen such as red pulp, marginal zone, and white pulp, but also fine structures of them, periarteriolar lymphocyte sheath (PALS), follicle, and germinal center were clearly pictured simultaneously. The distributions of subsets of dendritic cells (DC) and macrophages (M(phi) ) markers such as BM8, F4/80, MOMA2 and Mac3 around the marginal zone were imagined simultaneously. Their inhomogeneous expressions were clearly demonstrated. These results show the usefulness of the method in the study of the structure that consists of many kinds of cells and in the identification of cells characterized by multiple markers.

  18. Integrated imaging instrument for self-calibrated fluorescence protein microarrays

    NASA Astrophysics Data System (ADS)

    Reddington, A. P.; Monroe, M. R.; Ünlü, M. S.

    2013-10-01

    Protein microarrays, or multiplexed and high-throughput assays, monitor multiple protein binding events to facilitate the understanding of disease progression and cell physiology. Fluorescence imaging is a popular method to detect proteins captured by immobilized probes with high sensitivity and specificity. Reliability of fluorescence assays depends on achieving minimal inter- and intra-assay probe immobilization variation, an ongoing challenge for protein microarrays. Therefore, it is desirable to establish a label-free method to quantify the probe density prior to target incubation to calibrate the fluorescence readout. Previously, a silicon oxide on silicon chip design was introduced to enhance the fluorescence signal and enable interferometric imaging to self-calibrate the signal with the immobilized probe density. In this paper, an integrated interferometric reflectance imaging sensor and wide-field fluorescence instrument is introduced for sensitive and calibrated microarray measurements. This platform is able to analyze a 2.5 mm × 3.4 mm area, or 200 spots (100 μm diameter with 200 μm pitch), in a single field-of-view.

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

    SciTech Connect

    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.

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

    PubMed Central

    Cao, Hop S Tran; Kimura, Hiroaki; Kaushal, Sharmeela; Snyder, Cynthia S; Reynoso, Jose; Hoffman, Robert M; Bouvet, Michael

    2015-01-01

    Context 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. PMID:19287108

  1. Fluorescent Molecular Imaging and Dosimetry Tools in Photodynamic Therapy

    PubMed Central

    Pogue, Brian W.; Samkoe, Kimberley S.; Gibbs-Strauss, Summer L.; Davis, Scott C.

    2013-01-01

    Measurement of fluorescence and phosphorescence in vivo is readily used to quantify the concentration of specific species that are relevant to photodynamic therapy. However, the tools to make the data quantitatively accurate vary considerably between different applications. Sampling of the signal can be done with point samples, such as specialized fiber probes or from bulk regions with either imaging or sampling, and then in broad region image-guided manner. Each of these methods is described below, the application to imaging photosensitizer uptake is discussed, and developing methods to image molecular responses to therapy are outlined. PMID:20552350

  2. Fluorescence lifetime imaging of human skin and hair

    NASA Astrophysics Data System (ADS)

    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.

  3. Image-guided cancer surgery using near-infrared fluorescence.

    PubMed

    Vahrmeijer, Alexander L; Hutteman, Merlijn; van der Vorst, Joost R; van de Velde, Cornelis J H; Frangioni, John V

    2013-09-01

    Paradigm shifts in surgery arise when surgeons are empowered to perform surgery faster, better and less expensively than current standards. Optical imaging that exploits invisible near-infrared (NIR) fluorescent light (700-900 nm) has the potential to improve cancer surgery outcomes, minimize the time patients are under anaesthesia and lower health-care costs largely by way of its improved contrast and depth of tissue penetration relative to visible light. Accordingly, the past few years have witnessed an explosion of proof-of-concept clinical trials in the field. In this Review, we introduce the concept of NIR fluorescence imaging for cancer surgery, examine the clinical trial literature to date and outline the key issues pertaining to imaging system and contrast agent optimization. Although NIR seems to be superior to many traditional imaging techniques, its incorporation into routine care of patients with cancer depends on rigorous clinical trials and validation studies. PMID:23881033

  4. Handheld multispectral fluorescence lifetime imaging system for in vivo applications.

    PubMed

    Cheng, Shuna; Cuenca, Rodrigo M; Liu, Boang; Malik, Bilal H; Jabbour, Joey M; Maitland, Kristen C; Wright, John; Cheng, Yi-Shing Lisa; Jo, Javier A

    2014-03-01

    There is an increasing interest in the application of fluorescence lifetime imaging (FLIM) for medical diagnosis. Central to the clinical translation of FLIM technology is the development of compact and high-speed clinically compatible systems. We present a handheld probe design consisting of a small maneuverable box fitted with a rigid endoscope, capable of continuous lifetime imaging at multiple emission bands simultaneously. The system was characterized using standard fluorescent dyes. The performance was then further demonstrated by imaging a hamster cheek pouch in vivo, and oral mucosa tissue both ex vivo and in vivo, all using safe and permissible exposure levels. Such a design can greatly facilitate the evaluation of FLIM for oral cancer imaging in vivo. PMID:24688824

  5. Fluorescent Pluronic nanodots for in vivo two-photon imaging.

    PubMed

    Maurin, Mathieu; Vurth, Laeticia; Vial, Jean-Claude; Baldeck, Patrice; Marder, Seth R; Van der Sanden, Boudewijn; Stephan, Olivier

    2009-06-10

    We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 microm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h. PMID:19448291

  6. Fluorescent Pluronic nanodots for in vivo two-photon imaging

    NASA Astrophysics Data System (ADS)

    Maurin, Mathieu; Vurth, Laeticia; Vial, Jean-Claude; Baldeck, Patrice; Marder, Seth R.; Sanden, Boudewijn Van der; Stephan, Olivier

    2009-06-01

    We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 µm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h.

  7. Fluorescence imaging of metal ions implicated in diseases.

    PubMed

    Qian, Xuhong; Xu, Zhaochao

    2015-07-21

    Metal ions play an important role in various biological processes, their abnormal homeostasis in cells is related to many diseases, such as neurodegenerative disease, cancer and diabetes. Fluorescent imaging offers a unique route to detect metal ions in cells via a contactless and damage-free way with high spatial and temporal fidelity. Consequently, it represents a promising method to advance the understanding of physiological and pathological functions of metal ions in cell biology. In this highlight article, we will discuss recent advances in fluorescent imaging of metal ions by small-molecule sensors for understanding the role of metals in related diseases. We will also discuss challenges and opportunities for the design of small-molecule sensors for fluorescent detection of cellular metal ions as a potential method for disease diagnosis. PMID:25556818

  8. Fluorescence imaging of angiogenesis in green fluorescent protein-expressing tumors

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Baranov, Eugene; Jiang, Ping; Li, Xiao-Ming; Wang, Jin W.; Li, Lingna; Yagi, Shigeo; Moossa, A. R.; Hoffman, Robert M.

    2002-05-01

    The development of therapeutics for the control of tumor angiogenesis requires a simple, reliable in vivo assay for tumor-induced vascularization. For this purpose, we have adapted the orthotopic implantation model of angiogenesis by using human and rodent tumors genetically tagged with Aequorea victoria green fluorescent protein (GFP) for grafting into nude mice. Genetically-fluorescent tumors can be readily imaged in vivo. The non-luminous induced capillaries are clearly visible against the bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. Fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. High-level GFP-expressing tumor cell lines made it possible to acquire the high-resolution real-time fluorescent optical images of angiogenesis in both primary tumors and their metastatic lesions in various human and rodent tumor models by means of a light-based imaging system. Intravital images of angiogenesis onset and development were acquired and quantified from a GFP- expressing orthotopically-growing human prostate tumor over a 19-day period. Whole-body optical imaging visualized vessel density increasing linearly over a 20-week period in orthotopically-growing, GFP-expressing human breast tumor MDA-MB-435. Vessels in an orthotopically-growing GFP- expressing Lewis lung carcinoma tumor were visualized through the chest wall via a reversible skin flap. These clinically-relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological micro- environments.

  9. Deep UV Native Fluorescence Imaging of Antarctic Cryptoendolithic Communities

    NASA Technical Reports Server (NTRS)

    Storrie-Lombardi, M. C.; Douglas, S.; Sun, H.; McDonald, G. D.; Bhartia, R.; Nealson, K. H.; Hug, W. F.

    2001-01-01

    An interdisciplinary team at the Jet Propulsion Laboratory Center for Life Detection has embarked on a project to provide in situ chemical and morphological characterization of Antarctic cryptoendolithic microbial communities. We present here in situ deep ultraviolet (UV) native fluorescence and environmental scanning electron microscopy images transiting 8.5 mm into a sandstone sample from the Antarctic Dry Valleys. The deep ultraviolet imaging system employs 224.3, 248.6, and 325 nm lasers to elicit differential fluorescence and resonance Raman responses from biomolecules and minerals. The 224.3 and 248.6 nm lasers elicit a fluorescence response from the aromatic amino and nucleic acids. Excitation at 325 nm may elicit activity from a variety of biomolecules, but is more likely to elicit mineral fluorescence. The resultant fluorescence images provide in situ chemical and morphological maps of microorganisms and the associated organic matrix. Visible broadband reflectance images provide orientation against the mineral background. Environmental scanning electron micrographs provided detailed morphological information. The technique has made possible the construction of detailed fluorescent maps extending from the surface of an Antarctic sandstone sample to a depth of 8.5 mm. The images detect no evidence of microbial life in the superficial 0.2 mm crustal layer. The black lichen component between 0.3 and 0.5 mm deep absorbs all wavelengths of both laser and broadband illumination. Filamentous deep ultraviolet native fluorescent activity dominates in the white layer between 0.6 mm and 5.0 mm from the surface. These filamentous forms are fungi that continue into the red (iron-rich) region of the sample extending from 5.0 to 8.5 mm. Using differential image subtraction techniques it is possible to identify fungal nuclei. The ultraviolet response is markedly attenuated in this region, apparently from the absorption of ultraviolet light by iron-rich particles coating

  10. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA

    NASA Astrophysics Data System (ADS)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e.

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  11. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    PubMed

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. PMID:26629954

  12. Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

    PubMed Central

    Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

    2012-01-01

    The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

  13. Path method for reconstructing images in fluorescence optical tomography

    SciTech Connect

    Kravtsenyuk, Olga V; Lyubimov, Vladimir V; Kalintseva, Natalie A

    2006-11-30

    A reconstruction method elaborated for the optical diffusion tomography of the internal structure of objects containing absorbing and scattering inhomogeneities is considered. The method is developed for studying objects with fluorescing inhomogeneities and can be used for imaging of distributions of artificial fluorophores whose aggregations indicate the presence of various diseases or pathological deviations. (special issue devoted to multiple radiation scattering in random media)

  14. A Review of Indocyanine Green Fluorescent Imaging in Surgery

    PubMed Central

    Alander, Jarmo T.; Kaartinen, Ilkka; Laakso, Aki; Pätilä, Tommi; Spillmann, Thomas; Tuchin, Valery V.; Venermo, Maarit; Välisuo, Petri

    2012-01-01

    The purpose of this paper is to give an overview of the recent surgical intraoperational applications of indocyanine green fluorescence imaging methods, the basics of the technology, and instrumentation used. Well over 200 papers describing this technique in clinical setting are reviewed. In addition to the surgical applications, other recent medical applications of ICG are briefly examined. PMID:22577366

  15. Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy

    PubMed Central

    Mohan, Kavya; Purnapatra, Subhajit B.; Mondal, Partha Pratim

    2014-01-01

    We developed a multiple light-sheet microscopy (MLSM) system capable of 3D fluorescence imaging. Employing spatial filter in the excitation arm of a SPIM system, we successfully generated multiple light-sheets. This improves upon the existing SPIM system and is capable of 3D volume imaging by simultaneously illuminating multiple planes in the sample. Theta detection geometry is employed for data acquisition from multiple specimen layers. This detection scheme inherits many advantages including, background reduction, cross-talk free fluorescence detection and high-resolution at long working distance. Using this technique, we generated equi-intense light-sheets of thickness approximately with an inter-sheet separation of . Moreover, the light-sheets generated by MLSM is found to be 2 times thinner than the state-of-art SPIM system. Imaging of fluorescently coated yeast cells of size (encaged in Agarose gel-matrix) is achieved. Proposed imaging technique may accelerate the field of fluorescence microscopy, cell biology and biophotonics. PMID:24911061

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Fluorescent carbonaceous nanospheres as biological probe for noninvasive brain imaging.

    PubMed

    Qian, Jun; Ruan, Shaobo; Cao, Xi; Cun, Xingli; Chen, Jiantao; Shen, Shun; Jiang, Xinguo; He, Qin; Zhu, Jianhua; Gao, Huile

    2014-12-15

    Fluorescent carbonaceous nanospheres (CDs) have generated much excitement in bioimaging because of their impressive fluorescent properties and good biocompatibility. In this study, we evaluated the potential application of CDs in noninvasive brain imaging. A new kind of CDs was prepared by a heat treating method using glutamic acid and glucose as the precursors. The hydrated diameter and zeta potential of CDs were 101.1 nm (PDI=0.110) and -22.4 mV respectively. Palpable emission spectrum could be observed from 400 nm to 600 nm when excited at corresponding wavelength, suggesting CDs could be used as a noninvasive bio-probe for in vivo imaging. Additionally, several experiments indicated that CDs possess good serum stability and hemocompatibility with low cytotoxicity. In vitro, the CDs could be efficiently taken up by bEnd.3 cells in a concentration- and time-dependent manner. In vivo, CDs could be used for noninvasive brain imaging due to its high accumulation in brain region, which was demonstrated by in vivo imaging and ex vivo tissue imaging. Moreover, the fluorescent distribution in tissue slice showed CDs accumulated in brain with high intensity. In conclusion, CDs were prepared using a simple one-step method with unique optical and good biological properties and could be used for noninvasive brain imaging. PMID:25278360

  18. SIMA: Python software for analysis of dynamic fluorescence imaging data

    PubMed Central

    Kaifosh, Patrick; Zaremba, Jeffrey D.; Danielson, Nathan B.; Losonczy, Attila

    2014-01-01

    Fluorescence imaging is a powerful method for monitoring dynamic signals in the nervous system. However, analysis of dynamic fluorescence imaging data remains burdensome, in part due to the shortage of available software tools. To address this need, we have developed SIMA, an open source Python package that facilitates common analysis tasks related to fluorescence imaging. Functionality of this package includes correction of motion artifacts occurring during in vivo imaging with laser-scanning microscopy, segmentation of imaged fields into regions of interest (ROIs), and extraction of signals from the segmented ROIs. We have also developed a graphical user interface (GUI) for manual editing of the automatically segmented ROIs and automated registration of ROIs across multiple imaging datasets. This software has been designed with flexibility in mind to allow for future extension with different analysis methods and potential integration with other packages. Software, documentation, and source code for the SIMA package and ROI Buddy GUI are freely available at http://www.losonczylab.org/sima/. PMID:25295002

  19. Novel fluorescence molecular imaging of chemotherapy-induced intestinal apoptosis

    NASA Astrophysics Data System (ADS)

    Levin, Galit; Shirvan, Anat; Grimberg, Hagit; Reshef, Ayelet; Yogev-Falach, Merav; Cohen, Avi; Ziv, Ilan

    2009-09-01

    Chemotherapy-induced enteropathy (CIE) is one of the most serious complications of anticancer therapy, and tools for its early detection and monitoring are highly needed. We report on a novel fluorescence method for detection of CIE, based on molecular imaging of the related apoptotic process. The method comprises systemic intravenous administration of the ApoSense fluorescent biomarker (N,N'-didansyl-L-cystine DDC) in vivo and subsequent fluorescence imaging of the intestinal mucosa. In the reported proof-of-concept studies, mice were treated with either taxol+cyclophosphamide or doxil. DDC was administered in vivo at various time points after drug administration, and tracer uptake by ileum tissue was subsequently evaluated by ex vivo fluorescent microscopy. Chemotherapy caused marked and selective uptake of DDC in ileal epithelial cells, in correlation with other hallmarks of apoptosis (i.e., DNA fragmentation and Annexin-V binding). Induction of DDC uptake occurred early after chemotherapy, and its temporal profile was parallel to that of the apoptotic process, as assessed histologically. DDC may therefore serve as a useful tool for detection of CIE. Future potential integration of this method with fluorescent endoscopic techniques, or development of radio-labeled derivatives of DDC for emission tomography, may advance early diagnosis and monitoring of this severe adverse effect of chemotherapy.

  20. Fluorescence lifetime to image epidermal ionic concentrations

    NASA Astrophysics Data System (ADS)

    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.

  1. Linear and non-linear fluorescence imaging of neuronal activity

    NASA Astrophysics Data System (ADS)

    Fisher, Jonathan A. N.

    Optical imaging of neuronal activity offers new possibilities for understanding brain physiology. The predominant methods in neuroscience for measuring electrical activity require electrodes inserted into the tissue. Such methods, however, provide limited spatial information and are invasive. Optical methods are less physically invasive and offer the possibility for simultaneously imaging the activity of many neurons. In this thesis one- and two-photon fluorescence microscopy techniques were applied to several in vivo and in vitro mammalian preparations. Using one-photon absorption fluorescence microscopy and gradient index (GRIN) lens optics, cortical electrical activity in response to electric stimulation was resolved in three-dimensions at high-speed in the primary somatosensory cortex of the mouse in vivo using voltage-sensitive dyes. Imaging at depths up to 150 mum below the cortex surface, it was possible to resolve depth-dependent patterns of neuronal activity in response to cortical and thalamic electric stimulation. The patterns of activity were consistent with known cortical cellular architecture. In a qualitatively different set of experiments, one-photon fluorescence microscopy via voltage-sensitive dyes was successfully employed to image an in vitro preparation of the perfused rat brainstem during the process of respiratory rhythmogenesis. Imaging results yielded insights into the spatial organization of the central respiratory rhythm generation region in the ventrolateral medulla. A multifocal two-photon scanning microscope was constructed, and design and operation principles are described. Utilizing the novel device, anatomical and functional two-photon imaging via potentiometric dyes and calcium dyes is described, and the results of in vivo versus in vitro imaging are compared. Anatomical imaging results used either functional probe background fluorescence or green fluorescent protein (GFP) expression. Spectroscopic experiments measuring the two

  2. Fluorescence imaging of glutamate release in neurons

    SciTech Connect

    Wang, Ziqiang; Yeung, Edward S.

    1999-12-01

    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 charge-coupled device (CCD) imaging is down to {mu}M levels of glutamate with reasonable response time ({approx}30 s). The standard glutamate test shows a linear response over 3 orders of magnitude, from {mu}M to 0.1 mM range. The in vitro monitoring of glutamate release from cultured neuron cells demonstrated excellent spatial and temporal resolution. (c) 1999 Society for Applied Spectroscopy.

  3. Wide-field in vivo background free imaging by selective magnetic modulation of nanodiamond fluorescence

    PubMed Central

    Sarkar, Susanta K.; Bumb, Ambika; Wu, Xufeng; Sochacki, Kem A.; Kellman, Peter; Brechbiel, Martin W.; Neuman, Keir C.

    2014-01-01

    The sensitivity and resolution of fluorescence-based imaging in vivo is often limited by autofluorescence and other background noise. To overcome these limitations, we have developed a wide-field background-free imaging technique based on magnetic modulation of fluorescent nanodiamond emission. Fluorescent nanodiamonds are bright, photo-stable, biocompatible nanoparticles that are promising probes for a wide range of in vitro and in vivo imaging applications. Our readily applied background-free imaging technique improves the signal-to-background ratio for in vivo imaging up to 100-fold. This technique has the potential to significantly improve and extend fluorescent nanodiamond imaging capabilities on diverse fluorescence imaging platforms. PMID:24761300

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

    PubMed

    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

  5. Red Fluorescent Proteins: Advanced Imaging Applications and Future Design

    PubMed Central

    Shcherbakova, Daria M.; Subach, Oksana M.; Verkhusha, Vladislav V.

    2015-01-01

    In the past few years a large series of the advanced red-shifted fluorescent proteins (RFPs) has been developed. These enhanced RFPs provide new possibilities to study biological processes at the levels ranging from single molecules to whole organisms. Herein the relationship between the properties of the RFPs of different phenotypes and their applications to various imaging techniques are described. Existing and emerging imaging approaches are discussed for conventional RFPs, far-red FPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable and reversibly photo-switchable RFPs. Advantages and limitations of specific RFPs for each technique are presented. Recent progress in understanding the chemical transformations of red chromophores allows the future RFP phenotypes and their respective novel imaging applications to be foreseen. PMID:22851529

  6. Portable Fluorescence Imaging System for Hypersonic Flow Facilities

    NASA Technical Reports Server (NTRS)

    Wilkes, J. A.; Alderfer, D. W.; Jones, S. B.; Danehy, P. M.

    2003-01-01

    A portable fluorescence imaging system has been developed for use in NASA Langley s hypersonic wind tunnels. The system has been applied to a small-scale free jet flow. Two-dimensional images were taken of the flow out of a nozzle into a low-pressure test section using the portable planar laser-induced fluorescence system. Images were taken from the center of the jet at various test section pressures, showing the formation of a barrel shock at low pressures, transitioning to a turbulent jet at high pressures. A spanwise scan through the jet at constant pressure reveals the three-dimensional structure of the flow. Future capabilities of the system for making measurements in large-scale hypersonic wind tunnel facilities are discussed.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Compressive fluorescence microscopy for biological and hyperspectral imaging.

    PubMed

    Studer, Vincent; Bobin, Jérome; Chahid, Makhlad; Mousavi, Hamed Shams; Candes, Emmanuel; Dahan, Maxime

    2012-06-26

    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 redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy. PMID:22689950

  9. Infrared imaging of LED lighting tubes and fluorescent tubes

    NASA Astrophysics Data System (ADS)

    Siikanen, Sami; Kivi, Sini; Kauppinen, Timo; Juuti, Mikko

    2011-05-01

    The low energy efficiency of conventional light sources is mainly caused by generation of waste heat. We used infrared (IR) imaging in order to monitor the heating of both LED tube luminaires and ordinary T8 fluorescent tubes. The IR images showed clearly how the surface temperatures of the fluorescent tube ends quickly rose up to about +50...+70°C, whereas the highest surface temperatures seen on the LED tubes were only about +30...+40°C. The IR images demonstrated how the heat produced by the individual LED chips can be efficiently guided to the supporting structure in order to keep the LED emitters cool and hence maintain efficient operation. The consumed electrical power and produced illuminance were also recorded during 24 hour measurements. In order to assess the total luminous efficacy of the luminaires, separate luminous flux measurements were made in a large integrating sphere. The currently available LED tubes showed efficacies of up to 88 lm/W, whereas a standard "cool white" T8 fluorescent tube produced ca. 75 lm/W. Both lamp types gave ca. 110 - 130 lx right below the ceiling-mounted luminaire, but the LED tubes consume only 40 - 55% of the electric power compared to fluorescent tubes.

  10. Onychomycosis diagnosis using fluorescence and infrared imaging systems

    NASA Astrophysics Data System (ADS)

    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.

  11. Fluorescent supramolecular micelles for imaging-guided cancer therapy

    NASA Astrophysics Data System (ADS)

    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

  12. Performance validation of EMCCD and ICCD based near-infrared fluorescence imaging systems on a fluorescence solid phantom

    NASA Astrophysics Data System (ADS)

    Zhu, Banghe; Sevick-Muraca, Eva M.

    2012-03-01

    Near infrared (NIR) fluorescence imaging has been successfully applied for non-invasive assessment of both lymphatic architecture and function as well as potential disease markers of lymphatic dysfunction in clinical studies with intradermal injection of indocyanine green (ICG). For new "first-in-humans" NIR fluorescence imaging agents that need to be employed at far lower quantities, NIR fluorescence imaging devices with high measurement sensitivity are most favorable. However, the measurement sensitivity of NIR fluorescence imaging devices is limited by various parameters, including quantum efficiency of CCD chip, noise sources in the CCD camera, and the leakage of excitation light through optical filters. In this contribution, we present a quantum dot-based fluorescence solid phantom and its use for characterization of excitation light leakage and measurement sensitivity in both the intensified CCD (ICCD) and Electron Multiplying CCD (EMCCD) based NIR fluorescence imaging devices. The stability of the constructed quantum dot-based fluorescence solid phantom was first demonstrated and used to demonstrate higher measurement sensitivity compared of the ICCD as opposed to the EMCCD based NIR fluorescence imaging device when integration time were maintained less than 1.0 s. The phantom was used to assess the calculated transmission ratio, R, to minimize noise owing to excitation light leakage and show optimized filtering capabilities. The constructed quantum dot based solid phantom and the methodology for measuring parameters of transmission ratio and SNR can be used as a standard and quantifiable metric for installation and operational qualification of all NIR fluorescence imaging devices.

  13. The study of blue LED to induce fluorescence spectroscopy and fluorescence imaging for oral carcinoma detection

    NASA Astrophysics Data System (ADS)

    Zheng, Longjiang; Hu, Yuanting

    2009-07-01

    Fluorescence spectroscopy and fluorescence imaging diagnosis of malignant lesions provides us with a new method to diagnose diseases in precancerous stage. Early diagnosis of disease has significant importance in cancer treatment, because most cancers can be cured well in precancerous, especially when the diffusion of cancer is limited in a restricted region. In this study, Golden hamster models were applied to 5% 9, 10 dimethyl-1, 2-benzanthracene (DMBA) to induce hamster buccal cheek pouch carcinoma three times a week. Rose Bengal, which has been used in clinican for years and avoids visible side-effect to human was chosen as photosensitizer. 405 nm blue LED was used to induce the fluorescence of photosensitizer. After topical application of photosensitizer, characteristic red emission fluorescence peak was observed around 600nm. Similar, normal oral cavity has special luminescence around 480nm. Fluorescence spectroscopy technology is based on analysing emission peaks of photosensitizer in the areas of oral carcinoma, moreover, red-to-green (IR/IG) intensity ratio is also applied as a diagnostic algorithm. A CCD which is connected with a computer is used to take pictures at carcinoma areas through different filters. Fluorescence images from normal hamster buccal cheek pouch are compared with those from carcinogen-induced models of carcinoma, and morphological differences between normal and lesion tissue can be distinguished. The pictures are analyzed by Matlab and shown on the screen of computer. This paper demonstrates that Rose Bengal could be used as photosensitizer to detect oral carcinoma, and blue LED as excitation source could not only have a good effect to diagnose oral carcinoma, but also decrease cost greatly.

