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Sample records for advanced fluorescence imaging

  1. 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

  2. Recent advances on in vivo imaging with fluorescent proteins.

    PubMed

    Hoffman, Robert M

    2008-01-01

    In vivo imaging with green fluorescent protein (GFP) and other fluorescent proteins is revolutionizing cancer biology and other fields of in vivo biology (Hoffman, 2005; Hoffman and Yang, 2006a,b,c). Our laboratory pioneered the use of GFP for in vivo imaging in 1997 (Chishima et al., 1997). This chapter highlights recent developments from our laboratory on both macro and micro in vivo imaging by using fluorescent proteins.

  3. 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

  4. Targeted radionuclide and fluorescence dual-modality imaging of cancer: preclinical advances and clinical translation.

    PubMed

    Lütje, S; Rijpkema, M; Helfrich, W; Oyen, W J G; Boerman, O C

    2014-12-01

    In oncology, sensitive and reliable detection tumor tissue is crucial to prevent recurrences and to improve surgical outcome. Currently, extensive research is focused on the use of radionuclides as well as fluorophores to provide real-time guidance during surgery to aid the surgeon in the identification of malignant tissue. Particularly, dual-modality approaches combining radionuclide and near-infrared fluorescence (NIRF) imaging have shown promising results in preclinical studies. Radionuclide imaging allows sensitive intra-operative localization of tumor lesions using a gamma probe, whereas NIRF imaging allows more accurate real-time tumor delineation. Consequently, both radionuclide and NIRF imaging might complement each other, and dual-modality image-guided surgery may overcome limitations of the currently used single-modality imaging techniques. In this review, a comprehensive overview on recent preclinical advances in tumor-targeted radionuclide and fluorescence dual-modality imaging is provided. Subsequently, the clinical applicability of dual-modality image-guided surgery is discussed.

  5. Recent advances in near-infrared fluorescence-guided imaging surgery using indocyanine green.

    PubMed

    Namikawa, Tsutomu; Sato, Takayuki; Hanazaki, Kazuhiro

    2015-12-01

    Near-infrared (NIR) fluorescence imaging has better tissue penetration, allowing for the effective rejection of excitation light and detection deep inside organs. Indocyanine green (ICG) generates NIR fluorescence after illumination by an NIR ray, enabling real-time intraoperative visualization of superficial lymphatic channels and vessels transcutaneously. The HyperEye Medical System (HEMS) can simultaneously detect NIR rays under room light to provide color imaging, which enables visualization under bright light. Thus, NIR fluorescence imaging using ICG can provide for excellent diagnostic accuracy in detecting sentinel lymph nodes in cancer and microvascular circulation in various ischemic diseases, to assist us with intraoperative decision making. Including HEMS in this system could further improve the sentinel lymph node mapping and intraoperative identification of blood supply in reconstructive organs and ischemic diseases, making it more attractive than conventional imaging. Moreover, the development of new laparoscopic imaging systems equipped with NIR will allow fluorescence-guided surgery in a minimally invasive setting. Future directions, including the conjugation of NIR fluorophores to target specific cancer markers might be realistic technology with diagnostic and therapeutic benefits.

  6. Advanced fluorescence imaging endoscopy using an acousto-optic tuneable filter

    NASA Astrophysics Data System (ADS)

    Whelan, Maurice P.; Bouhifd, Mounir; Aprahamian, Marc

    2004-07-01

    Two novel prototype instruments for in vivo fluorescence-based medical diagnostics are described. The devices are based on an acousto-optic tuneable filter (AOTF) and can be easily attached to the eyepiece of most commercially available endoscopes. The instruments developed offer significant advantages over typical fixed-filter or filter-wheel fluorescence imaging systems in terms of flexibility, performance and diagnostic potential. Any filtering center-wavelength in the range from 450 to 700 nm can be rapidly selected either by random access or sequential tuning using simple commands delivered over a PC serial interface. In addition, both filtered and unfiltered light can be imaged to facilitate the direct association of fluorescence signals with specific anatomical sites. To demonstrate the system in vivo, a study of the diagnostic potential of fluorescence imaging for pancreatitis was conducted on rats. The aim was to detect extremely low-levels of endogenous protoporphyrin IX (PpIX) that has been shown to accumulate in early-stage diseased tissue undergoing an inflammatory response. Results show clearly that the device is effective in diagnosing mild pancreatitis in rats without the necessity of administering PpIX promoting agents such as ALA. Planning of human clinical trials is currently underway to demonstrate its potential as a tool for non-invasive early diagnosis of gastroenterological diseases.