  14. In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

    PubMed Central

    Fitoussi, Victor; Faye, Nathalie; Chamming's, Foucauld; Clement, Olivier; Cuenod, Charles-Andre; Fournier, Laure S.

    2013-01-01

    Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained. We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 μm in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools. The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability. PMID:24056503

  15. In vivo imaging of tumor angiogenesis using fluorescence confocal videomicroscopy.

    PubMed

    Fitoussi, Victor; Faye, Nathalie; Chamming's, Foucauld; Clement, Olivier; Cuenod, Charles-Andre; Fournier, Laure S

    2013-01-01

    Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained. We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 μm in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools. The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability. PMID:24056503

  16. Coded aperture imaging for fluorescent x-rays

    SciTech Connect

    Haboub, A.; MacDowell, A. A.; Marchesini, S.; Parkinson, D. Y.

    2014-06-15

    We employ a coded aperture pattern in front of a pixilated charge couple device detector to image fluorescent x-rays (6–25 KeV) from samples irradiated with synchrotron radiation. Coded apertures encode the angular direction of x-rays, and given a known source plane, allow for a large numerical aperture x-ray imaging system. The algorithm to develop and fabricate the free standing No-Two-Holes-Touching aperture pattern was developed. The algorithms to reconstruct the x-ray image from the recorded encoded pattern were developed by means of a ray tracing technique and confirmed by experiments on standard samples.

  17. In Vivo Metal Ion Imaging Using Fluorescent Sensors.

    PubMed

    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

  18. Artificial neural network approaches for fluorescence lifetime imaging techniques.

    PubMed

    Wu, Gang; Nowotny, Thomas; Zhang, Yongliang; Yu, Hong-Qi; Li, David Day-Uei

    2016-06-01

    A novel high-speed fluorescence lifetime imaging (FLIM) analysis method based on artificial neural networks (ANN) has been proposed. In terms of image generation, the proposed ANN-FLIM method does not require iterative searching procedures or initial conditions, and it can generate lifetime images at least 180-fold faster than conventional least squares curve-fitting software tools. The advantages of ANN-FLIM were demonstrated on both synthesized and experimental data, showing that it has great potential to fuel current revolutions in rapid FLIM technologies. PMID:27244414

  19. In situ fluorescence imaging of localized corrosion with a pH-sensitive imaging fiber.

    PubMed

    Panova, A A; Pantano, P; Walt, D R

    1997-04-15

    A fiber-optic pH-imaging sensor array capable of both visualizing remote corrosion sites and measuring local chemical concentrations at these sites was applied to realtime corrosion monitoring. The imaging fiber's distal face, containing an immobilized pH-sensitive fluorescent dye, was brought into contact with metal surfaces submerged in aqueous buffers and fluorescence images were acquired as a function of time. Heterogeneous fluorescence signals were observed due to both pH increases at cathodic surface sites and pH decreases at anodic surface sites. These fluorescence signals showed both localization and rates of corrosion activity. Three corrosion processes were investigated, galvanic corrosion at a copper/aluminum interface and crevice corrosion and pitting at a stainless steel surface. The spatial resolution of the technique was limited by proton/hydroxide diffusion and the diameter of the individually clad optical fibers comprising the imaging bundle. PMID:9109355

  20. Rapid imaging of surgical breast excisions using direct temporal sampling two photon fluorescent lifetime imaging

    PubMed Central

    Giacomelli, Michael G.; Sheikine, Yuri; Vardeh, Hilde; Connolly, James L.; Fujimoto, James G.

    2015-01-01

    Two photon fluorescent lifetime imaging is a modality that enables depth-sectioned, molecularly-specific imaging of cells and tissue using intrinsic contrast. However, clinical applications have not been well explored due to low imaging speed and limited field of view, which make evaluating large pathology samples extremely challenging. To address these limitations, we have developed direct temporal sampling two photon fluorescent lifetime imaging (DTS-FLIM), a method which enables a several order of magnitude increase in imaging speed by capturing an entire lifetime decay in a single fluorescent excitation. We use this greatly increased speed to perform a preliminary study using gigapixel-scale imaging of human breast pathology surgical specimens. PMID:26600997

  1. Fluorescent In Situ Hybridization in Suspension by Imaging Flow Cytometry.

    PubMed

    Maguire, Orla; Wallace, Paul K; Minderman, Hans

    2016-01-01

    The emergence of imaging flow cytometry (IFC) has brought novel applications exploiting its advantages over conventional flow cytometry and microscopy. One of the new applications is fluorescence in situ hybridization in suspension (FISH-IS). Conventional FISH is a slide-based approach in which the spotlike imagery resulting from hybridization with fluorescently tagged probes is evaluated by fluorescence microscopy. The FISH-IS approach evaluated by IFC enables the evaluation of tens to hundreds of thousands of cells in suspension and the analysis can be automated and standardized diminishing operator bias from the analysis. The high cell number throughput of FISH-IS improves the detection of rare events compared to conventional FISH. The applicability of FISH-IS is currently limited to detection of abnormal quantitative differences of hybridization targets such as occur in numerical chromosome abnormalities, deletions and amplifications.Here, we describe a protocol for FISH-IS using chromosome enumeration probes as an example. PMID:27460240

  2. Biochip Image Grid Normalization Absolute Signal Fluorescence Measurement Using

    Energy Science and Technology Software Center (ESTSC)

    2001-04-17

    This software was developed to measure absolute fluorescent intensities of gel pads on a microchip in units defined by a standard fluorescent slide. It can accomodate varying measurement conditions (e.g. exposure time, sensitivity of detector, resolution of detector, etc.) as well as fluorescent microscopes with non-uniform sensitivity across their field of view allowing the user to compare measurements done on different detectors with varying exposure times, sensitivities, and resolutions. The software is designed both tomore » operate Roper Scientific, Inc. cameras and to use image files produced by the program supplied with that equipment for its calculations. the intensity of the gel pad signal is computed so as to reduce background influence.« less

  3. Fluorescent ligand for human progesterone receptor imaging in live cells.

    PubMed

    Weinstain, Roy; Kanter, Joan; Friedman, Beth; Ellies, Lesley G; Baker, Michael E; Tsien, Roger Y

    2013-05-15

    We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core. PMID:23600997

  4. Clinical results of fluorescence lifetime imaging in ophthalmology

    NASA Astrophysics Data System (ADS)

    Schweitzer, D.; Quick, S.; Klemm, M.; Hammer, M.; Jentsch, S.; Dawczynski, J.; Becker, W.

    2009-07-01

    A laser scanner ophthalmoscope was developed for in vivo fluorescence lifetime measurements at the human retina. Measurements were performed in 30 degree fundus images. The fundus was excited by pulses of 75 ps (FWHM). The dynamic fluorescence was detected in two spectral channels K1(490-560nm), K2(560-700 nm) by time-correlated single photon counting. The decay of fluorescence was three-exponentially. Local and global alterations in lifetimes were found between healthy subjects and patients suffering from age-related macular degeneration, diabetic retinopathy, and vessel occlusion. The lifetimes T1, T2, and T3 in both channels are changed to longer values in AMD and diabetic retinopathy in comparison with healthy subjects. The lifetime T2 in K1 is most sensitive to metabolic alterations in branch arterial vessel occlusion.

  5. Visualizing photosynthesis through processing of chlorophyll fluorescence images

    NASA Astrophysics Data System (ADS)

    Daley, Paul F.; Ball, J. Timothy; Berry, Joseph A.; Patzke, Juergen; Raschke, Klaus E.

    1990-05-01

    Measurements of terrestrial plant photosynthesis frequently exploit sensing of gas exchange from leaves enclosed in gas-tight, climate controlled chambers. These methods are typically slow, and do not resolve variation in photosynthesis below the whole leaf level. A photosynthesis visualization technique is presented that uses images of leaves employing light from chlorophyll (Chl) fluorescence. Images of Chl fluorescence from whole leaves undergoing steady-state photosynthesis, photosynthesis induction, or response to stress agents were digitized during light flashes that saturated photochemical reactions. Use of saturating flashes permitted deconvolution of photochemical energy use from biochemical quenching mechanisms (qN) that dissipate excess excitation energy, otherwise damaging to the light harvesting apparatus. Combination of the digital image frames of variable fluorescence with reference frames obtained from the same leaves when dark-adapted permitted derivation of frames in which grey scale represented the magnitude of qN. Simultaneous measurements with gas-exchange apparatus provided data for non-linear calibration filters for subsequent rendering of grey-scale "images" of photosynthesis. In several experiments significant non-homogeneity of photosynthetic activity was observed following treatment with growth hormones, or shifts in light or humidity, and following infection by virus. The technique provides a rapid, non-invasive probe for stress physiology and plant disease detection.

  6. Fluorescence imaging to study cancer burden on lymph nodes

    NASA Astrophysics Data System (ADS)

    D'Souza, Alisha V.; Elliott, Jonathan T.; Gunn, Jason R.; Samkoe, Kimberley S.; Tichauer, Kenneth M.; Pogue, Brian W.

    2015-03-01

    Morbidity and complexity involved in lymph node staging via surgical resection and biopsy calls for staging techniques that are less invasive. While visible blue dyes are commonly used in locating sentinel lymph nodes, since they follow tumor-draining lymphatic vessels, they do not provide a metric to evaluate presence of cancer. An area of active research is to use fluorescent dyes to assess tumor burden of sentinel and secondary lymph nodes. The goal of this work was to successfully deploy and test an intra-nodal cancer-cell injection model to enable planar fluorescence imaging of a clinically relevant blue dye, specifically methylene blue along with a cancer targeting tracer, Affibody labeled with IRDYE800CW and subsequently segregate tumor-bearing from normal lymph nodes. This direct-injection based tumor model was employed in athymic rats (6 normal, 4 controls, 6 cancer-bearing), where luciferase-expressing breast cancer cells were injected into axillary lymph nodes. Tumor presence in nodes was confirmed by bioluminescence imaging before and after fluorescence imaging. Lymphatic uptake from the injection site (intradermal on forepaw) to lymph node was imaged at approximately 2 frames/minute. Large variability was observed within each cohort.

  7. A portable near-infrared fluorescence image overlay device for surgical navigation (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    McWade, Melanie A.

    2016-03-01

    A rise in the use of near-infrared (NIR) fluorescent dyes or intrinsic fluorescent markers for surgical guidance and tissue diagnosis has triggered the development of NIR fluorescence imaging systems. Because NIR wavelengths are invisible to the naked eye, instrumentation must allow surgeons to visualize areas of high fluorescence. Current NIR fluorescence imaging systems have limited ease-of-use because they display fluorescent information on remote display monitors that require surgeons to divert attention away from the patient to identify the location of tissue fluorescence. Furthermore, some systems lack simultaneous visible light imaging which provides valuable spatial context to fluorescence images. We have developed a novel, portable NIR fluorescence imaging approach for intraoperative surgical guidance that provides information for surgical navigation within the clinician's line of sight. The system utilizes a NIR CMOS detector to collect excited NIR fluorescence from the surgical field. Tissues with NIR fluorescence are overlaid with visible light to provide information on tissue margins directly on the surgical field. In vitro studies have shown this versatile imaging system can be applied to applications with both extrinsic NIR contrast agents such as indocyanine green and weaker sources of biological fluorescence such as parathyroid gland tissue. This non-invasive, portable NIR fluorescence imaging system overlays an image directly on tissue, potentially allowing surgical decisions to be made quicker and with greater ease-of-use than current NIR fluorescence imaging systems.

  8. Imaging cellular dynamics in vivo with multicolor fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2005-04-01

    The new field of in vivo cell biology is being developed with multi-colored fluorescent proteins. With the use of fluorescent proteins, the behavior of individual cells can be visualized in the living animal. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of the tumor-stroma cell-cell interaction especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. Another example is the color-coding of cells with RFP or GFP such that both cell types and their interaction can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo with fluorescent proteins. Mice, in which the regulatory elements of the stem-cell marker nestin drive GFP expression, can be used to visualize hair follicle stem cells including their ability to form hair follicles as well as blood vessels. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Dual-color cells also enable the in vivo imaging of cell and nuclear deformation as well as trafficking in capillaries in living animals. Multiple-color labeling of cells will enable multiple events to be simultaneously visualized in vivo including cell-cell interaction, gene expression, ion fluxes, protein and organelle trafficking, chromosome dynamics and numerous other processes currently still studied in vitro.

  9. Optimization of multiparameter radar estimates of rainfall

    NASA Technical Reports Server (NTRS)

    Chandrasekar, V.; Gorgucci, Eugenio; Scarchilli, Gianfranco

    1993-01-01

    The estimates of rainfall rate derived from a multiparameter radar based on reflectivity factor (R sub ZH), differential reflectivity (R sub DR), and specific differential propagation phase (R sub DP) have widely varying accuracies over the dynamic range of the natural occurrence of rainfall. This paper presents a framework to optimally combine the three estimates, R sub zH, R sub DR, and R sub DP, to derive the best estimate of rainfall using coherent multiparameter radars. The optimization procedure is demonstrated for application to multiparameter radar measurements at C band.

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

    PubMed

    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 <25 nm hydrodynamic size and their application as fluorescent cell labels. Hydrophobic 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

  11. [A tracking algorithm for live mitochondria in fluorescent microscopy images].

    PubMed

    Xu, Junmei; Li, Yang; Du, Sidan; Zhao, Kanglian

    2012-04-01

    Quantitative analysis of biological image data generally involves the detection of many pixel spots. In live mitochondria video image, for which fluorescent microscopy is often used, the signal-to-noise ratio (SNR) can be extremely low, making the detection and tracking of mitochondria particle difficult. It is especially not easy to get the movement curve when the movement of the mitochondria involves its self-move and the motion caused by the neuron. An tracking algorithm for live mitochondria is proposed in this paper. First the whole image sequence is frame-to-frame registered, in which the edge corners are chosen to be the feature points. Then the mitochondria particles are tracked by frame-to-frame displacement vector. The algorithm proposed has been applied to the dynamic image sequence including neuron and mitochondria, saving time without manually picking up the feature points. It provides an new method and reference for medical image processing and biotechnological research. PMID:22616189

  12. Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques

    PubMed Central

    Costantini, Lindsey; Snapp, Erik

    2013-01-01

    This UNIT describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using commercially available widefield and confocal laser scanning microscopes (CLSM). It has been long appreciated that the ER plays a number of key roles in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has been often restricted to biochemical assays that average the behaviors of millions of lysed cells or to imaging static fixed cells. Now, with new fluorescent protein reporter tools, highly sensitive commercial microscopes, and photobleaching techniques, it is possible to interrogate the behaviors of ER proteins, membranes, and stress pathways in single cells with exquisite spatial and temporal resolution. The ER presents a unique set of imaging challenges including the high mobility of ER membranes, a diverse range of dynamic ER structures, and the influence of post-translational modifications on fluorescent protein reporters. Solutions to these challenges are described and considerations for performing photobleaching assays, especially Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) for ER proteins will be discussed. In addition, ER reporters and ER-specific pharmacologic compounds are presented with a focus on misfolded secretory protein stress and the Unfolded Protein Response (UPR). PMID:24510787

  13. Fluorescent supramolecular micelles for imaging-guided cancer therapy.

    PubMed

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

    2016-03-01

    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. PMID:26881415

  14. Characterization of a Fluorescent Probe for Imaging Nitric Oxide

    PubMed Central

    Ghebremariam, Yohannes T; Huang, Ngan F; Kambhampati, Swetha; Volz, Katharina S; Joshi, Gururaj G; Anslyn, Eric V; Cooke, John P

    2014-01-01

    Background Nitric Oxide (NO), a potent vasodilator and anti-atherogenic molecule, is synthesized in various cell types including vascular endothelial cells (ECs). The biological importance of NO enforces the need to develop and characterize specific and sensitive probes. To date, several fluorophores, chromophores and colorimetric techniques have been developed to detect NO or its metabolites (NO2 and NO3) in biological fluids, viable cells or cell lysates. Methods Recently, a novel probe (NO550) has been developed and reported to detect NO in solution and in primary astrocytes and neuronal cells with a fluorescence signal arising from a non-fluorescent background. Results Here, we report further characterization of this probe by optimizing conditions for the detection and imaging of NO products in primary vascular endothelial cells, fibroblasts, embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)- derived endothelial cells (ESC-ECs. and iPSC-ECs respectively) in the absence and presence of pharmacological agents that modulate NO levels. In addition, we studied the stability of this probe in cells over time and evaluated its compartmentalization in reference to organelle-labeling dyes. Finally, we synthesized an inherently fluorescent diazo ring compound (AZO550) that is expected to form when the non-fluorescent NO550 reacts with cellular NO and compared its cellular distribution with that of NO550. Conclusion NO550 is a promising agent for imaging NO at baseline and in response to pharmacological agents that modulate its levels. PMID:24335468

  15. Evaluation of the reasons why freshly appearing citrus peel fluorescence during automatic inspection by fluorescent imaging technique

    NASA Astrophysics Data System (ADS)

    Momin, Md. Abdul; Kondo, Naoshi; Kuramoto, Makoto; Ogawa, Yuichi; Yamamoto, Kazuya; Shiigi, Tomoo; Ninomiya, Kazunori

    2011-07-01

    Defective unshu oranges (Citrus reticulate Blanco var. unshu) were sorted based on fluorescent imaging technique in a commercial packinghouse but fresh appearing unshu were rejected due to fluorescence appearing on their peel. We studied the various visible patterns based on colour, fluorescence and microscopic images, where even areas of the peel that are not obviously damaged can have fluorescence, to provide a categorization of fluorescence reasons. The categorization corresponded to: 1) hole and flow; 2) influenced by damaged or rotten fruits that have released peel oil onto it; 3) immature or poor peel quality; 4) whitish fluorescence due to agro-chemicals and 5) variation of the growing season. The identification of such patterns of fluorescence might be useful for citrus grading industry to take some initiatives to make the entire automated system more efficient.

  16. Proton-induced x-ray fluorescence CT imaging

    SciTech Connect

    Bazalova-Carter, Magdalena Xing, Lei; Ahmad, Moiz; Matsuura, Taeko; Takao, Seishin; Shirato, Hiroki; Umegaki, Kikuo; Matsuo, Yuto; Fahrig, Rebecca

    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

  17. Proton-induced x-ray fluorescence CT imaging

    PubMed Central

    Bazalova-Carter, Magdalena; Ahmad, Moiz; Matsuura, Taeko; Takao, Seishin; Matsuo, Yuto; Fahrig, Rebecca; Shirato, Hiroki; Umegaki, Kikuo; Xing, Lei

    2015-01-01

    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. Study on excitation and fluorescence spectrums of Japanese citruses to construct machine vision systems for acquiring fluorescent images

    NASA Astrophysics Data System (ADS)

    Momin, Md. Abdul; Kondo, Naoshi; Kuramoto, Makoto; Ogawa, Yuichi; Shigi, Tomoo

    2011-06-01

    Research was conducted to acquire knowledge of the ultraviolet and visible spectrums from 300 -800 nm of some common varieties of Japanese citrus, to investigate the best wave-lengths for fluorescence excitation and the resulting fluorescence wave-lengths and to provide a scientific background for the best quality fluorescent imaging technique for detecting surface defects of citrus. A Hitachi U-4000 PC-based microprocessor controlled spectrophotometer was used to measure the absorption spectrum and a Hitachi F-4500 spectrophotometer was used for the fluorescence and excitation spectrums. We analyzed the spectrums and the selected varieties of citrus were categorized into four groups of known fluorescence level, namely strong, medium, weak and no fluorescence.The level of fluorescence of each variety was also examined by using machine vision system. We found that around 340-380 nm LEDs or UV lamps are appropriate as lighting devices for acquiring the best quality fluorescent image of the citrus varieties to examine their fluorescence intensity. Therefore an image acquisition device was constructed with three different lighting panels with UV LED at peak 365 nm, Blacklight blue lamps (BLB) peak at 350 nm and UV-B lamps at peak 306 nm. The results from fluorescent images also revealed that the findings of the measured spectrums worked properly and can be used for practical applications such as for detecting rotten, injured or damaged parts of a wide variety of citrus.

  19. Development and integration of Raman imaging capabilities to Sandia National Laboratories hyperspectral fluorescence imaging instrument.

    SciTech Connect

    Timlin, Jerilyn Ann; Nieman, Linda T.

    2005-11-01

    Raman spectroscopic imaging is a powerful technique for visualizing chemical differences within a variety of samples based on the interaction of a substance's molecular vibrations with laser light. While Raman imaging can provide a unique view of samples such as residual stress within silicon devices, chemical degradation, material aging, and sample heterogeneity, the Raman scattering process is often weak and thus requires very sensitive collection optics and detectors. Many commercial instruments (including ones owned here at Sandia National Laboratories) generate Raman images by raster scanning a point focused laser beam across a sample--a process which can expose a sample to extreme levels of laser light and requires lengthy acquisition times. Our previous research efforts have led to the development of a state-of-the-art two-dimensional hyperspectral imager for fluorescence imaging applications such as microarray scanning. This report details the design, integration, and characterization of a line-scan Raman imaging module added to this efficient hyperspectral fluorescence microscope. The original hyperspectral fluorescence instrument serves as the framework for excitation and sample manipulation for the Raman imaging system, while a more appropriate axial transmissive Raman imaging spectrometer and detector are utilized for collection of the Raman scatter. The result is a unique and flexible dual-modality fluorescence and Raman imaging system capable of high-speed imaging at high spatial and spectral resolutions. Care was taken throughout the design and integration process not to hinder any of the fluorescence imaging capabilities. For example, an operator can switch between the fluorescence and Raman modalities without need for extensive optical realignment. The instrument performance has been characterized and sample data is presented.

  20. Neurotransmitter imaging in living cells based on native fluorescence detection

    SciTech Connect

    Tan, W.; Yeung, E.S. |; Parpura, V.; Haydon, P.G.

    1995-08-01

    A UV laser-based optical microscope and CCD detection system with high sensitivity has been developed to image neurotransmitters in living cells. We demonstrate the detection of serotonin that has been taken up into individual living glial cells (astrocytes) based on its native fluorescence. We found that the fluorescence intensity of astrocytes increased by up to 10 times after serotonin uptake. The temporal resolution of this detection system at 10{sup -4} M serotonin is as fast as 50 ms, and the spatial resolution is diffraction limited. This UV laser microscope imaging system shows promise for studies of spatial-temporal dynamics of neurotransmitter levels in living neurons and glia. 19 refs., 5 figs., 1 tab.

  1. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    NASA Astrophysics Data System (ADS)

    Kuo, Yung; Hsu, Tsung-Yuan; Wu, Yi-Chun; Hsu, Jui-Hung; Chang, Huan-Cheng

    2013-03-01

    The negatively charged nitrogen-vacancy (NV-) center in bulk diamond is a photostable fluorophore with a radiative lifetime of 11.6 ns at room temperature. The lifetime substantially increases to ~20 ns for diamond nanoparticles (size ~ 100 nm) suspended in water due to the change in refractive index of the surrounding medium of the NV- centers. This fluorescence decay time is much longer than that (typically 1 - 4 ns) of endogenous and exogenous fluorophores commonly used in biological imaging, making it possible to detect NV--containing nanodiamonds in vivo at the single particle level by fluorescence lifetime imaging microscopy (FLIM). We demonstrate the feasibility of this approach using Caenorhabditis elegans (C. elegans) as a model organism.

  2. Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors

    PubMed Central

    Hosny, Neveen A.; Mohamedi, Graciela; Rademeyer, Paul; Owen, Joshua; Wu, Yilei; Tang, Meng-Xing; Eckersley, Robert J.; Stride, Eleanor; Kuimova, Marina K.

    2013-01-01

    Encapsulated microbubbles are well established as highly effective contrast agents for ultrasound imaging. There remain, however, some significant challenges to fully realize the potential of microbubbles in advanced applications such as perfusion mapping, targeted drug delivery, and gene therapy. A key requirement is accurate characterization of the viscoelastic surface properties of the microbubbles, but methods for independent, nondestructive quantification and mapping of these properties are currently lacking. We present here a strategy for performing these measurements that uses a small fluorophore termed a “molecular rotor” embedded in the microbubble surface, whose fluorescence lifetime is directly related to the viscosity of its surroundings. We apply fluorescence lifetime imaging to show that shell viscosities vary widely across the population of the microbubbles and are influenced by the shell composition and the manufacturing process. We also demonstrate that heterogeneous viscosity distributions exist within individual microbubble shells even with a single surfactant component. PMID:23690599

  3. Computed tomography based spectral imaging for fluorescence microscopy

    NASA Astrophysics Data System (ADS)

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

    NASA Astrophysics Data System (ADS)

    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

    NASA Astrophysics Data System (ADS)

    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. Manganese doped fluorescent paramagnetic nanocrystals for dual-modal imaging.

    PubMed

    Sharma, Vijay Kumar; Gokyar, Sayim; Kelestemur, Yusuf; Erdem, Talha; Unal, Emre; Demir, Hilmi Volkan

    2014-12-10

    In this work, dual-modal (fluorescence and magnetic resonance) imaging capabilities of water-soluble, low-toxicity, monodisperse Mn-doped ZnSe nanocrystals (NCs) with a size (6.5 nm) below the optimum kidney cutoff limit (10 nm) are reported. Synthesizing Mn-doped ZnSe NCs with varying Mn(2+) concentrations, a systematic investigation of the optical properties of these NCs by using photoluminescence (PL) and time resolved fluorescence are demonstrated. The elemental properties of these NCs using X-ray photoelectron spectroscopy and inductive coupled plasma-mass spectroscopy confirming Mn(2+) doping is confined to the core of these NCs are also presented. It is observed that with increasing Mn(2+) concentration the PL intensity first increases, reaching a maximum at Mn(2+) concentration of 3.2 at% (achieving a PL quantum yield (QY) of 37%), after which it starts to decrease. Here, this high-efficiency sample is demonstrated for applications in dual-modal imaging. These NCs are further made water-soluble by ligand exchange using 3-mercaptopropionic acid, preserving their PL QY as high as 18%. At the same time, these NCs exhibit high relaxivity (≈2.95 mM(-1) s(-1)) to obtain MR contrast at 25 °C, 3 T. Therefore, the Mn(2+) doping in these water-soluble Cd-free NCs are sufficient to produce contrast for both fluorescence and magnetic resonance imaging techniques. PMID:25111198

  7. Compact instrument for fluorescence image-guided surgery

    NASA Astrophysics Data System (ADS)

    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.

    PubMed

    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. Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

    NASA Astrophysics Data System (ADS)

    Kim, Moon S.; Cho, Byoung-Kwan; Lefcourt, Alan M.; Chen, Yud-Ren; Kang, Sukwon

    2008-04-01

    We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.

  10. Particle Image Velocimetry Applications Using Fluorescent Dye-Doped Particles

    NASA Technical Reports Server (NTRS)

    Petrosky, Brian J.; Maisto, Pietro; Lowe, K. Todd; Andre, Matthieu A.; Bardet, Philippe M.; Tiemsin, Patsy I.; Wohl, Christopher J.; Danehy, Paul M.

    2015-01-01

    Polystyrene latex sphere particles are widely used to seed flows for velocimetry techniques such as Particle Image Velocimetry (PIV) and Laser Doppler Velocimetry (LDV). These particles may be doped with fluorescent dyes such that signals spectrally shifted from the incident laser wavelength may be detected via Laser Induced Fluorescence (LIF). An attractive application of the LIF signal is achieving velocimetry in the presence of strong interference from laser scatter, opening up new research possibilities very near solid surfaces or at liquid/gas interfaces. Additionally, LIF signals can be used to tag different fluid streams to study mixing. While fluorescence-based PIV has been performed by many researchers for particles dispersed in water flows, the current work is among the first in applying the technique to micron-scale particles dispersed in a gas. A key requirement for such an application is addressing potential health hazards from fluorescent dyes; successful doping of Kiton Red 620 (KR620) has enabled the use of this relatively safe dye for fluorescence PIV for the first time. In this paper, basic applications proving the concept of PIV using the LIF signal from KR620-doped particles are exhibited for a free jet and a twophase flow apparatus. Results indicate that while the fluorescence PIV techniques are roughly 2 orders of magnitude weaker than Mie scattering, they provide a viable method for obtaining data in flow regions previously inaccessible via standard PIV. These techniques have the potential to also complement Mie scattering signals, for example in multi-stream and/or multi-phase experiments.

  11. Fluorescence Imaging Study of Transition in Underexpanded Free Jets

    NASA Technical Reports Server (NTRS)

    Wilkes, Jennifer A.; Danehy, Paul M.; Nowak, Robert J.

    2005-01-01

    Planar laser-induced fluorescence (PLIF) is demonstrated to be a valuable tool for studying the onset of transition to turbulence. For this study, we have used PLIF of nitric oxide (NO) to image underexpanded axisymmetric free jets issuing into a low-pressure chamber through a smooth converging nozzle with a sonic orifice. Flows were studied over a range of Reynolds numbers and nozzle-exit-to-ambient pressure ratios with the aim of empirically determining criteria governing the onset of turbulence. We have developed an image processing technique, involving calculation of the standard deviation of the intensity in PLIF images, in order to aid in the identification of turbulence. We have used the resulting images to identify laminar, transitional and turbulent flow regimes. Jet scaling parameters were used to define a rescaled Reynolds number that incorporates the influence of a varying pressure ratio. An empirical correlation was found between transition length and this rescaled Reynolds number for highly underexpanded jets.

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Pathological diagnosis of bladder cancer by image analysis of hypericin induced fluorescence cystoscopic images

    NASA Astrophysics Data System (ADS)

    Kah, James C. Y.; Olivo, Malini C.; Lau, Weber K. O.; Sheppard, Colin J. R.

    2005-08-01

    Photodynamic diagnosis of bladder carcinoma based on hypericin fluorescence cystoscopy has shown to have a higher degree of sensitivity for the detection of flat bladder carcinoma compared to white light cystoscopy. The potential of the photosensitizer hypericin-induced fluorescence in performing non-invasive optical biopsy to grade bladder cancer in vivo using fluorescence cystoscopic image analysis without surgical resection for tissue biopsy is investigated in this study. The correlation between tissue fluorescence and histopathology of diseased tissue was explored and a diagnostic algorithm based on fluorescence image analysis was developed to classify the bladder cancer without surgical resection for tissue biopsy. Preliminary results suggest a correlation between tissue fluorescence and bladder cancer grade. By combining both the red-to-blue and red-to-green intensity ratios into a 2D scatter plot yields an average sensitivity and specificity of around 70% and 85% respectively for pathological cancer grading of the three different grades of bladder cancer. Therefore, the diagnostic algorithm based on colorimetric intensity ratio analysis of hypericin fluorescence cystoscopic images developed in this preliminary study shows promising potential to optically diagnose and grade bladder cancer in vivo.

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

    NASA Astrophysics Data System (ADS)

    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. Volumetric retinal fluorescence microscopic imaging with extended depth of field

    NASA Astrophysics Data System (ADS)

    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.

  17. Normalized fluorescence lifetime imaging for tumor identification and margin delineation

    NASA Astrophysics Data System (ADS)

    Sherman, Adria J.; Papour, Asael; Bhargava, Siddharth; Taylor, Zach; Grundfest, Warren S.; Stafsudd, Oscar M.

    2013-03-01

    Fluorescence lifetime imaging microscopy (FLIM) is a technique that has been proven to produce quantitative and qualitative differentiation and identification of substances with good specificity and sensitivity based on lifetime extracted information. This technique has shown the ability to also differentiate between a wide range of tissue types to identify malignant from benign tissue in vivo and ex vivo. However, the complexity, long duration and effort required to generate this information has limited the adoption of these techniques in a clinical setting. Our group has developed a time-resolved imaging system (patent pending) that does not require the extraction of lifetimes or use of complex curve fitting algorithms to display the needed information. The technique, entitled Lifetime Fluorescence Imaging (LFI, or NoFYI), converts fluorescence lifetime decay information directly into visual contrast. Initial studies using Fluorescein and Rhodamine-B demonstrated the feasibility of this approach. Subsequent studies demonstrated the ability to separate collagen and elastin powders. The technique uses nanosecond pulsed UV LEDs at 375 nm for average illumination intensities of ~4.5 μW on the tissue surface with detection by a gated CCD camera. To date, we have imaged 11 surgical head and neck squamous cell carcinoma and brain cancer biopsy specimens including 5 normal and 6 malignant samples. Images at multiple wavelengths clearly demonstrate differentiation between benign and malignant tissue, which was later confirmed by histology. Contrast was obtained between fluorophores with 35 μm spatial resolution and an SNR of ~30 dB allowing us to clearly define tumor margins in these highly invasive cancers. This method is capable of providing both anatomical and chemical information for the pathologist and the surgeon. These results suggest that this technology has a possible role in identifying tumors in tissue specimens and detecting tumor margins

  18. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy.

    PubMed

    Min, Wei; Lu, Sijia; Chong, Shasha; Roy, Rahul; Holtom, Gary R; Xie, X Sunney

    2009-10-22

    Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging. PMID:19847261

  19. De Novo-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters

    PubMed Central

    2015-01-01

    Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme–small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores. PMID:25209927

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

    PubMed Central

    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 both color information and fluorescence signal with high sensitivity under normal surgical lighting illumination. The experimental results show that ICG sample with concentration of 0.13 μM can be detected when the excitation irradiance is 3.92 mW/cm2 at an exposure time of 10 ms. PMID:25136502

  1. Selective Detection of Neurotransmitters by Fluorescence and Chemiluminescence Imaging

    SciTech Connect

    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.

  2. Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics

    PubMed Central

    Hayashi, Shinichi; Okada, Yasushi

    2015-01-01

    Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro­tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging. PMID:25717185

  3. Fluorescence resonance energy transfer imaging by maximum likelihood estimation

    NASA Astrophysics Data System (ADS)

    Zhang, Yupeng; Yuan, Yumin; Holmes, Timothy J.

    2004-06-01

    Fluorescence resonance energy transfer (FRET) is a fluorescence microscope imaging process involving nonradiative energy transfer between two fluorophores (the donor and the acceptor). FRET is used to detect the chemical interactions and, in some cases, measure the distance between molecules. Existing approaches do not always well compensate for bleed-through in excitation, cross-talk in emission detection and electronic noise in image acquisition. We have developed a system to automatically search for maximum-likelihood estimates of the FRET image, donor concentration and acceptor concentration. It also produces other system parameters, such as excitation/emission filter efficiency and FRET conversion factor. The mathematical model is based upon a Poisson process since the CCD camera is a photon-counting device. The main advantage of the approach is that it automatically compensates for bleed-through and cross-talk degradations. Tests are presented with synthetic images and with real data referred to as positive and negative controls, where FRET is known to occur and to not occur, respectively. The test results verify the claimed advantages by showing consistent accuracy in detecting FRET and by showing improved accuracy in calculating FRET efficiency.

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

    PubMed Central

    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

  5. Fluorescence lifetime imaging of endogenous biomarker of oxidative stress.

    PubMed

    Datta, Rupsa; Alfonso-García, Alba; Cinco, Rachel; Gratton, Enrico

    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 advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. Further, we performed spontaneous Raman spectral analysis at single points of the sample which provided molecular vibration information characteristics of lipid droplets. PMID:25993434

  6. Localizing heat-generating defects using fluorescent microthermal imaging

    SciTech Connect

    Tangyunyong, P.; Liang, A.Y.; Righter, A.W.; Barton, D.L.; Soden, J.M.

    1996-10-01

    Fluorescent microthermal imaging (FMI) involves coating a sample surface with a thin fluorescent film that, upon exposure to UV light source, emits temperature-dependent fluorescence. The principle behind FMI was thoroughly reviewed at the ISTFA in 1994. In two recent publications, we identified several factors in film preparation and data processing that dramatically improved the thermal resolution and sensitivity of FMI. These factors include signal averaging, the use of base mixture films, film stabilization and film curing. These findings significantly enhance the capability of FMI as a failure analysis tool. In this paper, we show several examples that use FMI to quickly localize heat-generating defects (``hot spots``). When used with other failure analysis techniques such as focused ion beam (FIB) cross sectioning and scanning electron microscope (SEM) imaging, we demonstrate that FMI is a powerful tool to efficiently identify the root cause of failures in complex ICs. In addition to defect localization, we use a failing IC to I determine the sensitivity of FMI (i.e., the lowest power that can be detected) in an ideal situation where the defects are very localized and near the surface.

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

    PubMed

    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. Small Portable Interchangeable Imager of Fluorescence for Fluorescence Guided Surgery and Research

    PubMed Central

    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

    2014-01-01

    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

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

    PubMed

    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

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

    PubMed Central

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

    2016-01-01

    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 [PalczewskaG., Nat Med. 20, 785 (2014)24952647 SharmaR., Biomed. Opt. Express 4, 1285 (2013)24009992]. PMID:27446697

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

    PubMed

    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

  12. Bioreductive fluorescent imaging agents: applications to tumour hypoxia.

    PubMed

    Elmes, Robert B P

    2016-07-12

    Large tumours contain regions with very low intracellular O2 concentrations. Known as hypoxia, this feature of tumours yields a highly reducing environment owing to the presence of numerous oxygen sensitive reductase enzymes. The development of new optical chemosensors for these various reductases presents an ideal approach to visualise areas of hypoxia or highly reducing environments. Critical to the success of such chemosensors is the design of probes containing a bioreductively activated moiety that either ensures the selective retention of fluorescence within a hypoxic tissue or a probe that irreversibly releases a reporter fluorophore. This Feature Article aims to summarise the fluorescent tools that have been developed to image tumour hypoxia and the various reductase enzymes associated with the bioreduction process. PMID:26924320

  13. Near-Infrared Fluorescent NanoGUMBOS for Biomedical Imaging

    SciTech Connect

    Bwambok, David; El-Zahab, Bilal; Challa, Santhosh; Li, Min; Chandler, Lin; Baker, Gary A; Warner, Isiah M

    2009-01-01

    Herein, we report on near-infrared (NIR) fluorescent nanoparticles generated from an emergent class of materials we refer to as a Group of Uniform Materials Based on Organic Salts (GUMBOS). GUMBOS are largely frozen ionic liquids, although the concept is more general and is also easily applied to solid ionic materials with melting points in excess of 100 C. Nanoparticles based on GUMBOS (nanoGUMBOS) derived from a NIR fluorophore are prepared using a reprecipitation method and evaluated for in vivo fluorescence imaging. Due to their uniformity, single-step preparation, and composite nature, nanoGUMBOS help to resolve issues with dye leakage problems innate to alternate cellular stains and unlock a myriad of applications for these materials, highlighting exciting possibilities for multifunctional nanoGUMBOS.

  14. Measurement of Nanoparticle Magnetic Hyperthermia Using Fluorescent Microthermal Imaging

    NASA Astrophysics Data System (ADS)

    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.

  15. Near Infrared Fluorescent NanoGUMBOS for Biomedical Imaging

    PubMed Central

    Bwambok, David K.; El-Zahab, Bilal; Challa, Santhosh K.; Li, Min; Chandler, Lin; Baker, Gary A.; Warner, Isiah M.

    2009-01-01

    Herein, we report on near infrared (NIR) fluorescent nanoparticles generated from an emergent class of materials we refer to as a Group of Uniform Materials Based on Organic Salts (GUMBOS). GUMBOS are largely frozen ionic liquids, although the concept is more general and is also easily applied to solid ionic materials with melting points in excess of 100 °C. Nanoparticles based on GUMBOS (nanoGUMBOS) derived from a NIR fluorophore are prepared using a reprecipitation method and evaluated for in vivo fluorescence imaging. Due to their uniformity, single-step preparation, and composite nature, nanoGUMBOS help to resolve issues with dye leakage problems innate to alternate cellular stains and unlock a myriad of applications for these materials, highlighting exciting possibilities for multifunctional nanoGUMBOS. PMID:19928781

  16. Multispectral fluorescence imaging technique for discrimination of cucumber (Cucumis Sativus) seed viability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, we developed a nondestructive method for discriminating viable cucumber (Cucumis sativus) seeds based on hyperspectral fluorescence imaging. The fluorescence spectra of cucumber seeds in the 420–700 nm range were extracted from hyperspectral fluorescence images obtained using 365 nm u...

  17. Real-time quantitative fluorescence imaging using a single snapshot optical properties technique for neurosurgical guidance

    NASA Astrophysics Data System (ADS)

    Valdes, Pablo A.; Angelo, Joseph; Gioux, Sylvain

    2015-03-01

    Fluorescence imaging has shown promise as an adjunct to improve the extent of resection in neurosurgery and oncologic surgery. Nevertheless, current fluorescence imaging techniques do not account for the heterogeneous attenuation effects of tissue optical properties. In this work, we present a novel imaging system that performs real time quantitative fluorescence imaging using Single Snapshot Optical Properties (SSOP) imaging. We developed the technique and performed initial phantom studies to validate the quantitative capabilities of the system for intraoperative feasibility. Overall, this work introduces a novel real-time quantitative fluorescence imaging method capable of being used intraoperatively for neurosurgical guidance.

  18. Compact Image Slicing Spectrometer (ISS) for hyperspectral fluorescence microscopy

    PubMed Central

    Gao, Liang; Kester, Robert T.; Tkaczyk, Tomasz S.

    2009-01-01

    An image slicing spectrometer (ISS) for microscopy applications is presented. Its principle is based on the redirecting of image zones by specially organized thin mirrors within a custom fabricated component termed an image slicer. The demonstrated prototype can simultaneously acquire a 140nm spectral range within its 2D field of view from a single image. The spectral resolution of the system is 5.6nm. The FOV and spatial resolution of the ISS depend on the selected microscope objective and for the results presented is 45×45μm2 and 0.45μm respectively. This proof-of-concept system can be easily improved in the future for higher (both spectral and spatial) resolution imaging. The system requires no scanning and minimal post data processing. In addition, the reflective nature of the image slicer and use of prisms for spectral dispersion make the system light efficient. Both of the above features are highly valuable for real time fluorescent-spectral imaging in biological and diagnostic applications. PMID:19654631

  19. Snapshot spectrally encoded fluorescence imaging through a fiber bundle

    PubMed Central

    Bedard, Noah

    2012-01-01

    Abstract. Fiber optic endomicroscopy is a valuable tool for clinical diagnostics and animal studies because it can capture images of tissue in vivo with subcellular resolution. Current configurations for endomicroscopes have either limited spatial resolution or require a scanning mechanism at the distal end of the fiber, which can slow imaging speed and increase the probe size. We present a novel configuration that provides high contrast 350×350 pixel images at 7.2 frames per second, without the need for mechanical scanning at the proximal or distal end of the fiber. The proof-of-concept benchtop system is tested in fluorescence mode and can resolve 1.5 µm features of a high resolution 1951 USAF target. PMID:23224159

  20. Snapshot spectrally encoded fluorescence imaging through a fiber bundle

    NASA Astrophysics Data System (ADS)

    Bedard, Noah; Tkaczyk, Tomasz S.

    2012-08-01

    Fiber optic endomicroscopy is a valuable tool for clinical diagnostics and animal studies because it can capture images of tissue in vivo with subcellular resolution. Current configurations for endomicroscopes have either limited spatial resolution or require a scanning mechanism at the distal end of the fiber, which can slow imaging speed and increase the probe size. We present a novel configuration that provides high contrast 350×350 pixel images at 7.2 frames per second, without the need for mechanical scanning at the proximal or distal end of the fiber. The proof-of-concept benchtop system is tested in fluorescence mode and can resolve 1.5 μm features of a high resolution 1951 USAF target.

  1. Multiparameter radar analysis using wavelets

    NASA Astrophysics Data System (ADS)

    Tawfik, Ben Bella Sayed

    Multiparameter radars have been used in the interpretation of many meteorological phenomena. Rainfall estimates can be obtained from multiparameter radar measurements. Studying and analyzing spatial variability of different rainfall algorithms, namely R(ZH), the algorithm based on reflectivity, R(ZH, ZDR), the algorithm based on reflectivity and differential reflectivity, R(KDP), the algorithm based on specific differential phase, and R(KDP, Z DR), the algorithm based on specific differential phase and differential reflectivity, are important for radar applications. The data used in this research were collected using CSU-CHILL, CP-2, and S-POL radars. In this research multiple objectives are addressed using wavelet analysis namely, (1)space time variability of various rainfall algorithms, (2)separation of convective and stratiform storms based on reflectivity measurements, (3)and detection of features such as bright bands. The bright band is a multiscale edge detection problem. In this research, the technique of multiscale edge detection is applied on the radar data collected using CP-2 radar on August 23, 1991 to detect the melting layer. In the analysis of space/time variability of rainfall algorithms, wavelet variance introduces an idea about the statistics of the radar field. In addition, multiresolution analysis of different rainfall estimates based on four algorithms, namely R(ZH), R( ZH, ZDR), R(K DP), and R(KDP, Z DR), are analyzed. The flood data of July 29, 1997 collected by CSU-CHILL radar were used for this analysis. Another set of S-POL radar data collected on May 2, 1997 at Wichita, Kansas were used as well. At each level of approximation, the detail and the approximation components are analyzed. Based on this analysis, the rainfall algorithms can be judged. From this analysis, an important result was obtained. The Z-R algorithms that are widely used do not show the full spatial variability of rainfall. In addition another intuitively obvious result

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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. PMID:26358823

  4. Another treatment of fluorescence polarization microspectroscopy and imaging.

    PubMed

    Fisz, Jacek J

    2009-04-16

    We here discuss a general (symmetry adapted) treatment for one-photon-excitation time-resolved fluorescence polarization microspectroscopy (TRFPM) at combined wide-angular excitation and detection apertures that correctly couples the principles of the optics of objective lenses with the principles of fluorescence spectroscopy with polarized light. The treatment is unified in the sense that it covers the electromagnetic description of focusing a linearly polarized beam of exciting light (diffraction theory, DT) and the description of the same problem in terms of the meridional plane properties (MPP) of the objective lenses (geometrical optics). It is shown that both approaches are quantitatively equivalent from the point of view of the polarization effects in typical TRFPM experiments on linear absorbers, despite the fact that in the MPP treatment the region of focus is treated as a pointlike object, while in the DT method the region of focus is characterized by a three-dimensional (3D) inhomogeneous electromagnetic field distribution, of generally ellipsoidal polarization at different points of the focus. This finding is essentially important from the point of view of the experimental practice because the MPP treatment is based on two very simple trigonometric expressions, in evident contrast to the DT method, in which the high-aperture focusing is described in terms of three complicated 3D integrals involving the Bessel functions of the first kind. A few words of comment are added on a similar problem in the case of nonlinear one-photon absorbers (e.g., chiral fluorophores). We discuss the synthetic fluorescence decays for the wide-field- and evanescent-wave-excitation confocal (or wide-field) detection fluorescence polarization microspectroscopy and imaging, which indicate the right experimental protocols for the kinetic and dynamic fluorescence polarization microspectroscopic studies. The manifestations of the effects resulting from the application of the wide

  5. Defining a Superlens Operating Regime for Imaging Fluorescent Molecules

    PubMed Central

    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 ( nm) or too far ( 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 structure (such as in the far

  6. A novel method for image denoising of fluorescence molecular imaging based on fuzzy C-Means clustering

    NASA Astrophysics Data System (ADS)

    An, Yu; Liu, Jie; Ye, Jinzuo; Mao, Yamin; Yang, Xin; Jiang, Shixin; Chi, Chongwei; Tian, Jie

    2015-03-01

    As an important molecular imaging modality, fluorescence molecular imaging (FMI) has the advantages of high sensitivity, low cost and ease of use. By labeling the regions of interest with fluorophore, FMI can noninvasively obtain the distribution of fluorophore in-vivo. However, due to the fact that the spectrum of fluorescence is in the section of the visible light range, there are mass of autofluorescence on the surface of the bio-tissues, which is a major disturbing factor in FMI. Meanwhile, the high-level of dark current for charge-coupled device (CCD) camera and other influencing factor can also produce a lot of background noise. In this paper, a novel method for image denoising of FMI based on fuzzy C-Means clustering (FCM) is proposed, because the fluorescent signal is the major component of the fluorescence images, and the intensity of autofluorescence and other background signals is relatively lower than the fluorescence signal. First, the fluorescence image is smoothed by sliding-neighborhood operations to initially eliminate the noise. Then, the wavelet transform (WLT) is performed on the fluorescence images to obtain the major component of the fluorescent signals. After that, the FCM method is adopt to separate the major component and background of the fluorescence images. Finally, the proposed method was validated using the original data obtained by in vivo implanted fluorophore experiment, and the results show that our proposed method can effectively obtain the fluorescence signal while eliminate the background noise, which could increase the quality of fluorescence images.

  7. Fluorescence and fluorescence-lifetime imaging microscopy (FLIM) to characterize yeast strains by autofluorescence

    NASA Astrophysics Data System (ADS)

    Bhatta, H.; Goldys, E. M.; Ma, J.

    2006-02-01

    We characterised populations of wild type baking and brewing yeast cells using intrinsic fluorescence and fluorescence lifetime microscopy, in order to obtain quantitative identifiers of different strains. The cell autofluorescence was excited at 405 nm and observed within 440-540 nm range where strong cell to cell variability was observed. The images were analyzed using customised public domain software, which provided information on cell size, intensity and texture-related features. In light of significant diversity of the data, statistical methods were utilized to assess the validity of the proposed quantitative identifiers for strain differentiation. The Kolmogorov-Smirnov test was applied to confirm that empirical distribution functions for size, intensity and entropy for different strains were statistically different. These characteristics were followed with culture age of 24, 48 and 72 h, (the latter corresponding to a stationary growth phase) and size, and to some extent entropy, were found to be independent of age. The fluorescence intensity presented a distinctive evolution with age, different for each of the examined strains. The lifetime analysis revealed a short decay time component of 1.4 ns and a second, longer one with the average value of 3.5 ns and a broad distribution. High variability of lifetime values within cells was observed however a lifetime texture feature in the studied strains was statistically different.