  7. Candida, fluorescent stain (image)

    MedlinePlus

    This microscopic film shows a fluorescent stain of Candida. Candida is a yeast (fungus) that causes mild disease, but in immunocompromised individuals it may cause life-threatening illness. (Image ...

  8. 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.

  9. Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging.

    PubMed

    Crick, Colin R; Noimark, Sacha; Peveler, William J; Bear, Joseph C; Ivanov, Aleksandar P; Edel, Joshua B; Parkin, Ivan P

    2016-01-01

    The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise composition of these materials is essential, especially in the design of surface catalysts, where the surface concentration of the active component determines the activity of the material. Antimicrobial materials which utilize nanoparticles are a particular focus of this technology. Recently swell encapsulation has emerged as a technique for inserting antimicrobial nanoparticles into a host polymer matrix. Swell encapsulation provides the advantage of localizing the incorporation to the external surfaces of materials, which act as the active sites of these materials. However, quantification of this nanoparticle uptake is challenging. Previous studies explore the link between antimicrobial activity and surface concentration of the active component, but this is not directly visualized. Here we show a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of CdSe/ZnS nanoparticles can be accurately visualized through cross-sectional fluorescence imaging. Using this method, we can quantify the uptake of nanoparticles via swell encapsulation and measure the surface concentration of encapsulated particles, which is key in optimizing the activity of functional materials. PMID:27500449

  10. 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

  11. 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.

  12. Multispectral fluorescence imaging of atherosclerosis

    SciTech Connect

    Davenport, C.M.C.

    1992-01-01

    Multispectral fluorescence imaging is a new diagnostic technique with the potential to provide improved detection and classification of atherosclerotic disease. This technique involves imaging the fluorescence response of a tissue region through a tunable band-pass filtering device. The result is a set of image in which each individual image is composed of the fluorescence emission within a specified band of wavelengths. Multispectral imaging combined with angioscopic technology allows direct access to important spectral information and spatial attributes providing the potential for more informed clinical decisions about which, if any, treatment modality is indicated. In this dissertation, the system requirements for an angioscopic system with multispectral imaging capability are identified. This analysis includes a description of the necessary optical components and their characteristics as well as the experimental determination of spectral radiance values for the fluorescence response of human aorta specimens and the estimation of anticipated signal-to-noise ratios for the spectral images. Other issues investigated include the number of spectral images required to provide good classification potential and the best normalization method to be utilized. Finally, the potential utility of the information contained within a multispectral data set is demonstrated. Two methods of utilizing the multispectral data are presented. The first method involves generating a ratio-image from the ratio of the intensities of two spectrally filtered images. The second method consists of using histologically verified training data to train a projector and then applying that projector to a set of spectral images. The result provides improved contrast image. White-light images (generated using an incandescent light source), total-fluorescence images (the fluorescence response without spectral filtering), ratio-images, and optimized contrast images are compared. T

  13. 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…

  14. 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 ...

  15. A hard x-ray scanning microprobe for fluorescence imaging and microdiffraction at the Advanced Photon Source

    SciTech Connect

    Cai, L.; Lai, B.; Yun, W.; Ilinski, P.; Legnini, D.; Maser, J.; Rodrigues, W.