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

    PubMed

    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

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

    PubMed Central

    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

  10. A CMOS In-Pixel CTIA High Sensitivity Fluorescence Imager

    PubMed Central

    Murari, Kartikeya; Etienne-Cummings, Ralph; Thakor, Nitish; Cauwenberghs, Gert

    2012-01-01

    Traditionally, charge coupled device (CCD) based image sensors have held sway over the field of biomedical imaging. Complementary metal oxide semiconductor (CMOS) based imagers so far lack sensitivity leading to poor low-light imaging. Certain applications including our work on animal-mountable systems for imaging in awake and unrestrained rodents require the high sensitivity and image quality of CCDs and the low power consumption, flexibility and compactness of CMOS imagers. We present a 132×124 high sensitivity imager array with a 20.1 μm pixel pitch fabricated in a standard 0.5 μ CMOS process. The chip incorporates n-well/p-sub photodiodes, capacitive transimpedance amplifier (CTIA) based in-pixel amplification, pixel scanners and delta differencing circuits. The 5-transistor all-nMOS pixel interfaces with peripheral pMOS transistors for column-parallel CTIA. At 70 fps, the array has a minimum detectable signal of 4 nW/cm2 at a wavelength of 450 nm while consuming 718 μA from a 3.3 V supply. Peak signal to noise ratio (SNR) was 44 dB at an incident intensity of 1 μW/cm2. Implementing 4×4 binning allowed the frame rate to be increased to 675 fps. Alternately, sensitivity could be increased to detect about 0.8 nW/cm2 while maintaining 70 fps. The chip was used to image single cell fluorescence at 28 fps with an average SNR of 32 dB. For comparison, a cooled CCD camera imaged the same cell at 20 fps with an average SNR of 33.2 dB under the same illumination while consuming over a watt. PMID:23136624

  11. Noninvasive imaging of focal atherosclerotic lesions using fluorescence molecular tomography

    NASA Astrophysics Data System (ADS)

    Maji, Dolonchampa; Solomon, Metasebya; Nguyen, Annie; Pierce, Richard A.; Woodard, Pamela K.; Akers, Walter J.; Achilefu, Samuel; Culver, Joseph P.; Abendschein, Dana R.; Shokeen, Monica

    2014-11-01

    Insights into the etiology of stroke and myocardial infarction suggest that rupture of unstable atherosclerotic plaque is the precipitating event. Clinicians lack tools to detect lesion instability early enough to intervene, and are often left to manage patients empirically, or worse, after plaque rupture. Noninvasive imaging of the molecular events signaling prerupture plaque progression has the potential to reduce the morbidity and mortality associated with myocardial infarction and stroke by allowing early intervention. Here, we demonstrate proof-of-principle in vivo molecular imaging of C-type natriuretic peptide receptor in focal atherosclerotic lesions in the femoral arteries of New Zealand white rabbits using a custom built fiber-based, fluorescence molecular tomography (FMT) system. Longitudinal imaging showed changes in the fluorescence signal intensity as the plaque progressed in the air-desiccated vessel compared to the uninjured vessel, which was validated by ex vivo tissue studies. In summary, we demonstrate the potential of FMT for noninvasive detection of molecular events leading to unstable lesions heralding plaque rupture.

  12. X-ray fluorescence microprobe imaging in biology and medicine.

    PubMed

    Paunesku, Tatjana; Vogt, Stefan; Maser, Jörg; Lai, Barry; Woloschak, Gayle

    2006-12-15

    Characteristic X-ray fluorescence is a technique that can be used to establish elemental concentrations for a large number of different chemical elements simultaneously in different locations in cell and tissue samples. Exposing the samples to an X-ray beam is the basis of X-ray fluorescence microscopy (XFM). This technique provides the excellent trace element sensitivity; and, due to the large penetration depth of hard X-rays, an opportunity to image whole cells and quantify elements on a per cell basis. Moreover, because specimens prepared for XFM do not require sectioning, they can be investigated close to their natural, hydrated state with cryogenic approaches. Until several years ago, XFM was not widely available to bio-medical communities, and rarely offered resolution better then several microns. This has changed drastically with the development of third-generation synchrotrons. Recent examples of elemental imaging of cells and tissues show the maturation of XFM imaging technique into an elegant and informative way to gain insight into cellular processes. Future developments of XFM-building of new XFM facilities with higher resolution, higher sensitivity or higher throughput will further advance studies of native elemental makeup of cells and provide the biological community including the budding area of bionanotechnology with a tool perfectly suited to monitor the distribution of metals including nanovectors and measure the results of interactions between the nanovectors and living cells and tissues. PMID:17006954

  13. Microbial biofilm detection on food contact surfaces by macro-scale fluorescence imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hyperspectral fluorescence imaging methods were utilized to evaluate the potential of multispectral fluorescence methods for detection of pathogenic biofilm formations on four types of food contact surface materials: stainless steel, high density polyethylene (HDPE) commonly used for cutting boards,...

  14. Multiparameter investigation of gravitational slip

    SciTech Connect

    Daniel, Scott F.; Caldwell, Robert R.; Cooray, Asantha; Serra, Paolo; Melchiorri, Alessandro

    2009-07-15

    A detailed analysis of gravitational slip, a new post-general relativity cosmological parameter characterizing the degree of departure of the laws of gravitation from general relativity on cosmological scales, is presented. This phenomenological approach assumes that cosmic acceleration is due to new gravitational effects; the amount of spacetime curvature produced per unit mass is changed in such a way that a universe containing only matter and radiation begins to accelerate as if under the influence of a cosmological constant. Changes in the law of gravitation are further manifest in the behavior of the inhomogeneous gravitational field, as reflected in the cosmic microwave background, weak lensing, and evolution of large-scale structure. The new parameter {pi}{sub 0} is naively expected to be of order unity. However, a multiparameter analysis, allowing for variation of all of the standard cosmological parameters, finds that {pi}{sub 0}=0.09{sub -0.59}{sup +0.74}(2{sigma}), where {pi}{sub 0}=0 corresponds to a cosmological constant plus cold dark matter universe under general relativity. Future probes of the cosmic microwave background (Planck) and large-scale structure (Euclid) may improve the limits by a factor of 4.

  15. Live Cell Imaging of a Fluorescent Gentamicin Conjugate

    PubMed Central

    Escobedo, Jorge O.; Chu, Yu-Hsuan; Wang, Qi; Steyger, Peter S.; Strongin, Robert M.

    2012-01-01

    Understanding cellular mechanisms of ototoxic and nephrotoxic drug uptake, intracellular distribution, and molecular trafficking across cellular barrier systems aids the study of potential uptake blockers that preserve sensory and renal function during critical life-saving therapy. Herein we report the design, synthesis characterization and evaluation of a fluorescent conjugate of the aminoglycoside antibiotic gentamicin. Live cell imaging results show the potential utility of this new material. Related gentamicin conjugates studied to date quench in live kindney cells, and have been largely restricted to use in fixed (delipidated) cells. PMID:22545403

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

    DOEpatents

    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.

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

    PubMed Central

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

    2013-01-01

    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

  18. Modelling of microcracks image treated with fluorescent dye

    NASA Astrophysics Data System (ADS)

    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

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

    NASA Technical Reports Server (NTRS)

    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.

  20. Fluorescence lifetime imaging with near-infrared dyes

    NASA Astrophysics Data System (ADS)

    Becker, Wolfgang; Shcheslavskiy, Vladislav

    2013-02-01

    Near-infrared (NIR) dyes are used as fluorescence markers in small-animal imaging and in diffuse optical tomography of the human brain. In these applications it is important to know whether the dyes bind to proteins or other tissue constituents, and whether their fluorescence lifetimes depend on the targets they are bound to. Unfortunately, neither the lasers nor the detectors of commonly used confocal and multiphoton laser scanning microscopes allow for excitation and detection of NIR fluorescence. We therefore upgraded existing confocal TCSPC FLIM systems with NIR lasers and NIR sensitive detectors. In multiphoton systems we used the Ti:Sa laser as a one-photon excitation source in combination with an NIR-sensitive detector in the confocal beam path. We tested a number of NIR dyes in biological tissue. Some of them showed clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information on the tissue constitution and on local biochemical parameters.

  1. Gold nanoclusters as contrast agents for fluorescent and X-ray dual-modality imaging.

    PubMed

    Zhang, Aili; Tu, Yu; Qin, Songbing; Li, Yan; Zhou, Juying; Chen, Na; Lu, Qiang; Zhang, Bingbo

    2012-04-15

    Multimodal imaging technique is an alternative approach to improve sensitivity of early cancer diagnosis. In this study, highly fluorescent and strong X-ray absorption coefficient gold nanoclusters (Au NCs) are synthesized as dual-modality imaging contrast agents (CAs) for fluorescent and X-ray dual-modality imaging. The experimental results show that the as-prepared Au NCs are well constructed with ultrasmall sizes, reliable fluorescent emission, high computed tomography (CT) value and fine biocompatibility. In vivo imaging results indicate that the obtained Au NCs are capable of fluorescent and X-ray enhanced imaging. PMID:22289255

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

    NASA Astrophysics Data System (ADS)

    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 (<114nm), high 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.

  3. Fluorescence imaging of chromosomal DNA using click chemistry.

    PubMed

    Ishizuka, Takumi; Liu, Hong Shan; Ito, Kenichiro; Xu, Yan

    2016-01-01

    Chromosome visualization is essential for chromosome analysis and genetic diagnostics. Here, we developed a click chemistry approach for multicolor imaging of chromosomal DNA instead of the traditional dye method. We first demonstrated that the commercially available reagents allow for the multicolor staining of chromosomes. We then prepared two pro-fluorophore moieties that served as light-up reporters to stain chromosomal DNA based on click reaction and visualized the clear chromosomes in multicolor. We applied this strategy in fluorescence in situ hybridization (FISH) and identified, with high sensitivity and specificity, telomere DNA at the end of the chromosome. We further extended this approach to observe several basic stages of cell division. We found that the click reaction enables direct visualization of the chromosome behavior in cell division. These results suggest that the technique can be broadly used for imaging chromosomes and may serve as a new approach for chromosome analysis and genetic diagnostics. PMID:27620982

  4. Detectors for single-molecule fluorescence imaging and spectroscopy

    PubMed Central

    MICHALET, X.; SIEGMUND, O.H.W.; VALLERGA, J.V.; JELINSKY, P.; MILLAUD, J.E.; WEISS, S.

    2010-01-01

    Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy. PMID:20157633

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics

    PubMed Central

    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

  7. Combining Optical Coherence Tomography with Fluorescence Molecular Imaging: Towards Simultaneous Morphology and Molecular Imaging

    PubMed Central

    Yuan, Shuai; Roney, Celeste A.; Wierwille, Jerry; Chen, Chao-Wei; Xu, Biying; Jiang, James; Ma, Hongzhou; Cable, Alex; Summers, Ronald M.; Chen, Yu

    2010-01-01

    Optical coherence tomography (OCT) provides high-resolution, cross-sectional imaging of tissue microstructure in situ and in real-time, while fluorescence molecular imaging (FMI) enables the visualization of basic molecular processes. There are great interests in combining these two modalities so that the tissue's structural and molecular information can be obtained simultaneously. This could greatly benefit biomedical applications such as detecting early diseases and monitoring therapeutic interventions. In this research, an optical system that combines OCT and FMI was developed. The system demonstrated that it could co-register en face OCT and FMI images with a 2.4 × 2.4 mm field of view. The transverse resolutions of OCT and FMI of the system are both ~10 μm. Capillary tubes filled with fluorescent dye Cy 5.5 in different concentrations under a scattering medium are used as the phantom. En face OCT images of the phantoms were obtained and successfully co-registered with FMI images that were acquired simultaneously. A linear relationship between FMI intensity and dye concentration was observed. The relationship between FMI intensity and target fluorescence tube depth measured by OCT images was also observed and compared with theoretical modeling. This relationship could help in correcting reconstructed dye concentration. Imaging of colon polyps of APCmin mouse model is presented as an example of biological applications of this co-registered OCT/FMI system. PMID:20009192

  8. Spectrally resolved fluorescence imaging of human colonic adenomas.

    PubMed

    Chwirot, B W; Kowalska, M; Sypniewska, N; Michniewicz, Z; Gradziel, M

    1999-06-01

    Native fluorescence (autofluorescence) of human tissues can be a valuable source of diagnostic information for detecting premalignant and malignant lesions in the human body. Digital imaging of autofluorescence may be useful for localization of such lesions during endoscopic examinations. Tissue fluorescence of 31 adenomatous polyps obtained from 16 patients has been excited in vitro using the 325 nm line of a He-Cd laser. Digital images of the autofluorescence are recorded in six spectral bands. This study provides new data about the spatial distributions of autofluorescence intensities emitted in different spectral bands by colonic adenomatous lesions and normal colonic mucosa. Areas characterized by autofluorescence intensity lower than in normal mucosa are found for a majority of the polyps under study. The observed patterns of spatial distribution differ for the different spectral bands and for different polypoid lesions. No inverse correlation is found between the emission intensity and the thickness of colonic mucosa. The results indicate the spectral bands most useful for diagnostic applications and demonstrate the complexity of the optical processes involved in shaping both the spectra and intensities of the autofluorescence. PMID:10515079

  9. New approach in prostate Gleason grading using fluorescence microscopic imaging

    NASA Astrophysics Data System (ADS)

    Alexandratou, Eleni; Yova, Dido; Gorpas, Dimitris

    2009-07-01

    Prostate cancer is a common disease among men with an increasing number of incidences during the last three decades. Histopathological grading of prostate cancer is based on tissue structural abnormalities. Gleason grading system is the gold standard and is based on the organization features of prostatic glands. However, till now there is an uncertainty assign Gleason grade to intermediate stages of the disease, Gleason 3 and Gleason 4. The aim of this study was to explore the possibility of introducing fluorescent probes in this prostate cancer Gleason grading problem. Propidium Iodide with cellular nuclei binding pattern and Alexa 488-WGA with selectivity in polysaccharides with sialic acid residues were finally chosen. Their localisation patterns were assessed using confocal microscopy. Their colocalisation degree was quantified using special developed algorithms of image processing and analysis. The introduced metrics of colocalisation were successfully used to correct classify samples in Gleason 3 and Gleason 4 grades. These metrics were found appropriate to correctly classify 93.10 % of the images into the two classes using the logistic algorithm. The integration of confocal microscopy along with fluorescent probes to pathologist routine, is an approach that cloud lead to prognostic advances.

  10. Fluorescence Lifetime Imaging of Membrane Lipid Order with a Ratiometric Fluorescent Probe

    PubMed Central

    Kilin, Vasyl; Glushonkov, Oleksandr; Herdly, Lucas; Klymchenko, Andrey; Richert, Ludovic; Mely, Yves

    2015-01-01

    To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N∗) and tautomer (T∗) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T∗ form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N∗ form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis. PMID:25992730

  11. Optical imaging for brain tissue characterization using relative fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Papour, Asael; Taylor, Zach; Sherman, Adria; Sanchez, Desiree; Lucey, Gregory; Liau, Linda; Stafsudd, Oscar; Yong, William; Grundfest, Warren

    2013-06-01

    An autofluorescence lifetime wide-field imaging system that can generate contrast in underlying tissue structures of normal and malignant brain tissue samples with video rate acquisition and processing time is presented. Images of the investigated tissues were acquired with high resolution (˜35 μm) using an algorithm to produce contrast based on differences in relative lifetimes. Sufficient contrast for delineation was produced without the computation of fluorescence decay times or Laguerre coefficients. The imaged tissues were sent for histological analysis that confirmed the detected imaged tissues morphological findings and correlations between relative lifetime maps and histology identified.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

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

    PubMed Central

    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

  15. Parallel-scan based microarray imager capable of simultaneous surface plasmon resonance and hyperspectral fluorescence imaging.

    PubMed

    Liu, Zhiyi; Yang, Lei; Liu, Le; Chong, Xinyuan; Guo, Jun; Ma, Suihua; Ji, Yanhong; He, Yonghong

    2011-12-15

    With the development of the microarray technology, demands for array detection techniques become higher and higher. For many microarrays, several biomolecular interactions occur simultaneously and the interplay of various factors that affect these interactions remains poorly understood. Detecting such interactions with a single technique can often be a difficult and complicated process. In this work we propose a combined technique which enables simultaneous angle-interrogation surface plasmon resonance (SPR) sensing and hyperspectral fluorescence imaging. This tandem technique offers two-dimensional imaging of the whole array plane. The refractive index information obtained from SPR sensing and the physicochemical properties obtained from fluorescence imaging provide a comprehensive analysis of biological events on the array-chip. In addition, SPR and fluorescence detection techniques confirm each other in experimental results to exclude false-positive or false-negative cases. In terms of SPR sensing performance, the refractive index resolution is 3.86×10(-6) refractive index units (RIU), and the detection limit is 10(4) cfu/ml of Escherichia coli bacteria. The resolving power and detection sensitivity of fluorescence imaging are approximately 20 μm and 0.61 fluors/μm(2), respectively. Finally, two model experiments, detecting the DNA hybridization and biotin-avidin interactions respectively, demonstrate the biomedical application of this system. PMID:21996322

  16. Fluorescence lifetime images of different green fluorescent proteins in fly brain

    NASA Astrophysics Data System (ADS)

    Lai, Sih-Yu; Lin, Y. Y.; Chiang, A. S.; Huang, Y. C.

    2009-02-01

    The mechanisms of learning and memory are the most important functions in an animal brain. Investigating neuron circuits and network maps in a brain is the first step toward understanding memory and learning behavior. Since Drosophila brain is the major model for understanding brain functions, we measure the florescence lifetimes of different GFP-based reporters expressed in a fly brain. In this work, two Gal4 drivers, OK 107 and MZ 19 were used. Intracellular calcium ([Ca2+]) concentration is an importation indicator of neuronal activity. Therefore, several groups have developed GFP-based calcium sensors, among which G-CaMP is the most popular and reliable. The fluorescence intensity of G-CaMP will increase when it binds to calcium ion; however, individual variation from different animals prevents quantitative research. In this work, we found that the florescence lifetime of G-CaMP will shrink from 1.8 ns to 1.0 ns when binding to Ca2+. This finding can potentially help us to understand the neuron circuits by fluorescence lifetime imaging microscopy (FLIM). Channelrhodopsin-2 (ChR2) is a light-activated ion-channel protein on a neuron cell membrane. In this work, we express ChR2 and G-CaMP in a fly brain. Using a pulsed 470-nm laser to activate the neurons, we can also record the fluorescence lifetime changes in the structure. Hence, we can trace and manipulate a specific circuit in this animal. This method provides more flexibility in brain research.

  17. Real-time computation of subdiffraction-resolution fluorescence images.

    PubMed

    Wolter, S; Schüttpelz, M; Tscherepanow, M; VAN DE Linde, S; Heilemann, M; Sauer, M

    2010-01-01

    In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with approximately 20 nm optical resolution can be completed in less than 10 s. PMID:20055915

  18. Fluorescence Imaging of the Cytoskeleton in Plant Roots.

    PubMed

    Dyachok, Julia; Paez-Garcia, Ana; Yoo, Cheol-Min; Palanichelvam, Karuppaiah; Blancaflor, Elison B

    2016-01-01

    During the past two decades the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis roots, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. For roots with a larger diameter such as Medicago truncatula, seeds are first germinated in moist paper, grown vertically in between plastic trays, and roots mounted on glass slides for confocal imaging. Parallel with our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use non-embedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development. PMID:26498783

  19. Motion corrected photoacoustic difference imaging of fluorescent contrast agents

    NASA Astrophysics Data System (ADS)

    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.

  20. Concurrent fluorescence macro-imaging across multiple spectral regions in the visible and the near infrared

    NASA Astrophysics Data System (ADS)

    Kazemzadeh, Farnoud; Haider, Shahid; Jin, Chao; Clausi, David A.; Wong, Alexander

    2015-09-01

    Fluorescent imaging, often synonymous with microscopic imaging, is an imaging modality whereby various features of a target are observed based on assignment of chemical labels. These labels are in most cases indirect tracers of specific structures or chemical compounds which cannot be otherwise identified. The tracers are excited by an illuminating source and they in turn emit light at specific wavelengths. This light is then captured by an imaging device and represented as an indirect observation of the specific feature in the sample. The process of excitation and imaging of the emitted light is performed sequentially and is proportional to the number of tracers or fluorescence species present in the sample. We present an imaging system that can image fluorescent tracers, in the visible and the near Infra-red, simultaneously. This system is capable of illuminating the target with different excitation light sources and capture the corresponding fluorescence images in one snapshot using a series of mirrors to capture different views of the sample. The simultaneously captured image are fused using a computational reconstruction process to present a coherent multispectral fluorescence image. The system is proposed for use in applications where the rapid enumeration of fluorescent species in a large field of view is paramount as opposed to their microscopic image in a narrow field of view. The system was tested using a controlled cocktail solution of four different types fluorescent microspheres and was able to enumerate the microspheres based on their different fluorescent signatures as captured by the system.

  1. The Chlorophyll Fluorescence Imaging Spectrometer (CFIS): A New Airborne Instrument for Quantifying Solar-Induced Fluorescence

    NASA Astrophysics Data System (ADS)

    Drewry, D.; Frankenberg, C.; Verma, M.; Berry, J. A.; Schimel, D.; Geier, S.; Schwochert, M.

    2015-12-01

    Recent demonstrations of the retrieval of vegetation solar-induced fluorescence (SIF) emission from satellite platforms have opened up the possibility of remotely monitoring photosynthetic function, in addition to the structural and biochemical parameters that characterize the current capabilities of vegetation observing systems. These satellite retrievals, from platforms such as GOSAT, GOME-2, and most recently NASA's Orbiting Carbon Observatory 2 (OCO-2), provide powerful evidence of the correlation between vegetation productivity and SIF at seasonal to annual timescales, and at spatial resolutions of tens to hundreds of kilometers. The Chlorophyll Fluorescence Imaging Spectrometer (CFIS) was recently developed for OCO-2 validation purposes and provides an airborne capability to help fill the spatial gap between leaf- or canopy-level observations of SIF flux and extensive satellite footprints. The flexibility of an airborne instrument likewise allows for studies of the temporal variability of SIF emission over consecutive days, or with meteorological variability throughout a day. CFIS is a high resolution (<0.1nm) spectrometer covering the 740-770nm wavelength range, optimized for SIF quantification. Here we present an overview of the instrument design and capabilities, along with the retrieval methodology. An evaluation of data collected during initial campaigns conducted during the spring and summer of 2015 are also presented, demonstrating variability within and between days for campaigns spanning multiple days in the Midwest US and Northern California. Results will be compared to OCO-2 data as well as flux-tower measurements made during the CFIS flights.

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

    PubMed

    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

  3. Multiparameter digitized video microscopy of toxic and hypoxic injury in single cells.

    PubMed Central

    Lemasters, J J; Gores, G J; Nieminen, A L; Dawson, T L; Wray, B E; Herman, B

    1990-01-01

    There is no clear picture of the critical events that lead to the transition from reversible to irreversible injury. Many studies have suggested that a rise in cytosolic free Ca2+ initiates plasma membrane bleb formation and a sequence of events that lead ultimately to cell death. In recent studies, we have measured changes in cytosolic free Ca2+, mitochondrial membrane potential, cytosolic pH, and cell surface blebbing in relation to the onset of irreversible injury and cell death following anoxic and toxic injury to single hepatocytes by using multiparameter digitized video microscopy (MDVM). MDVM is an emerging new technology that permits single living cells to be labeled with multiple probes whose fluorescence is responsive to specific cellular parameters of interest. Fluorescence images specific for each probe are collected over time, digitized, and stored. Image analysis and processing then permits quantitation of the spatial distribution of the various parameters with the single living cells. Our results indicate the following: The formation of plasma membrane blebs accompanies all types of injury in hepatocytes. Cell death is a rapid event initiated by rupture of a plasma membrane bleb, and it is coincident with the onset of irreversible injury. An increase of cytosolic free Ca2+ is not the stimulus for bleb formation or the final common pathway leading to cell death. A decrease of mitochondrial membrane potential precedes the loss of cell viability. Cytosolic pH falls by more than 1 pH unit during chemical hypoxia. This acidosis protects against the onset of cell death. Images FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 10. FIGURE 13. FIGURE 15. PMID:2190822

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

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

    SciTech Connect

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

    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}.

  7. Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging.

    PubMed

    Xu, Hao; Aylott, Jonathan W; Kopelman, Raoul

    2002-11-01

    Polyacrylamide-based, ratiometric, spherical, optical nanosensors, or polyacrylamide PEBBLEs (Probes Encapsulated By Biologically Localized Embedding), have been fabricated, aimed at real-time glucose imaging in intact biological systems, i.e. living cells. These nanosensors are prepared using a microemulsion polymerization process, and their average size is about 45 nm in diameter. The sensors incorporate glucose oxidase (GOx), an oxygen sensitive fluorescent indicator (Ru[dpp(SO3Na)2]3)Cl2, and an oxygen insensitive fluorescent dye, Oregon Green 488-dextran or Texas Red-dextran, as a reference for the purpose of ratiometric intensity measurements. The enzymatic oxidation of glucose to gluconic acid results in the local depletion of oxygen, which is measured by the oxygen sensitive ruthenium dye. The small size and inert matrix of these sensors allows them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. The PEBBLE matrix protects the enzyme and fluorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis. Conversely, the matrix also significantly reduces the toxicity of the indicator and reference dyes to the cells, so that a larger variety of dyes can be used in optimal fashion. Furthermore, the PEBBLE matrix enables the synergistic approach in which there is a steady state of local oxygen consumption, and this cannot be achieved by separately introducing free enzyme and dyes into a cell. The work presented here describes the production and characterization of glucose sensitive PEBBLEs, and their potential for intracellular glucose measurements. The sensor response is determined in terms of the linear range, ratiometric operation, response time, sensor stability, reversibility and immunity to interferences. PMID:12475037

  8. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy.

    PubMed

    Chen, Weili; Long, Kenneth D; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A; Cunningham, Brian T

    2014-11-21

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives. PMID:25265458

  9. Fluorescent Cy5 silica nanoparticles for cancer cell imaging

    NASA Astrophysics Data System (ADS)

    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.

  10. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy†

    PubMed Central

    Chen, Weili; Long, Kenneth D.; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A.; Cunningham, Brian T.

    2014-01-01

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives. PMID:25265458

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Performance comparison of different compact NIR fluorescent imaging systems with goggle display for intraoperative image-guidance

    NASA Astrophysics Data System (ADS)

    Gao, Shengkui; Mondal, Suman; Zhu, Nan; Liang, Rongguang; Achilefu, Samuel; Gruev, Viktor

    2015-03-01

    Near-infrared (NIR) fluorescent imaging system has been widely used for intraoperative image-guided application. In this paper, we present performance comparison from three compact NIR fluorescence imaging system prototypes with goggle display that we developed for intraoperative guidance: threshold detection based two camera system, feature matching based three cameras system and miniature beam-splitter single camera system. Their performance is evaluated according to sensitivity regarding different ICG concentrations, accuracy of image overlay between NIR-visible channels, compactness and practicability in intraoperative use. The comparison results show great potentials of using these NIR fluorescence imaging systems to improve user experience and surgical outcomes in intraoperative use.

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

    PubMed Central

    Li, David Day-Uei; Ameer-Beg, Simon; Arlt, Jochen; Tyndall, David; Walker, Richard; Matthews, Daniel R.; Visitkul, Viput; Richardson, Justin; Henderson, Robert K.

    2012-01-01

    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. PMID:22778606

  14. Development of a hyperspectral fluorescence lifetime imaging microscope and its application to tissue imaging

    NASA Astrophysics Data System (ADS)

    Owen, Dylan M.; Manning, Hugh B.; de Beule, Pieter; Talbot, Clifford; Requejo-Isidro, Jose; Dunsby, Chris; McGinty, James; Benninger, Richard K. P.; Elson, Dan S.; Munro, Ian; Galletly, Neil P.; Lever, M. Jon; Stamp, Gordon W.; Anand, Praveen; Neil, Mark A. A.; French, Paul M. W.

    2007-02-01

    We present the design, characterization and application of a novel, rapid, optically sectioned hyperspectral fluorescence lifetime imaging (FLIM) microscope. The system is based on a line scanning confocal configuration and uses a highspeed time-gated detector to extract lifetime information from many pixels in parallel. This allows the full spectraltemporal profiles of a fluorescence decay to be obtained from every pixel in an image. Line illumination and slit detection also gives the microscope a confocal optical sectioning ability. The system is applied to test samples and unstained biological tissue. In future, this microscope will be combined with recently-developed continuously electronically tunable, pulsed light sources based on tapered, micro-structured optical fibers. This will allow hyperspectral FLIM to be combined with the advantages of excitation spectroscopy to gain further insight into complex biological specimens including tissue and live cell imaging.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

    PubMed Central

    Discher, D E; Mohandas, N

    1996-01-01

    Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 PMID:8889146

  17. Live Imaging Fluorescent Proteins in Early Mouse Embryos

    PubMed Central

    Xenopoulos, Panagiotis; Nowotschin, Sonja; Hadjantonakis, Anna-Katerina

    2016-01-01

    Mouse embryonic development comprises highly dynamic and coordinated events that drive key cell lineage specification and morphogenetic events. These processes involve cellular behaviors including proliferation, migration, apoptosis, and differentiation, each of which is regulated both spatially and temporally. Live imaging of developing embryos provides an essential tool to investigate these coordinated processes in three-dimensional space over time. For this purpose, the development and application of genetically encoded fluorescent protein (FP) reporters has accelerated over the past decade allowing for the high-resolution visualization of developmental progression. Ongoing efforts are aimed at generating improved reporters, where spectrally distinct as well as novel FPs whose optical properties can be photomodulated, are exploited for live imaging of mouse embryos. Moreover, subcellular tags in combination with using FPs allow for the visualization of multiple subcellular characteristics, such as cell position and cell morphology, in living embryos. Here, we review recent advances in the application of FPs for live imaging in the early mouse embryo, as well as some of the methods used for ex utero embryo development that facilitate on-stage time-lapse specimen visualization. PMID:22341233

  18. Single aflatoxin contaminated corn kernel analysis with fluorescence hyperspectral image

    NASA Astrophysics Data System (ADS)

    Yao, Haibo; Hruska, Zuzana; Kincaid, Russell; Ononye, Ambrose; Brown, Robert L.; Cleveland, Thomas E.

    2010-04-01

    Aflatoxins are toxic secondary metabolites of the fungi Aspergillus flavus and Aspergillus parasiticus, among others. Aflatoxin contaminated corn is toxic to domestic animals when ingested in feed and is a known carcinogen associated with liver and lung cancer in humans. Consequently, aflatoxin levels in food and feed are regulated by the Food and Drug Administration (FDA) in the US, allowing 20 ppb (parts per billion) limits in food and 100 ppb in feed for interstate commerce. Currently, aflatoxin detection and quantification methods are based on analytical tests including thin-layer chromatography (TCL) and high performance liquid chromatography (HPLC). These analytical tests require the destruction of samples, and are costly and time consuming. Thus, the ability to detect aflatoxin in a rapid, nondestructive way is crucial to the grain industry, particularly to corn industry. Hyperspectral imaging technology offers a non-invasive approach toward screening for food safety inspection and quality control based on its spectral signature. The focus of this paper is to classify aflatoxin contaminated single corn kernels using fluorescence hyperspectral imagery. Field inoculated corn kernels were used in the study. Contaminated and control kernels under long wavelength ultraviolet excitation were imaged using a visible near-infrared (VNIR) hyperspectral camera. The imaged kernels were chemically analyzed to provide reference information for image analysis. This paper describes a procedure to process corn kernels located in different images for statistical training and classification. Two classification algorithms, Maximum Likelihood and Binary Encoding, were used to classify each corn kernel into "control" or "contaminated" through pixel classification. The Binary Encoding approach had a slightly better performance with accuracy equals to 87% or 88% when 20 ppb or 100 ppb was used as classification threshold, respectively.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Dual-modality, fluorescent, PLGA encapsulated bismuth nanoparticles for molecular and cellular fluorescence imaging and computed tomography

    NASA Astrophysics Data System (ADS)

    Swy, Eric R.; Schwartz-Duval, Aaron S.; Shuboni, Dorela D.; Latourette, Matthew T.; Mallet, Christiane L.; Parys, Maciej; Cormode, David P.; Shapiro, Erik M.

    2014-10-01

    Reports of molecular and cellular imaging using computed tomography (CT) are rapidly increasing. Many of these reports use gold nanoparticles. Bismuth has similar CT contrast properties to gold while being approximately 1000-fold less expensive. Herein we report the design, fabrication, characterization, and CT and fluorescence imaging properties of a novel, dual modality, fluorescent, polymer encapsulated bismuth nanoparticle construct for computed tomography and fluorescence imaging. We also report on cellular internalization and preliminary in vitro and in vivo toxicity effects of these constructs. 40 nm bismuth(0) nanocrystals were synthesized and encapsulated within 120 nm Poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles by oil-in-water emulsion methodologies. Coumarin-6 was co-encapsulated to impart fluorescence. High encapsulation efficiency was achieved ~70% bismuth w/w. Particles were shown to internalize within cells following incubation in culture. Bismuth nanocrystals and PLGA encapsulated bismuth nanoparticles exhibited >90% and >70% degradation, respectively, within 24 hours in acidic, lysosomal environment mimicking media and both remained nearly 100% stable in cytosolic/extracellular fluid mimicking media. μCT and clinical CT imaging was performed at multiple X-ray tube voltages to measure concentration dependent attenuation rates as well as to establish the ability to detect the nanoparticles in an ex vivo biological sample. Dual fluorescence and CT imaging is demonstrated as well. In vivo toxicity studies in rats revealed neither clinically apparent side effects nor major alterations in serum chemistry and hematology parameters. Calculations on minimal detection requirements for in vivo targeted imaging using these nanoparticles are presented. Indeed, our results indicate that these nanoparticles may serve as a platform for sensitive and specific targeted molecular CT and fluorescence imaging.Reports of molecular and cellular imaging using

  1. Inspection of fecal contamination on strawberries using fluorescence imaging

    NASA Astrophysics Data System (ADS)

    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.

  2. Measuring Agarwood Formation Ratio Quantitatively by Fluorescence Spectral Imaging Technique.

    PubMed

    Huang, Botao; Nguyen, Duykien; Liu, Tianyi; Jiang, Kaibin; Tan, Jinfen; Liu, Chunxin; Zhao, Jing; Huang, Shaowei

    2015-01-01

    Agarwood is a kind of important and precious traditional Chinese medicine. With the decreasing of natural agarwood, artificial cultivation has become more and more important in recent years. Quantifying the formation of agarwood is an essential work which could provide information for guiding cultivation and controlling quality. But people only can judge the amount of agarwood qualitatively by experience before. Fluorescence multispectral imaging method is presented to measure the agarwood quantitatively in this paper. A spectral cube from 450 nm to 800 nm was captured under the 365 nm excitation sources. The nonagarwood, agarwood, and rotten wood in the same sample were distinguished based on analyzing the spectral cube. Then the area ratio of agarwood to the whole sample was worked out, which is the quantitative information of agarwood area percentage. To our knowledge, this is the first time that the formation of agarwood was quantified accurately and nondestructively. PMID:26089935

  3. GPU acceleration of time-domain fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Wu, Gang; Nowotny, Thomas; Chen, Yu; Li, David Day-Uei

    2016-01-01

    Fluorescence lifetime imaging microscopy (FLIM) plays a significant role in biological sciences, chemistry, and medical research. We propose a graphic processing unit (GPU) based FLIM analysis tool suitable for high-speed, flexible time-domain FLIM applications. With a large number of parallel processors, GPUs can significantly speed up lifetime calculations compared to CPU-OpenMP (parallel computing with multiple CPU cores) based analysis. We demonstrate how to implement and optimize FLIM algorithms on GPUs for both iterative and noniterative FLIM analysis algorithms. The implemented algorithms have been tested on both synthesized and experimental FLIM data. The results show that at the same precision, the GPU analysis can be up to 24-fold faster than its CPU-OpenMP counterpart. This means that even for high-precision but time-consuming iterative FLIM algorithms, GPUs enable fast or even real-time analysis.

  4. Microbubble embedded with upconversion nanoparticles as a bimodal contrast agent for fluorescence and ultrasound imaging

    NASA Astrophysics Data System (ADS)

    Jin, Birui; Lin, Min; You, Minli; Zong, Yujin; Wan, Mingxi; Xu, Feng; Duan, Zhenfeng; Lu, Tianjian

    2015-08-01

    Bimodal imaging offers additional imaging signal thus finds wide spread application in clinical diagnostic imaging. Fluorescence/ultrasound bimodal imaging contrast agent using fluorescent dyes or quantum dots for fluorescence signal has emerged as a promising method, which however requires visible light or UV irradiation resulting in photobleaching, photoblinking, auto-fluorescence and limited tissue penetration depth. To surmount these problems, we developed a novel bimodal contrast agent using layer-by-layer assembly of upconversion nanoparticles onto the surface of microbubbles. The resulting microbubbles with average size of 2 μm provide enhanced ultrasound echo for ultrasound imaging and upconversion emission upon near infrared irradiation for fluorescence imaging. The developed bimodal contrast agent holds great potential to be applied in ultrasound target technique for targeted diseases diagnostics and therapy.

  5. Combined magnetic resonance, fluorescence, and histology imaging strategy in a human breast tumor xenograft model

    PubMed Central

    Jiang, Lu; Greenwood, Tiffany R.; Amstalden van Hove, Erika R.; Chughtai, Kamila; Raman, Venu; Winnard, Paul T.; Heeren, Ron; Artemov, Dmitri; Glunde, Kristine

    2014-01-01

    Applications of molecular imaging in cancer and other diseases frequently require combining in vivo imaging modalities, such as magnetic resonance and optical imaging, with ex vivo optical, fluorescence, histology, and immunohistochemical (IHC) imaging, to investigate and relate molecular and biological processes to imaging parameters within the same region of interest. We have developed a multimodal image reconstruction and fusion framework that accurately combines in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI), ex vivo brightfield and fluorescence microscopic imaging, and ex vivo histology imaging. Ex vivo brightfield microscopic imaging was used as an intermediate modality to facilitate the ultimate link between ex vivo histology and in vivo MRI/MRSI. Tissue sectioning necessary for optical and histology imaging required generation of a three-dimensional (3D) reconstruction module for 2D ex vivo optical and histology imaging data. We developed an external fiducial marker based 3D reconstruction method, which was able to fuse optical brightfield and fluorescence with histology imaging data. Registration of 3D tumor shape was pursued to combine in vivo MRI/MRSI and ex vivo optical brightfield and fluorescence imaging data. This registration strategy was applied to in vivo MRI/MRSI, ex vivo optical brightfield/fluorescence, as well as histology imaging data sets obtained from human breast tumor models. 3D human breast tumor data sets were successfully reconstructed and fused with this platform. PMID:22945331

  6. Imaging Multimodalities for Dissecting Alzheimer's Disease: Advanced Technologies of Positron Emission Tomography and Fluorescence Imaging

    PubMed Central

    Shimojo, Masafumi; Higuchi, Makoto; Suhara, Tetsuya; Sahara, Naruhiko

    2015-01-01

    The rapid progress in advanced imaging technologies has expanded our toolbox for monitoring a variety of biological aspects in living subjects including human. In vivo radiological imaging using small chemical tracers, such as with positron emission tomography, represents an especially vital breakthrough in the efforts to improve our understanding of the complicated cascade of neurodegenerative disorders including Alzheimer's disease (AD), and it has provided the most reliable visible biomarkers for enabling clinical diagnosis. At the same time, in combination with genetically modified animal model systems, the most recent innovation of fluorescence imaging is helping establish diverse applications in basic neuroscience research, from single-molecule analysis to animal behavior manipulation, suggesting the potential utility of fluorescence technology for dissecting the detailed molecular-based consequence of AD pathophysiology. In this review, our primary focus is on a current update of PET radiotracers and fluorescence indicators beneficial for understanding the AD cascade, and discussion of the utility and pitfalls of those imaging modalities for future translational research applications. We will also highlight current cutting-edge genetic approaches and discuss how to integrate individual technologies for further potential innovations. PMID:26733795

  7. Statistical image segmentation for the detection of skin lesion borders in UV fluorescence excitation

    NASA Astrophysics Data System (ADS)

    Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Franco, Walfre

    2016-04-01

    The skin contains several fluorescent molecules or fluorophores that serve as markers of structure, function and composition. UV fluorescence excitation photography is a simple and effective way to image specific intrinsic fluorophores, such as the one ascribed to tryptophan which emits at a wavelength of 345 nm upon excitation at 295 nm, and is a marker of cellular proliferation. Earlier, we built a clinical UV photography system to image cellular proliferation. In some samples, the naturally low intensity of the fluorescence can make it difficult to separate the fluorescence of cells in higher proliferation states from background fluorescence and other imaging artifacts -- like electronic noise. In this work, we describe a statistical image segmentation method to separate the fluorescence of interest. Statistical image segmentation is based on image averaging, background subtraction and pixel statistics. This method allows to better quantify the intensity and surface distributions of fluorescence, which in turn simplify the detection of borders. Using this method we delineated the borders of highly-proliferative skin conditions and diseases, in particular, allergic contact dermatitis, psoriatic lesions and basal cell carcinoma. Segmented images clearly define lesion borders. UV fluorescence excitation photography along with statistical image segmentation may serve as a quick and simple diagnostic tool for clinicians.

  8. Detection of fecal residue on poultry carcasses by laser induced fluorescence imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feasibility of fluorescence imaging technique for the detection of diluted fecal matters from various parts of the digestive tract, including colon, ceca, small intestine, and duodenum, on chicken carcasses was investigated. One of the challenges for using fluorescence imaging for inspection of agri...

  9. ASSESSING THE MATURITY OF APPLES BY INTEGRATING HYPERSPECTRAL REFLECTANCE AND FLUORESCENCE IMAGING TECHNIQUES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fluorescence and reflectance are two different forms of light interaction with the matter, and they can be complementary in measuring fruit maturity and quality. In this research, a hyperspectral imaging system was used to acquire both reflectance and fluorescence images from 'Golden Delicious' appl...

  10. MULTISPECTRAL LASER-INDUCED FLUORESCENCE IMAGING SYSTEM FOR LARGE BIOLOGICAL SAMPLES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Presented is a detailed description of a common aperture, multispectral laser-induced fluorescence imaging system developed to allow detection of fecal matter on agricultural products. With an expanded, 355 nm, Nd:YAG laser beam as the excitation source, fluorescence emission images in the blue, gr...

  11. Detection of fecal residue on poultry carcasses by laser induced fluorescence imaging techniques

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potential use of laser-induced fluorescence imaging techniques was investigated for the detection of diluted fecal matters from various parts of the digestive tract, including colon, ceca, small intestine, and duodenum, on poultry carcasses. One of the challenges for using fluorescence imaging f...

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

    PubMed

    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

  13. Automated platform for multiparameter stimulus response studies of metabolic activity at the single-cell level

    NASA Astrophysics Data System (ADS)

    Ashili, Shashanka P.; Kelbauskas, Laimonas; Houkal, Jeff; Smith, Dean; Tian, Yanqing; Youngbull, Cody; Zhu, Haixin; Anis, Yasser H.; Hupp, Michael; Lee, Kristen B.; Kumar, Ashok V.; Vela, Juan; Shabilla, Andrew; Johnson, Roger H.; Holl, Mark R.; Meldrum, Deirdre R.

    2011-02-01

    We have developed a fully automated platform for multiparameter characterization of physiological response of individual and small numbers of interacting cells. The platform allows for minimally invasive monitoring of cell phenotypes while administering a variety of physiological insults and stimuli by means of precisely controlled microfluidic subsystems. It features the capability to integrate a variety of sensitive intra- and extra-cellular fluorescent probes for monitoring minute intra- and extra-cellular physiological changes. The platform allows for performance of other, post- measurement analyses of individual cells such as transcriptomics. Our method is based on the measurement of extracellular metabolite concentrations in hermetically sealed ~200-pL microchambers, each containing a single cell or a small number of cells. The major components of the system are a) a confocal laser scan head to excite and detect with single photon sensitivity the emitted photons from sensors; b) a microfluidic cassette to confine and incubate individual cells, providing for dynamic application of external stimuli, and c) an integration module consisting of software and hardware for automated cassette manipulation, environmental control and data collection. The custom-built confocal scan head allows for fluorescence intensity detection with high sensitivity and spatial confinement of the excitation light to individual pixels of the sensor area, thus minimizing any phototoxic effects. The platform is designed to permit incorporation of multiple optical sensors for simultaneous detection of various metabolites of interest. The modular detector structure allows for several imaging modalities, including high resolution intracellular probe imaging and extracellular sensor readout. The integrated system allows for simulation of physiologically relevant microenvironmental stimuli and simultaneous measurement of the elicited phenotypes. We present details of system design, system

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

    NASA Astrophysics Data System (ADS)

    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.

  15. Cell nuclear features for classification from fluorescence images

    NASA Astrophysics Data System (ADS)

    Heynen, Susanne; Hunter, Edward; Price, Jeffrey H.

    2000-04-01

    In clinical cytology, nuclear features play an important role in cell and tissue classification. To increase efficiency and decrease subjectivity of cytological results, automation of the analytic process has been proposed and discussed by many authors. This automation can be achieved by estimating the probability of occurrence of a certain class given particular features of a microscope specimen. In this paper, feature sets that might be used as inputs for mathematical cytological classification algorithms are reviewer. The primary goal was to determine the important properties of these features sets, i.e., are there mathematically efficient features that provide a more or less compete description of the cell. Under what conditions will these feature then result in optimal classification of the cells using quantitative fluorescence staining. And how would these mathematical features relate to conventional features that a human observer understands. Example human observer features are size, shape, and chromaticity o the cell nucleus while example mathematical features are image moments. If the cell image can be completely reconstructed from the feature set, then it should be possible to derive the conventional features used by human observers from the mathematical feature set for presentation to clinicians. Finally, the suitability of different mathematical decision making algorithms like probabilistic reasoning, clustering or neural networks are also briefly evaluated in the context of a mathematically complete feature set.

  16. Multimodality imaging probe for positron emission tomography and fluorescence imaging studies.

    PubMed

    Pandey, Suresh K; Kaur, Jasmeet; Easwaramoorthy, Balu; Shah, Ankur; Coleman, Robert; Mukherjee, Jogeshwar

    2014-01-01

    Our goal is to develop multimodality imaging agents for use in cell tracking studies by positron emission tomography (PET) and optical imaging (OI). For this purpose, bovine serum albumin (BSA) was complexed with biotin (histologic studies), 5(6)-carboxyfluorescein, succinimidyl ester (FAM SE) (OI studies), and diethylenetriamine pentaacetic acid (DTPA) for chelating gallium 68 (PET studies). For synthesis of BSA-biotin-FAM-DTPA, BSA was coupled to (+)-biotin N-hydroxysuccinimide ester (biotin-NHSI). BSA-biotin was treated with DTPA-anhydride and biotin-BSA-DTPA was reacted with FAM. The biotin-BSA-DTPA-FAM was reacted with gallium chloride 3 to 5 mCi eluted from the generator using 0.1 N HCl and was passed through basic resin (AG 11 A8) and 150 μCi (100 μL, pH 7-8) was incubated with 0.1 mg of FAM conjugate (100 μL) at room temperature for 15 minutes to give 68Ga-BSA-biotin-DTPA-FAM. A shaved C57 black mouse was injected with FAM conjugate (50 μL) at one flank and FAM-68Ga (50 μL, 30 μCi) at the other. Immediately after injection, the mouse was placed in a fluorescence imaging system (Kodak In-Vivo F, Bruker Biospin Co., Woodbridge, CT) and imaged (λex: 465 nm, λem: 535 nm, time: 8 seconds, Xenon Light Source, Kodak). The same mouse was then placed under an Inveon microPET scanner (Siemens Medical Solutions, Knoxville, TN) injected (intravenously) with 25 μCi of 18F and after a half-hour (to allow sufficient bone uptake) was imaged for 30 minutes. Molecular weight determined using matrix-associated laser desorption ionization (MALDI) for the BSA sample was 66,485 Da and for biotin-BSA was 67,116 Da, indicating two biotin moieties per BSA molecule; for biotin-BSA-DTPA was 81,584 Da, indicating an average of 30 DTPA moieties per BSA molecule; and for FAM conjugate was 82,383 Da, indicating an average of 1.7 fluorescent moieties per BSA molecule. Fluorescence imaging clearly showed localization of FAM conjugate and FAM-68Ga at respective flanks of the mouse

  17. Fluorescence-lifetime molecular imaging can detect invisible peritoneal ovarian tumors in bloody ascites

    PubMed Central

    Nakajima, Takahito; Sano, Kohei; Sato, Kazuhide; Watanabe, Rira; Harada, Toshiko; Hanaoka, Hirofumi; Choyke, Peter L; Kobayashi, Hisataka

    2014-01-01

    Blood contamination, such as bloody ascites or hemorrhages during surgery, is a potential hazard for clinical application of fluorescence imaging. In order to overcome this problem, we investigate if fluorescence-lifetime imaging helps to overcome this problem. Samples were prepared at concentrations ranging 0.3–2.4 μm and mixed with 0–10% of blood. Fluorescence intensities and lifetimes of samples were measured using a time-domain fluorescence imager. Ovarian cancer SHIN3 cells overexpressing the D-galactose receptor were injected into the peritoneal cavity 2.5 weeks before the experiments. Galactosyl serum albumin-rhodamine green (GSA-RhodG), which bound to the D-galactose receptor and was internalized thereafter, was administered intraperitoneally to peritoneal ovarian cancer-bearing mice with various degrees of bloody ascites. In vitro study showed a linear correlation between fluorescence intensity and probe concentration (r2 > 0.99), whereas the fluorescence lifetime was consistent (range, 3.33 ± 0.15–3.75 ± 0.04 ns). By adding 10% of blood to samples, fluorescence intensities decreased to <1%, while fluorescence lifetimes were consistent. In vivo fluorescence lifetime of GSA-RhodG stained tumors was longer than the autofluorescence lifetime (threshold, 2.87 ns). Tumor lesions under hemorrhagic peritonitis were not depicted using fluorescence intensity imaging; however, fluorescence-lifetime imaging clearly detected tumor lesions by prolonged lifetimes. In conclusion, fluorescence-lifetime imaging with GSA-RhodG depicted ovarian cancer lesions, which were invisible in intensity images, in hemorrhagic ascites. PMID:24479901

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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 < 3 mm depth. Unfortunately, patient 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.