    1999-11-02

    A hard x-ray scanning microprobe based on zone plate optics and undulator radiation, in the energy region from 6 to 20 keV, has reached a focal spot size (FWHM) of 0.15 {micro}m (v) x 0.6 {micro}m (h), and a photon flux of 4 x 10{sup 9} photons/sec/0.01%BW. Using a slit 44 meters upstream to create a virtual source, a circular beam spot of 0.15 {micro}m in diameter can be obtained with a photon flux of one order of magnitude less. During fluorescence mapping of trace elements in a single human ovarian cell, the microprobe exhibited an imaging sensitivity for Pt (L{sub a} line) of 80 attograms/{micro}m{sup 2} for a count rate of 10 counts per second. The x-ray microprobe has been used to map crystallographic strain and multiquantum well thickness in micro-optoelectronic devices produced with the selective area growth technique.

  16. A hard x-ray scanning microprobe for fluorescence imaging and microdiffraction at the advanced photon source

    NASA Astrophysics Data System (ADS)

    Cai, Z.; Lai, B.; Yun, W.; Ilinski, P.; Legnini, D.; Maser, J.; Rodrigues, W.

    2000-05-01

    A hard x-ray scanning microprobe based on zone plate optics and undulator radiation, in the energy region from 6 to 20 keV, has reached a focal spot size (FWHM) of 0.15 μm(v)×0.6 μm(h), and a photon flux of 4×109photons/sec/0.01%BW. Using a slit 44 meters upstream to create a virtual source, a circular beam spot of 0.15 μm in diameter can be obtained with a photon flux of one order of magnitude less. During fluorescence mapping of trace elements in a single human ovarian cell, the microprobe exhibited an imaging sensitivity for Pt (Lα line) of 80 attograms/μm2 for a count rate of 10 counts per second. The x-ray microprobe has been used to map crystallographic strain and multiquantum well thickness in micro-optoelectronic devices produced with the selective area growth technique.

  17. 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

  18. 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

  19. 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

  20. Advancing biomedical imaging

    PubMed Central

    Weissleder, Ralph; Nahrendorf, Matthias

    2015-01-01

    Imaging reveals complex structures and dynamic interactive processes, located deep inside the body, that are otherwise difficult to decipher. Numerous imaging modalities harness every last inch of the energy spectrum. Clinical modalities include magnetic resonance imaging (MRI), X-ray computed tomography (CT), ultrasound, and light-based methods [endoscopy and optical coherence tomography (OCT)]. Research modalities include various light microscopy techniques (confocal, multiphoton, total internal reflection, superresolution fluorescence microscopy), electron microscopy, mass spectrometry imaging, fluorescence tomography, bioluminescence, variations of OCT, and optoacoustic imaging, among a few others. Although clinical imaging and research microscopy are often isolated from one another, we argue that their combination and integration is not only informative but also essential to discovering new biology and interpreting clinical datasets in which signals invariably originate from hundreds to thousands of cells per voxel. PMID:26598657

  1. 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.

  2. Advanced Fluorescence Protein-Based Synapse-Detectors

    PubMed Central

    Lee, Hojin; Oh, Won Chan; Seong, Jihye; Kim, Jinhyun

    2016-01-01

    The complex information-processing capabilities of the central nervous system emerge from intricate patterns of synaptic input-output relationships among various neuronal circuit components. Understanding these capabilities thus requires a precise description of the individual synapses that comprise neural networks. Recent advances in fluorescent protein engineering, along with developments in light-favoring tissue clearing and optical imaging techniques, have rendered light microscopy (LM) a potent candidate for large-scale analyses of synapses, their properties, and their connectivity. Optically imaging newly engineered fluorescent proteins (FPs) tagged to synaptic proteins or microstructures enables the efficient, fine-resolution illumination of synaptic anatomy and function in large neural circuits. Here we review the latest progress in fluorescent protein-based molecular tools for imaging individual synapses and synaptic connectivity. We also identify associated technologies in gene delivery, tissue processing, and computational image analysis that will play a crucial role in bridging the gap between synapse- and system-level neuroscience. PMID:27445785