  20. Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

    NASA Technical Reports Server (NTRS)

    Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

  1. TCSPC based approaches for multiparameter detection in living cells

    NASA Astrophysics Data System (ADS)

    Jahn, Karolina; Buschmann, Volker; Koberling, Felix; Hille, Carsten

    2014-03-01

    In living cells a manifold of processes take place simultaneously. This implies a precise regulation of intracellular ion homeostasis. In order to understand their spatio-temporal pattern comprehensively, the development of multiplexing concepts is essential. Due to the multidimensional characteristics of fluorescence dyes (absorption and emission spectra, decay time, anisotropy), the highly sensitive and non-invasive fluorescence microscopy is a versatile tool for realising multiplexing concepts. A prerequisite are analyte-specific fluorescence dyes with low cross-sensitivity to other dyes and analytes, respectively. Here, two approaches for multiparameter detection in living cells are presented. Insect salivary glands are well characterised secretory active tissues which were used as model systems to evaluate multiplexing concepts. Salivary glands secrete a KCl-rich or NaCl-rich fluid upon stimulation which is mainly regulated by intracellular Ca2+ as second messenger. Thus, pairwise detection of intracellular Na+, Cl- and Ca2+ with the fluorescent dyes ANG2, MQAE and ACR were tested. Therefore, the dyes were excited simultaneously (2-photon excitation) and their corresponding fluorescence decay times were recorded within two spectral ranges using time-correlated singlephoton counting (TCSPC). A second approach presented here is based on a new TCSPC-platform covering decay time detection from picoseconds to milliseconds. Thereby, nanosecond decaying cellular fluorescence and microsecond decaying phosphorescence of Ruthenium-complexes, which is quenched by oxygen, were recorded simultaneously. In both cases changes in luminescence decay times can be linked to changes in analyte concentrations. In consequence of simultaneous excitation as well as detection, it is possible to get a deeper insight into spatio-temporal pattern in living tissues.

  2. Compact whole-body fluorescent imaging of nude mice bearing EGFP expressing tumor

    NASA Astrophysics Data System (ADS)

    Chen, Yanping; Xiong, Tao; Chu, Jun; Yu, Li; Zeng, Shaoqun; Luo, Qingming

    2005-01-01

    Issue of tumor has been a hotspot of current medicine. It is important for tumor research to detect tumors bearing in animal models easily, fast, repetitively and noninvasivly. Many researchers have paid their increasing interests on the detecting. Some contrast agents, such as green fluorescent protein (GFP) and Discosoma red fluorescent protein (Dsred) were applied to enhance image quality. Three main kinds of imaging scheme were adopted to visualize fluorescent protein expressing tumors in vivo. These schemes based on fluorescence stereo microscope, cooled charge-coupled-device (CCD) or camera as imaging set, and laser or mercury lamp as excitation light source. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes. The authors set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a Kodak digital camera (model DC290), fluorescence filters(B and G2;HB Optical, Shenyang, Liaoning, P.R. China) and a mercury 50-W lamp power supply (U-LH50HG;Olympus Optical, Japan) as excitation light source. The EGFP was excited directly by mercury lamp with D455/70 nm band-pass filter and fluorescence was recorded by digital camera with 520nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For half a year our experiments suggested the imaging scheme was feasible. Whole-body fluorescence optical imaging for fluorescent expressing tumors in nude mouse is an ideal tool for antitumor, antimetastatic, and antiangiogenesis drug screening.

  3. Time-resolved imaging system for fluorescence-guided surgery with lifetime imaging capability

    NASA Astrophysics Data System (ADS)

    Powolny, F.; Homicsko, K.; Sinisi, R.; Bruschini, Claudio E.; Grigoriev, E.; Homulle, H.; Prior, John O.; Hanahan, D.; Dubikovskaya, E.; Charbon, E.

    2014-05-01

    We present a single-photon camera for fluorescence imaging, with a time resolution better than 100ps, capable of providing both intensity and lifetime images. the camera was fabricated in standard CMOS technology. With this FluoCam we show the possibility to study sub-nanosecond fluorescence mechanisms. The FluoCam was used to characterize a near-infrared probe, indocyanine green, conjugated with multimeric cyclic pentapeptide (cRGD). The fluorescent probe-conjugated was used to target and mark tumors with better specificity, in particular aiming at targeting the integrins αvβ3 and αvβ5. As a first step towards clinical studies, preliminary results obtained in-vivo are presented. The first envisioned clinical application would be image-guided surgical oncology to help the surgeon to remove tumor tissue by a better discrimination from normal tissues and also to improve the detection of metastatic lymph nodes. A further application could be the in-vivo determination of the αvβ3 and αvβ5 targets to select patients for therapy with RGD chemotherapy conjugates.

  4. Image-based separation of reflective and fluorescent components using illumination variant and invariant color.

    PubMed

    Zhang, Cherry; Sato, Imari

    2013-12-01

    Traditionally, researchers tend to exclude fluorescence from color appearance algorithms in computer vision and image processing because of its complexity. In reality, fluorescence is a very common phenomenon observed in many objects, from gems and corals, to different kinds of writing paper, and to our clothes. In this paper, we provide detailed theories of fluorescence phenomenon. In particular, we show that the color appearance of fluorescence is unaffected by illumination in which it differs from ordinary reflectance. Moreover, we show that the color appearance of objects with reflective and fluorescent components can be represented as a linear combination of the two components. A linear model allows us to separate the two components using images taken under unknown illuminants using independent component analysis (ICA). The effectiveness of the proposed method is demonstrated using digital images of various fluorescent objects. PMID:24136427

  5. Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

    PubMed Central

    Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

    2016-01-01

    We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

  6. Endoscopic detection of early malignancies in the upper gastrointestinal tract using laser-induced fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Sukowski, Uwe; Ebert, Bernd; Ortner, Marianne; Zumbusch, Katharina; Mueller, Karsten; Fleige, Barbara; Lochs, Herbert; Rinneberg, Herbert H.

    2001-01-01

    Fluorescence images were recorded simultaneously with white light images to detect dyspasia or early malignancies during regular endoscopy of the upper gastrointestinal tract, after topical administration of 5-aminolaevulinic acid. Biopsies were taken at locations where fluorescence intensity were high compared with the mean fluorescence intensity of the image. Prompt and delayed fluorescence spectra of biopsies were subsequently recorded ex vivo, and normalized fluorescence intensities of Protoporphyrin IX derived from these spectra were compared with routine histology. In contrast to routine endoscopy, one early carcinoma and one signet-ring carcinoma were found in the stomach, and malignancies in a duodenal polyp. In addition, intestinal metaplasia could be visualized in the stomach of two patients, which had not been detected in biopsies taken prior to fluorescence endoscopy.

  7. Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection.

    PubMed

    Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

    2016-06-01

    We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

  8. Singlet oxygen phosphorescence lifetime imaging based on a fluorescence lifetime imaging microscope.

    PubMed

    Tian, Wenming; Deng, Liezheng; Jin, Shengye; Yang, Heping; Cui, Rongrong; Zhang, Qing; Shi, Wenbo; Zhang, Chunlei; Yuan, Xiaolin; Sha, Guohe

    2015-04-01

    The feasibility of singlet oxygen phosphorescence (SOP) lifetime imaging microscope was studied on a modified fluorescence lifetime imaging microscope (FLIM). SOP results from the infrared radiative transition of O2(a(1)Δg → X(3)Σg(-)) and O2(a(1)Δg) was produced in a C60 powder sample via photosensitization process. To capture the very weak SOP signal, a dichroic mirror was placed between the objective and tube lens of the FLIM and used to divide the luminescence returning from the sample into two beams: the reflected SOP beam and the transmitted photoluminescence of C60 (C60-PL) beam. The C60-PL beam entered the scanner of the FLIM and followed the normal optical path of the FLIM, while the SOP steered clear of the scanner and directly entered a finely designed SOP detection channel. Confocal C60-PL images and nonconfocal SOP images were then simultaneously obtained by using laser-scanning mode. Experimental results show that (1) under laser-scanning mode, the obstacle to confocal SOP imaging is the infrared-incompatible scanner, which can be solved by using an infrared-compatible scanner. Confocal SOP imaging is also expected to be realized under stage-scanning mode when the laser beam is parked and meanwhile a pinhole is added into the SOP detection channel. (2) A great challenge to SOP imaging is its extraordinarily long imaging time, and selecting only a few interesting points from fluorescence images to measure their SOP time-dependent traces may be a correct compromise. PMID:25781060

  9. Fluorescence imaging of experimental rheumatoid arthritis in vivo using a fast flying-spot scanner

    NASA Astrophysics Data System (ADS)

    Berger, J.; Voigt, J.; Seifert, F.; Ebert, B.; Macdonald, R.; Gemeinhardt, I.; Gemeinhardt, O.; Schnorr, J.; Taupitz, M.; Vater, A.; Vollmer, S.; Licha, K.; Schirner, M.

    2007-07-01

    We have developed a flying-spot scanner for fluorescence imaging of rheumatoid arthritis in the near infrared (NIR) spectral range following intravenous administration of contrast agents. The new imaging system has been characterized with respect to linearity, dynamic range and spatial resolution with the help of fluorescent phantoms. In vivo experiments were performed on an animal model of rheumatoid arthritis. Finally, NIR-fluorescence images of early stages of joint inflammation have been compared with findings from contrast enhanced MR imaging and histology.

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

    SciTech Connect

    Berg, W.; Ko, K.

    1992-12-01

    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.

  11. Cryo-imaging of fluorescently labeled single cells in a mouse

    NASA Astrophysics Data System (ADS)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

    2009-02-01

    We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

    PubMed

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

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    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

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

    SciTech Connect

    Sokolov, Vladimir V; Filonenko, E V; Telegina, L V; Boulgakova, N N; Smirnov, V V

    2002-11-30

    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)

  18. Noninvasive imaging in vivo with fluorescent proteins from centimeters to micrometers

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Jiang, Ping; Al-Zaid, Manal; Hoffman, Robert M.

    2008-02-01

    Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many applications in small animals. Our laboratory pioneered in vivo imaging with fluorescent proteins (1) including noninvasive whole-body imaging (2). Whole-body imaging with fluorescent proteins depends in large part on the brightness of the protein. Brighter, red-shifted proteins can make whole-body imaging more sensitive due to reduced absorption by tissues and less scatter. Non-invasive imaging with fluorescent proteins has been shown to be able to quantitatively track tumor growth and metastasis, gene expression, angiogenesis, and bacterial infection (3) even at subcellular resolution depending on the position of the cells in the animal. Interference by skin autofluorescence is kept to a minimum with the use of proper filters. To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of green fluorescent protein (GFP)-expressing mice transplanted with dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Various in vivo phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow as well as drug response. This imageable technology should lead to many new insights of in vivo cancer cell biology.

  19. Multispectral laser-induced fluorescence imaging system for large biological samples

    NASA Astrophysics Data System (ADS)

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

    2003-07-01

    A laser-induced fluorescence imaging system developed to capture multispectral fluorescence emission images simultaneously from a relatively large target object is described. With an expanded, 355-nm Nd:YAG laser as the excitation source, the system captures fluorescence emission images in the blue, green, red, and far-red regions of the spectrum centered at 450, 550, 678, and 730 nm, respectively, from a 30-cm-diameter target area in ambient light. Images of apples and of pork meat artificially contaminated with diluted animal feces have demonstrated the versatility of fluorescence imaging techniques for potential applications in food safety inspection. Regions of contamination, including sites that were not readily visible to the human eye, could easily be identified from the images.

  20. Whole-body Fluorescent Optical Imaging Based on Power Light Emitting Diode.

    PubMed

    Chen, Yanping; Xiong, Tao; Yu, Li; Zeng, Shaoqun; Luo, Qingming

    2005-01-01

    With complex configuration, the general whole-body fluorescence optical imaging system is power-consuming for it is mainly composed of laser or mercury lamp, filter and fiber-optic cable. In this paper we aimed at setting up a compact imaging system based on power light emitting diode (LED). We first discussed fluorescence excitation efficiency of mercury lamp and LED. Then we developed a compact prototype whole-body fluorescence optical imaging system based on power LED. With the prototype, we monitored the dynamic course of green fluorescence protein (GFP) expressing tumors in the same intact nude mice. We also recorded the temporal behavior of the infectious process of GFP-expressing bacteria from outside intact infected animals. This study puts forward a platform for monitoring tumor growth. The experiment reveals that it is doable to substitute power LED for mercury lamp for whole-body fluorescence optical imaging. PMID:17282471

  1. Status of the fluorescent screens and image processing for the APS linac

    SciTech Connect

    Berg, W.; Ko, K.

    1993-11-01

    Ten fluorescent screens and cameras determine the relative position and image profile of the beam in both the electron and positron linacs at the Advanced Photon Source (APS). The timing techniques used to capture the beam image allow direct synchronization to the electron gun trigger to minimize timing uncertainties. This paper discusses the design and status of the APS linac fluorescent screen assemblies and imaging system.

  2. Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

    PubMed

    Mondal, Samsuzzoha; Rakshit, Ananya; Pal, Suranjana; Datta, Ankona

    2016-07-15

    Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling. PMID:27082310

  3. Comparison of iodine K-edge subtraction and fluorescence subtraction imaging in an animal system

    NASA Astrophysics Data System (ADS)

    Zhang, H.; Zhu, Y.; Bewer, B.; Zhang, L.; Korbas, M.; Pickering, I. J.; George, G. N.; Gupta, M.; Chapman, D.

    2008-09-01

    K-Edge Subtraction (KES) utilizes the discontinuity in the X-ray absorption across the absorption edge of the selected contrast element and creates an image of the projected density of the contrast element from two images acquired just above and below the K-edge of the contrast element. KES has proved to be powerful in coronary angiography, micro-angiography, bronchography, and lymphatic imaging. X-ray fluorescence imaging is a successful technique for the detection of dilute quantities of elements in specimens. However, its application at high X-ray energies (e.g. at the iodine K-edge) is complicated by significant Compton background, which may enter the energy window set for the contrast material's fluorescent X-rays. Inspired by KES, Fluorescence Subtraction Imaging (FSI) is a technique for high-energy (>20 keV) fluorescence imaging using two different incident beam energies just above and below the absorption edge of a contrast element (e.g. iodine). The below-edge image can be assumed as a "background" image, which includes Compton scatter and fluorescence from other elements. The above-edge image will contain nearly identical spectral content as the below-edge image but will contain the additional fluorescence of the contrast element. This imaging method is especially promising with thick objects with dilute contrast materials, significant Compton background, and/or competing fluorescence lines from other materials. A quality factor is developed to facilitate the comparison. The theoretical value of the quality factor sets the upper limit that an imaging method can achieve when the noise is Poisson limited. The measured value of this factor makes two or more imaging methods comparable. Using the Hard X-ray Micro-Analysis (HXMA) beamline at the Canadian Light Source (CLS), the techniques of FSI and KES were critically compared, with reference to radiation dose, image acquisition time, resolution, signal-to-noise ratios, and quality factor.

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

    PubMed

    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

  5. A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

    PubMed Central

    Poland, Simon P.; Krstajić, Nikola; Monypenny, James; Coelho, Simao; Tyndall, David; Walker, Richard J.; Devauges, Viviane; Richardson, Justin; Dutton, Neale; Barber, Paul; Li, David Day-Uei; Suhling, Klaus; Ng, Tony; Henderson, Robert K.; Ameer-Beg, Simon M.

    2015-01-01

    We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. PMID:25780724

  6. Photoactivation and Imaging of Optical Highlighter Fluorescent Proteins

    PubMed Central

    Patterson, George H.

    2011-01-01

    A major advance in the microscopic study of cells and tissues is the introduction of photoactivatable fluorescent proteins which can specifically mark proteins of interest within a living cell. Fluorescent proteins are now available that allow a pool of molecules to be “turned on” by photoactivation. This unit discusses technical aspects for the general use of photoactivatable fluorescent proteins and introduces some specific applications in the concluding remarks. PMID:21732309

  7. Diffuse-dynamic multiparameter diffractometry: A review

    NASA Astrophysics Data System (ADS)

    Molodkin, V. B.; Shpak, A. P.; Kovalchuk, M. V.; Nosik, V. L.; Machulin, V. F.

    2010-12-01

    The results reported at the Conference on Application of X-Rays, Synchrotron Radiation, Neutrons, and Electrons in Nano-, Bio-, Information-, and Cognitive Technologies (RSNE-NBIC 2009) are briefly reviewed. This review is based on a cycle of studies [1-6] where a new method for studying the structure of real crystals—diffuse-dynamic multiparameter diffractometry (DDMD)—was proposed and substantiated.

  8. Two-photon excited fluorescence microendoscopic imaging using a GRIN lens

    NASA Astrophysics Data System (ADS)

    Yan, Wei; Peng, Xiao; Lin, Danying; Wang, Qi; Gao, Jian; Zhou, Jie; Ye, Tong; Qu, Junle; Niu, Hanben

    2015-03-01

    With the rapid development of life sciences, there is an increasing demand for intravital fluorescence imaging of small animals. However, large dimensions and limited working distances of objective lenses in traditional fluorescence microscopes have limited the imaging applications mostly to superficial tissues. To overcome this disadvantage, researchers have developed the graded-index (GRIN) probes with small diameters for imaging internal organs of small animals in a minimally invasive fashion. Here, we present the development of a fluorescence endoscopic imaging system based on a GRIN lens using two-photon excitation. Experimental results showed that this system could perform dynamic fluorescence microendoscopic imaging and monitor the blood flow in anesthetized living mice using two-photon excitation.

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

    PubMed Central

    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

  10. Laser line-scan fluorescence and multispectral imaging of coral reef environments

    NASA Astrophysics Data System (ADS)

    Strand, Michael P.; Coles, Bryan W.; Nevis, Andrew J.; Regan, Richard F.

    1997-02-01

    During the summer of 1996 a series of field trials were conducted in the Florida Keys and Bahama Islands to evaluate the ability of a unique laser line scan system to measure and map the fluorescent characteristics of coral reef environments. Typical fluorescence maps that were obtained are presented and compared with monochrome and RGB color images of the same reefs. The monochrome images were obtained with the laser line scan system simuftaneously with the fluorescent maps. The RGB images, which were also obtained with the laser line scan system, were recorded in the same location on a subsequent thai.

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

    PubMed

    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

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

    PubMed Central

    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

  13. Flexible imaging payload for real-time fluorescent biological imaging in parabolic, suborbital and space analog environments

    NASA Astrophysics Data System (ADS)

    Bamsey, Matthew T.; Paul, Anna-Lisa; Graham, Thomas; Ferl, Robert J.

    2014-10-01

    Fluorescent imaging offers the ability to monitor biological functions, in this case biological responses to space-related environments. For plants, fluorescent imaging can include general health indicators such as chlorophyll fluorescence as well as specific metabolic indicators such as engineered fluorescent reporters. This paper describes the Flex Imager a fluorescent imaging payload designed for Middeck Locker deployment and now tested on multiple flight and flight-related platforms. The Flex Imager and associated payload elements have been developed with a focus on 'flexibility' allowing for multiple imaging modalities and change-out of individual imaging or control components in the field. The imaging platform is contained within the standard Middeck Locker spaceflight form factor, with components affixed to a baseplate that permits easy rearrangement and fine adjustment of components. The Flex Imager utilizes standard software packages to simplify operation, operator training, and evaluation by flight provider flight test engineers, or by researchers processing the raw data. Images are obtained using a commercial cooled CCD image sensor, with light-emitting diodes for excitation and a suite of filters that allow biological samples to be imaged over wavelength bands of interest. Although baselined for the monitoring of green fluorescent protein and chlorophyll fluorescence from Arabidopsis samples, the Flex Imager payload permits imaging of any biological sample contained within a standard 10 cm by 10 cm square Petri plate. A sample holder was developed to secure sample plates under different flight profiles while permitting sample change-out should crewed operations be possible. In addition to crew-directed imaging, autonomous or telemetric operation of the payload is also a viable operational mode. An infrared camera has also been integrated into the Flex Imager payload to allow concurrent fluorescent and thermal imaging of samples. The Flex Imager has been

  14. Evaluation of Chemical Fluorescent Dyes as a Protein Conjugation Partner for Live Cell Imaging

    PubMed Central

    Hayashi-Takanaka, Yoko; Stasevich, Timothy J.; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2014-01-01

    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). PMID:25184362

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

    PubMed Central

    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) results. Methods An OI module was developed for a preclinical SPECT system (U-SPECT, MILabs, Utrecht, the Netherlands). The applicability of the module for bioluminescence and fluorescence imaging was evaluated in both a phantom and in an in vivo setting using mice implanted with a 4 T1-luc + tumor. A combination of a fluorescent dye and radioactive moiety was used to directly relate the optical images of the module to the SPECT findings. Bioluminescence imaging (BLI) was compared to the localization of the fluorescence signal in the tumors. Results Both the phantom and in vivo mouse studies showed that superficial fluorescence signals could be imaged accurately. The SPECT and bioluminescence images could be used to place the fluorescence findings in perspective, e.g. by showing tracer accumulation in non-target organs such as the liver and kidneys (SPECT) and giving a semi-quantitative read-out for tumor spread (bioluminescence). Conclusions We developed a fully integrated multimodal platform that provides complementary registered imaging of bioluminescent, fluorescent, and SPECT signatures in a single scanning session with a single dose of anesthesia. In our view, integration of these modalities helps to improve data interpretation of optical findings in relation to radionuclide images. PMID:25386389

  16. Dual-Modal Nanoprobes for Imaging of Mesenchymal Stem Cell Transplant by MRI and Fluorescence Imaging

    PubMed Central

    Hong, Kyung Ah; Lin, Shunmei; Lee, Yuwon; Cha, Jinmyung; Lee, Jin-Kyu; Hong, Cheol Pyo; Han, Bong Soo; Jung, Sung Il; Kim, Seung Hyup; Yoon, Kang Sup

    2009-01-01

    Objective To determine the feasibility of labeling human mesenchymal stem cells (hMSCs) with bifunctional nanoparticles and assessing their potential as imaging probes in the monitoring of hMSC transplantation. Materials and Methods The T1 and T2 relaxivities of the nanoparticles (MNP@SiO2[RITC]-PEG) were measured at 1.5T and 3T magnetic resonance scanner. Using hMSCs and the nanoparticles, labeling efficiency, toxicity, and proliferation were assessed. Confocal laser scanning microscopy and transmission electron microscopy were used to specify the intracellular localization of the endocytosed iron nanoparticles. We also observed in vitro and in vivo visualization of the labeled hMSCs with a 3T MR scanner and optical imaging. Results MNP@SiO2(RITC)-PEG showed both superparamagnetic and fluorescent properties. The r1 and r2 relaxivity values of the MNP@SiO2(RITC)-PEG were 0.33 and 398 mM-1 s-1 at 1.5T, respectively, and 0.29 and 453 mM-1 s-1 at 3T, respectively. The effective internalization of MNP@SiO2(RITC)-PEG into hMSCs was observed by confocal laser scanning fluorescence microscopy. The transmission electron microscopy images showed that MNP@SiO2(RITC)-PEG was internalized into the cells and mainly resided in the cytoplasm. The viability and proliferation of MNP@SiO2(RITC)-PEG-labeled hMSCs were not significantly different from the control cells. MNP@SiO2(RITC)-PEG-labeled hMSCs were observed in vitro and in vivo with optical and MR imaging. Conclusion MNP@SiO2(RITC)-PEG can be a useful contrast agent for stem cell imaging, which is suitable for a bimodal detection by MRI and optical imaging. PMID:19885318

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

  20. Wavefront sensorless approaches to adaptive optics for in vivo fluorescence imaging of mouse retina

    NASA Astrophysics Data System (ADS)

    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.

  1. Image navigation as a means to expand the boundaries of fluorescence-guided surgery.

    PubMed

    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

    2012-05-21

    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. PMID:22547491

  2. Image navigation as a means to expand the boundaries of fluorescence-guided surgery

    NASA Astrophysics Data System (ADS)

    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.

  3. Single-Step Assembly of Multimodal Imaging Nanocarriers: MRI and Long-Wavelength Fluorescence Imaging.

    PubMed

    Pinkerton, Nathalie M; Gindy, Marian E; Calero-DdelC, Victoria L; Wolfson, Theodore; Pagels, Robert F; Adler, Derek; Gao, Dayuan; Li, Shike; Wang, Ruobing; Zevon, Margot; Yao, Nan; Pacheco, Carlos; Therien, Michael J; Rinaldi, Carlos; Sinko, Patrick J; Prud'homme, Robert K

    2015-06-24

    Magnetic resonance imaging (MRI)- and near-infrared (NIR)-active, multimodal composite nanocarriers (CNCs) are prepared using a simple one-step process, flash nanoprecipitation (FNP). The FNP process allows for the independent control of the hydrodynamic diameter, co-core excipient and NIR dye loading, and iron oxide-based nanocrystal (IONC) content of the CNCs. In the controlled precipitation process, 10 nm IONCs are encapsulated into poly(ethylene glycol) (PEG) stabilized CNCs to make biocompatible T2 contrast agents. By adjusting the formulation, CNC size is tuned between 80 and 360 nm. Holding the CNC size constant at an intensity weighted average diameter of 99 ± 3 nm (PDI width 28 nm), the particle relaxivity varies linearly with encapsulated IONC content ranging from 66 to 533 × 10(-3) m(-1) s(-1) for CNCs formulated with 4-16 wt% IONC. To demonstrate the use of CNCs as in vivo MRI contrast agents, CNCs are surface functionalized with liver-targeting hydroxyl groups. The CNCs enable the detection of 0.8 mm(3) non-small cell lung cancer metastases in mice livers via MRI. Incorporating the hydrophobic, NIR dye tris-(porphyrinato)zinc(II) into CNCs enables complementary visualization with long-wavelength fluorescence at 800 nm. In vivo imaging demonstrates the ability of CNCs to act both as MRI and fluorescent imaging agents. PMID:25925128

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

    NASA Technical Reports Server (NTRS)

    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.

  5. INTEGRATION OF HYPERSPECTRAL REFLECTANCE AND LASER-INDUCED FLUORESCENCE IMAGING FOR ASSESSING APPLE MATURITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fluorescence and reflectance are two different forms of light interaction with matter, and they can be complementary in measuring fruit quality and condition. The objective of this research was to develop an integrated hyperspectral reflectance and fluorescence imaging system for measuring apple mat...

  6. Multispectral fluorescence imaging for detection of bovine feces on Romaine lettuce and baby spinach leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Integration of Hyperspectral Reflectance and Fluorescence Imaging for Assessing Apple Maturity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fluorescence and reflectance are two different forms of light interaction with matter, and they can be complementary in measuring fruit quality and condition. The objective of this research was to develop an integrated hyperspectral reflectance and fluorescence imaging technique for measuring apple ...

  8. Fluorescence spectroscopy as an aid to imaging latent fingermarks in the ultraviolet.

    PubMed

    Bramble, S K

    1996-11-01

    Two- and three-dimensional fluorescence spectroscopic data have been recorded from sebum-rich latent fingermarks on quartz and white card. The fingermark residue was found to fluoresce between 310 to 380 nm and have an excitation range between 260 to 300 nm. The data are used to describe the results observed when imaging the inherent ultraviolet photoluminescence of latent fingermarks. PMID:8914294

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

    NASA Astrophysics Data System (ADS)

    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. Sensitive and selective tumor imaging with novel and highly activatable fluorescence strategies

    NASA Astrophysics Data System (ADS)

    Urano, Yasuteru

    2008-02-01

    Nowadays, several tumor imaging modalities such as MRI, PET and fluorescence imaging techniques have been extensively investigated. One of the central problems associated with these conventional tumor-targeted imaging methods, however, is the fact that the signal contrast between tumor and surrounding tissues relies on the efficient targeting to the tumor and the rapid sequestration or excretion of unbound agent. Among these modalities, only fluorescence imaging technique has a significant feature, in that great signal activation could be achieved which potentially leads to the selective imaging of cancer with higher tumor-to-background ratio. In this symposium, I will present some examples of fluorescence cancer imaging based on highly activatable strategies with using precisely designed novel fluorescence probes. Recently, we developed highly sensitive fluorescence probes for β-galactosidase which is applicable for living cell system. By utilizing these probes, we could establish a novel and highly activatable strategy for sensitive and selective optical imaging of imbedded tumor in the peritoneum. We took a two step procedure in that a lectin is used to localize β-galactosidase to cancer cells as an activating enzyme, and subsequent administration of a highly-sensitive fluorescence probe for the enzyme have afforded remarkable fluorescence activation selectively in tumor mass. Since the tumor-targeted enzyme can catalyze numerous substrate turnovers, a great number of fluorescent molecules could be produced and hence the rapid and sensitive detection of tumor in vivo with high tumor-to-background ratio could be achieved. Moreover, the consequent close-up investigation using fluorescence microscopy revealed that cancer microfoci as small as 200 μm could be successfully visualized.

  11. Fluorescence tomography characterization for sub-surface imaging with protoporphyrin IX

    PubMed Central

    Kepshire, Dax; Davis, Scott C.; Dehghani, Hamid; Paulsen, Keith D.; Pogue, Brian W.

    2009-01-01

    Optical imaging of fluorescent objects embedded in a tissue simulating medium was characterized using non-contact based approaches to fluorescence remittance imaging (FRI) and sub-surface fluorescence diffuse optical tomography (FDOT). Using Protoporphyrin IX as a fluorescent agent, experiments were performed on tissue phantoms comprised of typical in-vivo tumor to normal tissue contrast ratios, ranging from 3.5:1 up to 10:1. It was found that tomographic imaging was able to recover interior inclusions with high contrast relative to the background; however, simple planar fluorescence imaging provided a superior contrast to noise ratio. Overall, FRI performed optimally when the object was located on or close to the surface and, perhaps most importantly, FDOT was able to recover specific depth information about the location of embedded regions. The results indicate that an optimal system for localizing embedded fluorescent regions should combine fluorescence reflectance imaging for high sensitivity and sub-surface tomography for depth detection, thereby allowing more accurate localization in all three directions within the tissue. PMID:18545571

  12. Determination of the modulation transfer function for a time-gated fluorescence imaging system.

    PubMed

    Gundy, Sarah; Van der Putten, Wil; Shearer, Andy; Buckton, Daniel; Ryder, Alan G

    2004-01-01

    The use of fluorescence for cancer detection is currently under investigation. Presently, steady-state fluorescence detection methods are in use, but have limitations due to poor contrast between the fluorescence of the tumor and background autofluorescence. Improved contrast can be obtained with time-resolved techniques because of the differing lifetimes between autofluorescence and exogenous photosensitizers that selectively accumulate within tumor tissue. An imaging system is constructed using a fast-gated (200-ps) charge-coupled device (CCD) camera and a pulsed 635-nm laser diode. To characterize the ability of the system to transfer object contrast to an image, the modulation transfer function (MTF) of the system is acquired by employing an extended knife-edge technique. A knife-edge target is assembled by drilling a rectangular well into a block of polymethyl methacrylate (PMMA). The imaging system records images of the photosensitizer, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS), within the well. AlPcTS was chosen to test the system because of its strong absorption of 635-nm, high fluorescence yield, and relatively long fluorescence lifetime (approximately 7.5 ns). The results show that the system is capable of resolving 10(-4) M AlPcTS fluorescence as small as 1 mm. The findings of this study contribute to the development of a time-gated imaging system using fluorescence lifetimes. PMID:15568941

  13. Cancer detection using NIR elastic light scattering and tissue fluorescence imaging

    SciTech Connect

    Demos, S G; Staggs, M; Radousky, H B; Gandour-Edwards, R; deVere White, R

    2000-12-04

    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.

  14. DETECTION OF BACTERIAL BIOFILM ON STAINLESS STEEL BY HYPERSPECTRAL FLUORESCENCE IMAGING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, hyperspectral fluorescence imaging techniques were investigated for detection of microbial biofilm on stainless steel plates typically used to manufacture food processing equipment. Stainless steel coupons were immersed in bacterium cultures consisting of nonpathogenic E. coli, Pseudo...

  15. Early detection of tumor masses by in vivo hematoporphyrin-mediated fluorescence imaging

    NASA Astrophysics Data System (ADS)

    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.

  16. Quantitative spatial frequency fluorescence imaging in the sub-diffusive domain for image-guided glioma resection

    PubMed Central

    Sibai, Mira; Veilleux, Israel; Elliott, Jonathan T.; Leblond, Frederic; Wilson, Brian C.

    2015-01-01

    Intraoperative 5- aminolevulinic acid induced-Protoporphyrin IX (PpIX) fluorescence guidance enables maximum safe resection of glioblastomas by providing surgeons with real-time tumor optical contrast. However, visual assessment of PpIX fluorescence is subjective and limited by the distorting effects of light attenuation and tissue autofluorescence. We have previously shown that non-invasive point measurements of absolute PpIX concentration identifies residual tumor that is otherwise non-detectable. Here, we extend this approach to wide-field quantitative fluorescence imaging by implementing spatial frequency domain imaging to recover tissue optical properties across the field-of-view in phantoms and ex vivo tissue. PMID:26713206

  17. Quantitative spatial frequency fluorescence imaging in the sub-diffusive domain for image-guided glioma resection.

    PubMed

    Sibai, Mira; Veilleux, Israel; Elliott, Jonathan T; Leblond, Frederic; Wilson, Brian C

    2015-12-01

    Intraoperative 5- aminolevulinic acid induced-Protoporphyrin IX (PpIX) fluorescence guidance enables maximum safe resection of glioblastomas by providing surgeons with real-time tumor optical contrast. However, visual assessment of PpIX fluorescence is subjective and limited by the distorting effects of light attenuation and tissue autofluorescence. We have previously shown that non-invasive point measurements of absolute PpIX concentration identifies residual tumor that is otherwise non-detectable. Here, we extend this approach to wide-field quantitative fluorescence imaging by implementing spatial frequency domain imaging to recover tissue optical properties across the field-of-view in phantoms and ex vivo tissue. PMID:26713206

  18. Time efficient methods for scanning a fluorescent membrane with a fluorescent microscopic imager for the quality assurance of food

    NASA Astrophysics Data System (ADS)

    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.

  19. Injectant mole-fraction imaging in compressible mixing flows using planar laser-induced iodine fluorescence

    NASA Technical Reports Server (NTRS)

    Hartfield, Roy J., Jr.; Abbitt, John D., III; Mcdaniel, James C.

    1989-01-01

    A technique is described for imaging the injectant mole-fraction distribution in nonreacting compressible mixing flow fields. Planar fluorescence from iodine, seeded into air, is induced by a broadband argon-ion laser and collected using an intensified charge-injection-device array camera. The technique eliminates the thermodynamic dependence of the iodine fluorescence in the compressible flow field by taking the ratio of two images collected with identical thermodynamic flow conditions but different iodine seeding conditions.

  20. Synthesis of a Targeted Biarsenical Cy3-Cy5 Affinity Probe for Superresolution Fluorescence Imaging

    SciTech Connect

    Fu, Na; Xiong, Yijia; Squier, Thomas C.

    2012-11-01

    Photoswitchable fluorescent probes capable of the targeted labeling of tagged proteins are of significant interest due to their ability to enable in situ imaging of protein complexes within native biomolecular assemblies. Here we describe the synthesis of a fluorescent probe (AsCy3Cy5), and demonstrate the targeted labeling and super-resolution imaging of a tagged protein within a supramolecular protein complex.

  1. Green Synthesis of Bifunctional Fluorescent Carbon Dots from Garlic for Cellular Imaging and Free Radical Scavenging.

    PubMed

    Zhao, Shaojing; Lan, Minhuan; Zhu, Xiaoyue; Xue, Hongtao; Ng, Tsz-Wai; Meng, Xiangmin; Lee, Chun-Sing; Wang, Pengfei; Zhang, Wenjun

    2015-08-12

    Nitrogen and sulfur codoped carbon dots (CDs) were prepared from garlic by a hydrothermal method. The as-prepared CDs possess good water dispersibility, strong blue fluorescence emission with a fluorescent quantum yield of 17.5%, and excellent photo and pH stabilities. It is also demonstrated that the fluorescence of CDs are resistant to the interference of metal ions, biomolecules, and high ionic strength environments. Combining with low cytotoxicity properties, CDs could be used as an excellent fluorescent probe for cellular multicolor imaging. Moreover, the CDs were also demonstrated to exhibit favorable radical scavenging activity. PMID:26193082

  2. X-ray fluorescence imaging with synchrotron radiation

    SciTech Connect

    Rivers, M.L.

    1987-01-01

    The micro-distribution of trace elements is of great interest in fields such as geochemistry, biology and material science. The synchrotron x-ray fluorescence microprobe provides a technique to quantitatively measure trace element compositions at individual points and to construct semiquantitative two dimensional maps of trace element compositions. This paper describes an x-ray fluorescence system used at the National Synchrotron Light Source.

  3. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

    NASA Astrophysics Data System (ADS)

    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.

  4. Fluorescent imaging of single nanoparticles and viruses on a smart phone.

    PubMed

    Wei, Qingshan; Qi, Hangfei; Luo, Wei; Tseng, Derek; Ki, So Jung; Wan, Zhe; Göröcs, Zoltán; Bentolila, Laurent A; Wu, Ting-Ting; Sun, Ren; Ozcan, Aydogan

    2013-10-22

    Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments. PMID:24016065

  5. Rapid, Noninvasive Screening for Perturbations of Metabolism and Plant Growth Using Chlorophyll Fluorescence Imaging1

    PubMed Central

    Barbagallo, Romina P.; Oxborough, Kevin; Pallett, Kenneth E.; Baker, Neil R.

    2003-01-01

    A rapid, noninvasive technique involving imaging of chlorophyll fluorescence parameters for detecting perturbations of leaf metabolism and growth in seedlings is described. Arabidopsis seedlings were grown in 96-well microtitre plates for 4 d and then treated with eight herbicides with differing modes of action to induce perturbations in a range of different metabolic processes. Imaging of chlorophyll fluorescence emissions from 96 seedlings growing on a microtitre plate enabled images of a number of fluorescence parameters to be rapidly and simultaneously produced for the plants in each well. Herbicideinduced perturbations in metabolism, even in metabolic reactions not directly associated with photosynthetic metabolism, were detected from the changes in the images of fluorescence parameters considerably before any visual effects on seedling growth were observed. Evaluations of seedling growth were made from measurements of the area of chlorophyll fluorescence emission in images of plants growing in the 96-well plates. Decreased seedling growth related directly to herbicideinduced changes in the imaged chlorophyll fluorescence parameters. The applicability of this rapid-screening technique for metabolic perturbations in monocotyledonous species was demonstrated by treating Agrostis tenuis seedlings with Imazapyr, an inhibitor of branched-chain amino acid synthesis. PMID:12805581

  6. Fluorescent Imaging of Single Nanoparticles and Viruses on a Smart Phone

    PubMed Central

    Wei, Qingshan; Qi, Hangfei; Luo, Wei; Tseng, Derek; Ki, So Jung; Wan, Zhe; Göröcs, Zoltán; Bentolila, Laurent A.; Wu, Ting-Ting; Sun, Ren; Ozcan, Aydogan

    2014-01-01

    Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments. PMID:24016065

  7. Wide field fluorescence imaging in narrow passageways using scanning fiber endoscope technology

    NASA Astrophysics Data System (ADS)

    Lee, Cameron M.; Chandler, John E.; Seibel, Eric J.

    2010-02-01

    An ultrathin scanning fiber endoscope (SFE) has been developed for high resolution imaging of regions in the body that are commonly inaccessible. The SFE produces 500 line color images at 30 Hz frame rate while maintaining a 1.2-1.7 mm outer diameter. The distal tip of the SFE houses a 9 mm rigid scan engine attached to a highly flexible tether (minimum bend radius < 8 mm) comprised of optical fibers and electrical wires within a protective sheath. Unlike other ultrathin technologies, the unique characteristics of this system have allowed the SFE to navigate narrow passages without sacrificing image quality. To date, the SFE has been used for in vivo imaging of the bile duct, esophagus and peripheral airways. In this study, the standard SFE operation was tailored to capture wide field fluorescence images and spectra. Green (523 nm) and blue (440 nm) lasers were used as illumination sources, while the white balance gain values were adjusted to accentuate red fluorescence signal. To demonstrate wide field fluorescence imaging of small lumens, the SFE was inserted into a phantom model of a human pancreatobiliary tract and navigated to a custom fluorescent target. Both wide field fluorescence and standard color images of the target were captured to demonstrate multimodal imaging.

  8. In-vivo validation of fluorescence lifetime imaging (FLIm) of coronary arteries in swine

    NASA Astrophysics Data System (ADS)

    Bec, Julien; Ma, Dinglong; Yankelevich, Diego R.; Gorpas, Dimitris S.; Ferrier, William T.; Southard, Jeffrey; Marcu, Laura

    2015-02-01

    We report a scanning imaging system that enables high speed multispectral fluorescence lifetime imaging (FLIm) of coronary arteries. This system combines a custom low profile (3 Fr) imaging catheter using a 200 μm core side viewing UV-grade silica fiber optic, an acquisition system able to measure fluorescence decays over four spectral bands at 20 kHz and a fast data analysis and display module. In vivo use of the system has been optimized, with particular emphasis on clearing blood from the optical pathway. A short acquisition time (5 seconds for a 20 mm long coronary segment) enabled data acquisition during a bolus saline solution injection through the 7 Fr catheter guide. The injection parameters were precisely controlled using a power injector and optimized to provide good image quality while limiting the bolus injection duration and volume (12 cc/s, 80 cc total volume). The ability of the system to acquire data in vivo was validated in healthy swine by imaging different sections of the left anterior descending (LAD) coronary. A stent coated with fluorescent markers was placed in the LAD and imaged, demonstrating the ability of the system to discriminate in vivo different fluorescent features and structures from the vessel background fluorescence using spectral and lifetime information. Intensity en face images over the four bands of the instrument were available within seconds whereas lifetime images were computed in 2 minutes, providing efficient feedback during the procedure. This successful demonstration of FLIm in coronaries enables future study of atherosclerotic cardiovascular diseases.

  9. Near-Infrared Fluorescence Imaging in Humans with Indocyanine Green: A Review and Update

    PubMed Central

    Marshall, Milton V.; Rasmussen, John C.; Tan, I-Chih; Aldrich, Melissa B.; Adams, Kristen E.; Wang, Xuejuan; Fife, Caroline E.; Maus, Erik A.; Smith, Latisha A.; Sevick-Muraca, Eva M.

    2010-01-01

    Near-infrared (NIR) fluorescence imaging clinical studies have been reported in the literature with six different devices that employ various doses of indocyanine green (ICG) as a non-specific contrast agent. To date, clinical applications range from (i) angiography, intraoperative assessment of vessel patency, and tumor/metastasis delineation following intravenous administration of ICG, and (ii) imaging lymphatic architecture and function following subcutaneous and intradermal ICG administration. In the latter case, NIR fluorescence imaging may enable new discoveries associated with lymphatic function due to (i) a unique niche that is not met by any other conventional imaging technology and (ii) its exquisite sensitivity enabling high spatial and temporal resolution. Herein, we (i) review the basics of clinical NIR fluorescence imaging, (ii) survey the literature on clinical application of investigational devices using ICG fluorescent contrast, (iii) provide an update of non-invasive dynamic lymphatic imaging conducted with our FDPM device, and finally, (iv) comment on the future NIR fluorescence imaging for non-invasive and intraoperative use given recent demonstrations showing capabilities for imaging following microdose administration of contrast agent. PMID:22924087

  10. Biosynthesis of fluorescent gold nanoclusters for in vitro and in vivo tumor imaging

    NASA Astrophysics Data System (ADS)

    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.

  11. Pseudo-color enhanced x-ray fluorescence imaging of the Archimedes Palimpsest

    NASA Astrophysics Data System (ADS)

    Bergmann, Uwe; Knox, Keith T.

    2009-01-01

    A combination of x-ray fluorescence and image processing has been shown to recover text characters written in iron gall ink on parchment, even when obscured by gold paint. Several leaves of the Archimedes Palimpsest were imaged using rapid-scan, x-ray fluorescence imaging performed at the Stanford Synchrotron Radiation Lightsource of the SLAC National Accelerator Laboratory. A simple linear show-through model is shown to successfully separate different layers of text in the x-ray images, making the text easier to read by the scholars.

  12. Aminopeptidase N/CD13 targeting fluorescent probes: synthesis and application to tumor cell imaging.

    PubMed

    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

  13. Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo

    NASA Astrophysics Data System (ADS)

    Larson, Daniel R.; Zipfel, Warren R.; Williams, Rebecca M.; Clark, Stephen W.; Bruchez, Marcel P.; Wise, Frank W.; Webb, Watt W.

    2003-05-01

    The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

  14. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo.

    PubMed

    Larson, Daniel R; Zipfel, Warren R; Williams, Rebecca M; Clark, Stephen W; Bruchez, Marcel P; Wise, Frank W; Webb, Watt W

    2003-05-30

    The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales. PMID:12775841

  15. A new indicator in early drought diagnosis of cucumber with chlorophyll fluorescence imaging

    NASA Astrophysics Data System (ADS)

    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.

  16. Wide field-of-view lens-free fluorescent imaging on a chip

    PubMed Central

    Coskun, Ahmet F.; Su, Ting-Wei

    2010-01-01

    We demonstrate an on-chip fluorescent detection platform that can simultaneously image fluorescent micro-objects or labeled cells over an ultra-large field-of-view of 2.5 cm × 3.5 cm without the use of any lenses, thin-film filters and mechanical scanners. Such a wide field-of-view lensless fluorescent imaging modality, despite its limited resolution, might be very important for high-throughput screening applications as well as for detection and counting of rare cells within large-area microfluidic devices. PMID:20379564

  17. Applying fluorescence lifetime imaging microscopy to evaluate the efficacy of anticancer drugs

    NASA Astrophysics Data System (ADS)

    Kawanabe, Satoshi; Araki, Yoshie; Uchimura, Tomohiro; Imasaka, Totaro

    2015-06-01

    Fluorescence lifetime imaging microscopy was applied to evaluate the efficacy of anticancer drugs. A decrease in the fluorescence lifetime of the nucleus in apoptotic cancer cells stained by SYTO 13 dye was detected after treatment with antitumor antibiotics such as doxorubicin or epirubicin. It was confirmed that the change in fluorescence lifetime occurred earlier than morphological changes in the cells. We found that the fluorescence lifetime of the nucleus in the cells treated with epirubicin decreased more rapidly than that of the cells treated with doxorubicin. This implies that epirubicin was more efficacious than doxorubicin in the treatment of cancer cells. The change in fluorescence lifetime was, however, not indicated when the cells were treated with cyclophosphamide. The decrease in fluorescence lifetime was associated with the processes involving caspase activation and chromatin condensation. Therefore, this technique would provide useful information about apoptotic cells, particularly in the early stages.

  18. Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Kremers, Gert-Jan; van Munster, Erik B.; Goedhart, Joachim; Gadella, Theodorus W. J.

    2008-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is a quantitative microscopy technique for imaging nanosecond decay times of fluorophores. In the case of frequency-domain FLIM, several methods have been described to resolve the relative abundance of two fluorescent species with different fluorescence decay times. Thus far, single-frequency FLIM methods generally have been limited to quantifying two species with monoexponential decay. However, multiexponential decays are the norm rather than the exception, especially for fluorescent proteins and biological samples. Here, we describe a novel method for determining the fractional contribution in each pixel of an image of a sample containing two (multiexponentially) decaying species using single-frequency FLIM. We demonstrate that this technique allows the unmixing of binary mixtures of two spectrally identical cyan or green fluorescent proteins, each with multiexponential decay. Furthermore, because of their spectral identity, quantitative images of the relative molecular abundance of these fluorescent proteins can be generated that are independent of the microscope light path. The method is rigorously tested using samples of known composition and applied to live cell microscopy using cells expressing multiple (multiexponentially decaying) fluorescent proteins. PMID:18359789

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Fluorescence-enhanced optical tomography and nuclear imaging system for small animals

    NASA Astrophysics Data System (ADS)

    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.

  1. Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.

    PubMed

    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

  2. Fluorescence microscopy imaging of cells with a plasmonic dish integrally molded

    NASA Astrophysics Data System (ADS)

    Tawa, Keiko; Sasakawa, Chisato; Fujita, Tsuyoshi; Kiyosue, Kazuyuki; Hosokawa, Chie; Nishii, Junji; Oike, Makoto; Kakinuma, Norihiro

    2016-03-01

    A plastic dish with a wavelength-scale periodic structure at a bottom panel was integrally molded and coated with thin metal films. The integrally molded dish called plasmonic dish was applied to bioimaging under a fluorescence microscope. On the plasmonic substrate, the enhanced electric field based on a grating-coupled surface plasmon resonance (GC-SPR) can provide an enhanced fluorescence. In this study, two kinds of cells, human embryonic kidney (HEK) cells and neuronal cells, were observed in our plasmonic dish. Fluorescence images of HEK cells were above 10 times brighter than those obtained on a conventional glass-bottomed dish. Neuronal cells were successfully cultured for 10 d on the plasmonic dish integrally molded, and in fluorescence images with transmitted light, a higher contrast was obtained than in epifluorescence images. The plasmonic dish integrally molded, as well as that fabricated by the UV nanoimprint method, was also found to be useful for sensitive bioimaging.

  3. Fluorescence microscopy imaging with a Fresnel zone plate array based optofluidic microscope

    PubMed Central

    Han, Chao; Lee, Lap Man; Yang, Changhuei

    2013-01-01

    We report the implementation of an on-chip microscope system, termed fluorescence optofluidic microscope (FOFM), which is capable of fluorescence microscopy imaging of samples in fluid media. The FOFM employs an array of Fresnel zone plates (FZP) to generate an array of focused light spots within a microfluidic channel. As a sample flows through the channel and across the array of focused light spots, the fluorescence emissions are collected by a filter-coated CMOS sensor, which serves as the channel's floor. The collected data can then be processed to render fluorescence microscopy images at a resolution determined by the focused light spot size (experimentally measured as 0.65 μm FWHM). In our experiments, our established resolution was 1.0 μm due to Nyquist criterion consideration. As a demonstration, we show that such a system can be used to image the cell nuclei stained by Acridine Orange and cytoplasm labeled by Qtracker®. PMID:21935556

  4. Simultaneous fluorescence and positron emission tomography for in vivo imaging of small animals

    NASA Astrophysics Data System (ADS)

    Zhang, Bin; Liu, Shuangquan; Liu, Fei; Zhang, Xiaochun; Xu, Yanyan; Luo, Jianwen; Shan, Baoci; Bai, Jing

    2011-12-01

    Simultaneous positron emission tomography (PET) and fluorescence tomography (FT) for in vivo imaging of small animals is proposed by a dual-modality system. This system combines a charge-coupled device-based near-infrared fluorescence imaging with a planar detector pair-based PET. With [18F]-2-fluoro-2-deoxy-d-glucose radioactive tracer and the protease activated fluorescence probe, on the one hand, the simultaneous metabolic activity and protease activity in tumor region are revealed by the PET and FT, respectively. On the other hand, the protease activity both on the surface layer and the deep tissue of the tumor is provided by the fluorescence reflection imaging and FT, respectively.

  5. Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells.

    PubMed

    Wang, Xiaohua; Li, Xiaojuan; Deng, Xin; Luu, Doan-Trung; Maurel, Christophe; Lin, Jinxing

    2015-12-01

    Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5-6 h. PMID:26584445

  6. Hyperspectral imaging fluorescence excitation scanning for detecting colorectal cancer: pilot study

    NASA Astrophysics Data System (ADS)

    Leavesley, Silas J.; Wheeler, Mikayla; Lopez, Carmen; Baker, Thomas; Favreau, Peter F.; Rich, Thomas C.; Rider, Paul F.; Boudreaux, Carole W.

    2016-03-01

    Optical spectroscopy and hyperspectral imaging have shown the theoretical potential to discriminate between cancerous and non-cancerous tissue with high sensitivity and specificity. To date, these techniques have not been able to be effectively translated to endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents a new technology that may be well-suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The objective of this pilot study was to evaluate the changes in the fluorescence excitation spectrum of resected specimen pairs of colorectal adenocarcinoma and normal colorectal mucosa. Patients being treated for colorectal adenocarcinoma were enrolled. Representative adenocarcinoma and normal colonic mucosa specimens were collected from each case. Specimens were flash frozen in liquid nitrogen. Adenocarcinoma was confirmed by histologic evaluation of H&E permanent sections. Hyperspectral image data of the fluorescence excitation of adenocarcinoma and surrounding normal tissue were acquired using a custom microscope configuration previously developed in our lab. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation spectral range of 390-450 nm. We conclude that fluorescence excitation-scanning hyperspectral imaging may offer an alternative approach for differentiating adenocarcinoma and surrounding normal mucosa of the colon. Future work will focus on expanding the number of specimen pairs analyzed and will utilize fresh tissues where possible, as flash freezing and reconstituting tissues may have altered the autofluorescence properties.

  7. Development of a time-gated fluorescence lifetime microscope for in vivo corneal metabolic imaging

    NASA Astrophysics Data System (ADS)

    Silva, Susana F.; Batista, Ana; Castejón, Olga C.; Quadrado, Maria João.; Domingues, José Paulo; Morgado, Miguel

    2015-07-01

    Metabolic imaging can be a valuable tool in the early diagnosis of corneal diseases. Cell metabolic changes can be assessed through non-invasive optical methods due to the autofluorescence of metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Both molecules exhibit double exponential fluorescence decays, with well-separated short and long lifetime components, which are related to their protein-bound and free states. Corneal metabolism can be monitored by measuring the relative contribution of these two components. Here we report on the development of a fluorescence lifetime imaging microscope for in vivo measurement of FAD fluorescence lifetimes in corneal cells. The microscope is based on one-photon fluorescence excitation, through a pulsed blue diode laser. Fluorescence lifetime imaging is achieved using the Time-Gated technique. Structured illumination is used to improve the low axial resolution of wide-field time-gated FLIM. A Digital Micromirror Device (DMD) is used to produce the sinusoidal patterns required by structural illumination. The DMD control is integrated with the acquisition software of the imaging system which is based on an ultra-high speed gated image intensifier coupled to a CCD camera. We present preliminary results concerning optical and timing performance of the fluorescence lifetime microscope. Preliminary tests with ex-vivo bovine corneas are also described.

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

    PubMed

    Lu, Yujie; Zhu, Banghe; Shen, Haiou; Rasmussen, John C; Wang, Ge; Sevick-Muraca, Eva M

    2010-08-21

    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 match quantitation provided by nuclear imaging. In this paper, a frequency-domain parallel adaptive finite element solver is developed with simplified spherical harmonics (SP(N)) approximations. To fully evaluate the performance of the SP(N) approximations, a fast time-resolved tetrahedron-based Monte Carlo fluorescence simulator suitable for complex heterogeneous geometries is developed using a convolution strategy to realize the simulation of the fluorescence excitation and emission. The validation results show that high-order SP(N) can effectively correct the modeling errors of the diffusion equation, especially when the tissues have high absorption characteristics or when high modulation frequency measurements are used. Furthermore, the parallel adaptive mesh evolution strategy improves the modeling precision and the simulation speed significantly on a realistic digital mouse phantom. This solver is a promising platform for fluorescence molecular tomography using high-order approximations to the radiative transfer equation. PMID:20671350

  9. Lensless wide-field fluorescent imaging on a chip using compressive decoding of sparse objects

    PubMed Central

    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 an on-chip fiber-optic faceplate and is delivered to a wide field-of-view opto-electronic sensor array for lensless recording of fluorescent spots corresponding to the samples. A compressive sampling based optimization algorithm is then used to rapidly reconstruct the sparse distribution of fluorescent sources to achieve ~10 µm spatial resolution over the entire active region of the sensor-array, i.e., over an imaging field-of-view of >8 cm2. Such a wide-field lensless fluorescent imaging platform could especially be significant for high-throughput imaging cytometry, rare cell analysis, as well as for micro-array research. PMID:20588904

  10. Spectral and fluorescence imaging of immune system and tissue response to an immunogenic agent

    NASA Astrophysics Data System (ADS)

    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.

  11. Rapid Imaging of Latent Fingerprints Using Biocompatible Fluorescent Silica Nanoparticles.

    PubMed

    Kim, Young-Jae; Jung, Hak-Sung; Lim, Joohyun; Ryu, Seung-Jin; Lee, Jin-Kyu

    2016-08-16

    Fluorescent silica nanoparticles (FSNPs) are synthesized through the Stöber method by incorporating silane-modified organic dye molecules. The modified fluorescent organic dye molecule is able to be prepared by allylation and hydrosilylation reactions. The optical properties of as-prepared FSNPs are shown the similar optical properties of PR254A (allylated Pigment Red 254) and have outstanding photostability. The polyvinylpyrrolidone (PVP) is introduced onto the surface of FSNP to enhance the binding affinity of PVP-coated FSNP for latent fingerprints (LFPs) detection. The simple preparation and easy control of surface properties of FSNPs show potential as a fluorescent labeling material for enhanced latent fingerprint detection on hydrophilic and hydrophobic substrates in forensic science for individual identification. PMID:27452188

  12. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    SciTech Connect

    Cuneo, Kyle C.; Mito, Jeffrey K.; Javid, Melodi P.; Ferrer, Jorge M.; Kim, Yongbaek; Lee, W. David; Bawendi, Moungi G.; Brigman, Brian E.; Kirsch, David G.

    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.

  13. Quantitative method to assess caries via fluorescence imaging from the perspective of autofluorescence spectral analysis

    NASA Astrophysics Data System (ADS)

    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 <0.66, 0.66-1.06, 1.06-1.62, and >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.

  14. eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM).

    PubMed

    Schmitt, Franz-Josef; Thaa, Bastian; Junghans, Cornelia; Vitali, Marco; Veit, Michael; Friedrich, Thomas

    2014-09-01

    The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO

  15. Fluorescence molecular tomographic image reconstruction based on reduced measurement data

    NASA Astrophysics Data System (ADS)

    Zou, Wei; Wang, Jiajun; Feng, David Dagan; Fang, Erxi

    2015-07-01

    The analysis of fluorescence molecular tomography is important for medical diagnosis and treatment. Although the quality of reconstructed results can be improved with the increasing number of measurement data, the scale of the matrices involved in the reconstruction of fluorescence molecular tomography will also become larger, which may slow down the reconstruction process. A new method is proposed where measurement data are reduced according to the rows of the Jacobian matrix and the projection residual error. To further accelerate the reconstruction process, the global inverse problem is solved with level-by-level Schur complement decomposition. Simulation results demonstrate that the speed of the reconstruction process can be improved with the proposed algorithm.

  16. Optical imaging of fluorescent carbon biomarkers using artificial neural networks

    NASA Astrophysics Data System (ADS)

    Dolenko, Tatiana A.; Burikov, Sergey A.; Vervald, Alexey M.; Vlasov, Igor I.; Dolenko, Sergey A.; Laptinskiy, Kirill A.; Rosenholm, Jessica M.; Shenderova, Olga A.

    2014-11-01

    The principle possibility of extraction of fluorescence of nanoparticles in the presence of background autofluorescence of a biological environment using neural network algorithms is demonstrated. It is shown that the methods used allow detection of carbon nanoparticles fluorescence against the background of the autofluorescence of egg white with a sufficiently low concentration detection threshold (not more than 2 μg/ml for carbon dots and 3 μg/ml for nanodiamonds). It was also shown that the use of the input data compression can further improve the accuracy of solving the inverse problem by 1.5 times.

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

    PubMed

    Sakhalkar, H S; Dewhirst, M; Oliver, T; Cao, Y; Oldham, M

    2007-04-21

    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

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

    NASA Astrophysics Data System (ADS)

    Sakhalkar, H. S.; Dewhirst, M.; Oliver, T.; Cao, Y.; Oldham, M.

    2007-04-01

    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

  19. Fluorescence endoscopic imaging study of anastomotic recurrence of Crohn's disease after right ileocolonic resection

    NASA Astrophysics Data System (ADS)

    Mordon, Serge R.; Maunoury, Vincent; Klein, Olivier; Colombel, Jean-Frederic

    1995-12-01

    Crohn's disease is an inflammatory bowel disease of unknown etiology. Vasculitis is hypothesized but it was never demonstrated in vivo. This study aimed to evaluate the vascular mucosa perfusion using fluorescence imaging in 13 patients who had previously undergone eileocolonic resection and who agreed to participate in a prospective endoscopic study of anastomotic recurrence. This anastomotic recurrence rate is known to be high (73% after 1 year follow-up) and is characterized by ulcerations. The fluorescence study was started with an I.V. bolus injection of sodium fluorescein. The pre-anastomotic mucosa was endoscopically examined with blue light that stimulates fluorescein fluorescence. Fluorescence emission was recorded with an ultra-high-sensitivity camera connected to the endoscope via an interference filter (520 - 560 nm). A uniform fluorescence was observed a few seconds after the injection and lasted for 15 min in healthy subjects. In case of recurrence, the centers of the ulcerations displayed a very low fluorescence indicating localized ischemia. In contrast, the rims of the ulcers revealed brighter fluorescent images than those of normal mucosa. The anastomotic ulcerations of Crohn's disease recurrence exhibit a high fluorescence intensity at their margins indicating an increased mucosal blood flow and/or enhanced transcapillary diffusion. These findings support the hypothesis of a primary vasculitis in Crohn's disease.

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

    SciTech Connect

    Chotimarkorn, Chatchawan; Nagasaka, Reiko; Ushio, Hideki . E-mail: hushio@s.kaiyodai.ac.jp; Ohshima, Toshiaki; Matsunaga, Shigeki

    2005-12-16

    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, {sup 1}H NMR, and {sup 13}C 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.

  1. Wide field-of-view fluorescence image deconvolution with aberration-estimation from Fourier ptychography

    PubMed Central

    Chung, Jaebum; Kim, Jinho; Ou, Xiaoze; Horstmeyer, Roarke; Yang, Changhuei

    2016-01-01

    This paper presents a method to simultaneously acquire an aberration-corrected, wide field-of-view fluorescence image and a high-resolution coherent bright-field image using a computational microscopy method. First, the procedure applies Fourier ptychographic microscopy (FPM) to retrieve the amplitude and phase of a sample, at a resolution that significantly exceeds the cutoff spatial frequency of the microscope objective lens. At the same time, redundancy within the set of acquired FPM bright-field images offers a means to estimate microscope aberrations. Second, the procedure acquires an aberrated fluorescence image, and computationally improves its resolution through deconvolution with the estimated aberration map. An experimental demonstration successfully improves the bright-field resolution of fixed, stained and fluorescently tagged HeLa cells by a factor of 4.9, and reduces the error caused by aberrations in a fluorescence image by up to 31%, over a field of view of 6.2 mm by 9.3 mm. For optimal deconvolution, we show the fluorescence image needs to have a signal-to-noise ratio of at least ~18. PMID:26977345

  2. Wide field-of-view fluorescence image deconvolution with aberration-estimation from Fourier ptychography.

    PubMed

    Chung, Jaebum; Kim, Jinho; Ou, Xiaoze; Horstmeyer, Roarke; Yang, Changhuei

    2016-02-01

    This paper presents a method to simultaneously acquire an aberration-corrected, wide field-of-view fluorescence image and a high-resolution coherent bright-field image using a computational microscopy method. First, the procedure applies Fourier ptychographic microscopy (FPM) to retrieve the amplitude and phase of a sample, at a resolution that significantly exceeds the cutoff spatial frequency of the microscope objective lens. At the same time, redundancy within the set of acquired FPM bright-field images offers a means to estimate microscope aberrations. Second, the procedure acquires an aberrated fluorescence image, and computationally improves its resolution through deconvolution with the estimated aberration map. An experimental demonstration successfully improves the bright-field resolution of fixed, stained and fluorescently tagged HeLa cells by a factor of 4.9, and reduces the error caused by aberrations in a fluorescence image by up to 31%, over a field of view of 6.2 mm by 9.3 mm. For optimal deconvolution, we show the fluorescence image needs to have a signal-to-noise ratio of at least ~18. PMID:26977345

  3. Evaluation and compensation of fluorescence for spectral imaging of art materials and historical documents

    NASA Astrophysics Data System (ADS)

    Christens-Barry, William A.; Boydston, Kenneth; Easton, Roger L., Jr.

    2010-01-01

    Imaging techniques have been developed to better account for fluorescent emission that may accompany reflected light during capture and processing of high color-fidelity imagery from art works and important historical documents. This approach is based on sequential capture of monochrome images of the object or scene, each illuminated by a narrow spectral band from a bank of light-emitting diodes (LED's) in the ultraviolet, visible, and near-infrared spectral regions. These images contain color reference materials in the field of view, and are augmented by images in which bandpass filters are placed in the capture path. Processing of these images allows the separate contributions of reflectance and fluorescence emission in narrow wavelength bands to be recognized and quantified, which allows accounting for any fluorescence contributions during nominal reflectance imaging so that adjustments may be made in subsequent rendering. This paper describes the apparatus, capture procedures, and processing techniques that are employed. The impact of fluorescence on color fidelity during reproduction, based on deltaE calculations, is calculated and discussed for highly fluorescent pastels.

  4. Low-cost fluorescence microscopy for point-of-care cell imaging

    NASA Astrophysics Data System (ADS)

    Lochhead, Michael J.; Ives, Jeff; Givens, Monique; Delaney, Marie; Moll, Kevin; Myatt, Christopher J.

    2010-02-01

    Fluorescence microscopy has long been a standard tool in laboratory medicine. Implementation of fluorescence microscopy for near-patient diagnostics, however, has been limited due to cost and complexity associated with traditional fluorescence microscopy techniques. There is a particular need for robust, low-cost imaging in high disease burden areas in the developing world, where access to central laboratory facilities and trained staff is limited. Here we describe a point-of-care assay that combines a disposable plastic cartridge with an extremely low cost fluorescence imaging instrument. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements. A two-color cell imager is presented, with magnification optimized for enumeration of immunostained human T cells. To demonstrate the system, peripheral blood mononuclear cells were stained with fluorescently labeled anti-human-CD4 and anti-human-CD3 antibodies. Registered images were used to generate fractional CD4+ and CD3+ staining and enumeration results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.

  5. Stepwise multi-photon activation fluorescence reveals a new method of melanoma imaging for dermatologists

    NASA Astrophysics Data System (ADS)

    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.

  6. Image analysis in fluorescence microscopy: Bacterial dynamics as a case study

    PubMed Central

    van Teeffelen, Sven; Shaevitz, Joshua W.; Gitai, Zemer

    2012-01-01

    Fluorescence microscopy is the primary tool for studying complex processes inside individual living cells. Technical advances in both molecular biology and microscopy have made it possible to image cells from many genetic and environmental backgrounds. These images contain a vast amount of information, but this information is often hidden behind various sources of noise, convoluted with other information, and stochastic in nature. Accessing the desired biological information therefore requires new tools of computational image analysis and modeling. Here, we review some of the recent advances in computational analysis of images obtained from fluorescence microscopy, focusing on bacterial systems. We emphasize techniques that are readily available to molecular and cell biologists but also point out examples where problem-specific image analyses are necessary. Thus, image analysis is not only a toolkit to be applied to new images but is an integral part of the design and implementation of a microscopy experiment. PMID:22415868

  7. BioMEMS for multiparameter clinical monitoring

    NASA Astrophysics Data System (ADS)

    Moser, Isabella

    2003-01-01

    For diabetes patients glucose monitoring means an important improvement of their life quality and additionally it is a $3-billion-a-year business. Continuous glucose monitoring provides gapless glucose level control, an early warning of hypoglycemia, and is intended to control insulin pumps. An upgrading to multi-parameter monitoring would not only benefit patients with severe metabolism defects but also the metabolism of diabetes patient could be better controlled by monitoring an additional parameter like lactate. Multi-parameter monitoring devices are not commercially available, one of the complications in the integration of different biosensors using the same detecting molecule for all analytes is chemical cross talk between adjacent amperometric biosensors. Recently some integrated biosensors were published but either they were not mass producible or they were realized in an expensive silicon based technology. In addition to it most of them were not tested under monitoring conditions but their integration principles will be discussed. As an example a low cost multi- parameter microsystem and some applications of it in clinical diagnosis will be presented. Also an overlook of non-invasive methods and (minimal) invasive methods will be given with a focus on microdialysis.

  8. Benzothiadiazole Derivatives as Fluorescence Imaging Probes: Beyond Classical Scaffolds.

    PubMed

    Neto, Brenno A D; Carvalho, Pedro H P R; Correa, Jose R

    2015-06-16

    This Account describes the origins, features, importance, and trends of the use of fluorescent small-molecule 2,1,3-benzothiadiazole (BTD) derivatives as a new class of bioprobes applied to bioimaging analyses of several (live and fixed) cell types. BTDs have been successfully used as probes for a plethora of biological analyses for only a few years, and the impressive responses obtained by using this important class of heterocycle are fostering the development of new fluorescent BTDs and expanding the biological applications of such derivatives. The first use of a fluorescent small-molecule BTD derivative as a selective cellular probe dates back to 2010, and since then impressive advances have been described by us and others. The well-known limitations of classical scaffolds urged the development of new classes of bioprobes. Although great developments have been achieved by using classical scaffolds such as coumarins, BODIPYs, fluoresceins, rhodamines, cyanines, and phenoxazines, there is still much to be done, and BTDs aim to succeed where these dyes have shown their limitations. Important organelles and cell components such as nuclear DNA, mitochondria, lipid droplets, and others have already been successfully labeled by fluorescent small-molecule BTD derivatives. New technological systems that use BTDs as the fluorophores for bioimaging experiments have been described in recent scientific literature. The successful application of BTDs as selective bioprobes has led some groups to explore their potential for use in studying membrane pores or tumor cells under hypoxic conditions. Finally, BTDs have also been used as fluorescent tags to investigate the action mechanism of some antitumor compounds. The attractive photophysical data typically observed for π-extended BTD derivatives is fostering interest in the use of this new class of bioprobes. Large Stokes shifts, large molar extinction coefficients, high quantum yields, high stability when stored in solution or

  9. Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

    PubMed

    Gong, Haibiao; Kovar, Joy L; Baker, Brenda; Zhang, Aihua; Cheung, Lael; Draney, Daniel R; Corrêa, Ivan R; Xu, Ming-Qun; Olive, D Michael

    2012-01-01

    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. PMID:22479502

  10. Red to far-red multispectral fluorescence image fusion for detection of fecal contamination on apples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Image overlay solution based on threshold detection for a compact near infrared fluorescence goggle system.

    PubMed

    Gao, Shengkui; Mondal, Suman B; Zhu, Nan; Liang, RongGuang; Achilefu, Samuel; Gruev, Viktor

    2015-01-01

    Near infrared (NIR) fluorescence imaging has shown great potential for various clinical procedures, including intraoperative image guidance. However, existing NIR fluorescence imaging systems either have a large footprint or are handheld, which limits their usage in intraoperative applications. We present a compact NIR fluorescence imaging system (NFIS) with an image overlay solution based on threshold detection, which can be easily integrated with a goggle display system for intraoperative guidance. The proposed NFIS achieves compactness, light weight, hands-free operation, high-precision superimposition, and a real-time frame rate. In addition, the miniature and ultra-lightweight light-emitting diode tracking pod is easy to incorporate with NIR fluorescence imaging. Based on experimental evaluation, the proposed NFIS solution has a lower detection limit of 25 nM of indocyanine green at 27 fps and realizes a highly precise image overlay of NIR and visible images of mice in vivo. The overlay error is limited within a 2-mm scale at a 65-cm working distance, which is highly reliable for clinical study and surgical use. PMID:25607724

  12. Image overlay solution based on threshold detection for a compact near infrared fluorescence goggle system

    NASA Astrophysics Data System (ADS)

    Gao, Shengkui; Mondal, Suman B.; Zhu, Nan; Liang, RongGuang; Achilefu, Samuel; Gruev, Viktor

    2015-01-01

    Near infrared (NIR) fluorescence imaging has shown great potential for various clinical procedures, including intraoperative image guidance. However, existing NIR fluorescence imaging systems either have a large footprint or are handheld, which limits their usage in intraoperative applications. We present a compact NIR fluorescence imaging system (NFIS) with an image overlay solution based on threshold detection, which can be easily integrated with a goggle display system for intraoperative guidance. The proposed NFIS achieves compactness, light weight, hands-free operation, high-precision superimposition, and a real-time frame rate. In addition, the miniature and ultra-lightweight light-emitting diode tracking pod is easy to incorporate with NIR fluorescence imaging. Based on experimental evaluation, the proposed NFIS solution has a lower detection limit of 25 nM of indocyanine green at 27 fps and realizes a highly precise image overlay of NIR and visible images of mice in vivo. The overlay error is limited within a 2-mm scale at a 65-cm working distance, which is highly reliable for clinical study and surgical use.

  13. Image overlay solution based on threshold detection for a compact near infrared fluorescence goggle system

    PubMed Central

    Gao, Shengkui; Mondal, Suman B.; Zhu, Nan; Liang, RongGuang; Achilefu, Samuel; Gruev, Viktor

    2015-01-01

    Abstract. Near infrared (NIR) fluorescence imaging has shown great potential for various clinical procedures, including intraoperative image guidance. However, existing NIR fluorescence imaging systems either have a large footprint or are handheld, which limits their usage in intraoperative applications. We present a compact NIR fluorescence imaging system (NFIS) with an image overlay solution based on threshold detection, which can be easily integrated with a goggle display system for intraoperative guidance. The proposed NFIS achieves compactness, light weight, hands-free operation, high-precision superimposition, and a real-time frame rate. In addition, the miniature and ultra-lightweight light-emitting diode tracking pod is easy to incorporate with NIR fluorescence imaging. Based on experimental evaluation, the proposed NFIS solution has a lower detection limit of 25 nM of indocyanine green at 27 fps and realizes a highly precise image overlay of NIR and visible images of mice in vivo. The overlay error is limited within a 2-mm scale at a 65-cm working distance, which is highly reliable for clinical study and surgical use. PMID:25607724

  14. Fluorescent-Guided Surgical Resection of Glioma with Targeted Molecular Imaging Agents: A Literature Review.

    PubMed

    Craig, Sonya E L; Wright, James; Sloan, Andrew E; Brady-Kalnay, Susann M

    2016-06-01

    The median life expectancy after a diagnosis of glioblastoma is 15 months. Although chemotherapeutics may someday cure glioblastoma by killing the highly dispersive malignant cells, the most important contribution that clinicians can currently offer to improve survival is by maximizing the extent of resection and providing concurrent chemo-radiation, which has become standard. Strides have been made in this area with the advent and implementation of methods of improved intraoperative tumor visualization. One of these techniques, optical fluorescent imaging with targeted molecular imaging agents, allows the surgeon to view fluorescently labeled tumor tissue during surgery with the use of special microscopy, thereby highlighting where to resect and indicating when tumor-free margins have been obtained. This advantage is especially important at the difficult-to-observe margins where tumor cells infiltrate normal tissue. Targeted fluorescent agents also may be valuable for identifying tumor versus nontumor tissue. In this review, we briefly summarize nontargeted fluorescent tumor imaging agents before discussing several novel targeted fluorescent agents being developed for glioma imaging in the context of fluorescent-guided surgery or live molecular navigation. Many of these agents are currently undergoing preclinical testing. As the agents become available, however, it is necessary to understand the strengths and weaknesses of each. PMID:26915698

  15. Comparative study reveals better far-red fluorescent protein for whole body imaging

    PubMed Central

    Luker, K.E.; Pata, P.; Shemiakina, I.I.; Pereverzeva, A.; Stacer, A.C.; Shcherbo, D.S.; Pletnev, V.Z.; Skolnaja, M.; Lukyanov, K.A.; Luker, G.D.; Pata, I.; Chudakov, D.M.

    2015-01-01

    Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals. PMID:26035795

  16. MACROscopic imaging of tumor xenografts using fluorescence, phase contrast, and transmitted light

    NASA Astrophysics Data System (ADS)

    Constantinou, Paul; Nicklee, Trudey; Hedley, David W.; Wilson, Brian C.; Damaskinos, Savvas

    2004-10-01

    Recent advances in imaging technology have contributed greatly to biological science. Confocal fluorescence microscopes (CFM) can acquire 2D and 3D images of biological samples such as live or fixed cells and tissues. Specimens that are large (e.g., a 10 mm x 10 mm tissue section) and overfill the field of view (FOV) of typical microscope objectives require use of image tiling to cover the entire specimen. This can be time consuming and cause artefacts in the composite image. The MACROscope system (Biomedical Photometrics Inc, Waterloo, Canada), is a confocal device with a 22 mm x 70 mm FOV; ideal for imaging large tissue sections in a single frame. The system used here is a prototype capable of simultaneous acquisition from up to three detection channels. Fluorescence images of SiHa mouse tumour xenografts stained with CD31-Cy3, showing blood vessel location, and EF5-Cy5, showing areas of tissue hypoxia, were collected. Differential phase contrast (DPC) images of the same section were also recorded to show tissue morphology. Finally, RGB transmitted light images of human tongue and pancreas tissues were obtained. This new device avoids the need for image tiling and provides simultaneous imaging of multiple fluorescently-labeled tissue specific markers in large biological samples. This enables time- and cost-efficient high-throughput screening of (immuno)histopathological samples. This device may also serve in the imaging of high-throughput DNA and tissue arrays.

  17. Photon-counting H33D detector for biological fluorescence