  3. Advanced Fluorescence Protein-Based Synapse-Detectors.

    PubMed

    Lee, Hojin; Oh, Won Chan; Seong, Jihye; Kim, Jinhyun

    2016-01-01

    The complex information-processing capabilities of the central nervous system emerge from intricate patterns of synaptic input-output relationships among various neuronal circuit components. Understanding these capabilities thus requires a precise description of the individual synapses that comprise neural networks. Recent advances in fluorescent protein engineering, along with developments in light-favoring tissue clearing and optical imaging techniques, have rendered light microscopy (LM) a potent candidate for large-scale analyses of synapses, their properties, and their connectivity. Optically imaging newly engineered fluorescent proteins (FPs) tagged to synaptic proteins or microstructures enables the efficient, fine-resolution illumination of synaptic anatomy and function in large neural circuits. Here we review the latest progress in fluorescent protein-based molecular tools for imaging individual synapses and synaptic connectivity. We also identify associated technologies in gene delivery, tissue processing, and computational image analysis that will play a crucial role in bridging the gap between synapse- and system-level neuroscience. PMID:27445785

  4. 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).

  5. Time-resolved fluorescence anisotropy imaging.

    PubMed

    Suhling, Klaus; Levitt, James; Chung, Pei-Hua

    2014-01-01

    Fluorescence can be characterized by its intensity, position, wavelength, lifetime, and polarization. The more of these features are acquired in a single measurement, the more can be learned about the sample, i.e., the microenvironment of the fluorescence probe. Polarization-resolved fluorescence lifetime imaging-time-resolved fluorescence anisotropy imaging, TR-FAIM-allows mapping of viscosity or binding or of homo-FRET which can indicate dimerization or generally oligomerization.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Advanced imaging system

    NASA Technical Reports Server (NTRS)

    1992-01-01

    This document describes the Advanced Imaging System CCD based camera. The AIS1 camera system was developed at Photometric Ltd. in Tucson, Arizona as part of a Phase 2 SBIR contract No. NAS5-30171 from the NASA/Goddard Space Flight Center in Greenbelt, Maryland. The camera project was undertaken as a part of the Space Telescope Imaging Spectrograph (STIS) project. This document is intended to serve as a complete manual for the use and maintenance of the camera system. All the different parts of the camera hardware and software are discussed and complete schematics and source code listings are provided.

  11. Nonlinear fluorescence imaging by photoinduced charge separation

    NASA Astrophysics Data System (ADS)

    Mochizuki, Kentaro; Shi, Lanting; Mizukami, Shin; Yamanaka, Masahito; Tanabe, Mamoru; Gong, Wei-Tao; Palonpon, Almar F.; Kawano, Shogo; Kawata, Satoshi; Kikuchi, Kazuya; Fujita, Katsumasa

    2015-04-01

    Manipulation of the optical property of fluorescent probes has been a powerful strategy to establish super-resolution microscopy. We describe a new strategy to realize a probe with a nonlinear fluorescence response by using photoinduced charge separation. In this scheme, the first photon is used for the generation of the charge-separated state and the second photon is for fluorescence excitation. This stepwise two-photon absorption was confirmed by detection of a second-order nonlinear fluorescence response. Transient absorption spectra studies and simulation indicate that fluorescence is emitted through the photophysical pathways we proposed. Fluorescence imaging of biological cells showed marked improvements in image contrast and resolution, demonstrating the usefulness of the fluorescent probe in laser scanning confocal microscopy.

  12. NIR fluorescent ytterbium compound for in vivo fluorescence molecular imaging.

    PubMed

    Aita, Kazuki; Temma, Takashi; Kuge, Yuji; Seki, Koh-ichi; Saji, Hideo

    2010-01-01

    We have developed a new NIR fluorescent probe based on an ytterbium(III) (E)-1-(pyridin-2-yl-diazenyl)naphthalen-2-ol (PAN) complex. This probe emits near-infrared luminescence derived from the Yb ion through excitation of the PAN moiety with visible light (lambda(ex)= 530 nm, lambda(em)= 975 nm). The results support the possible utility of the probe for in vivo fluorescence molecular imaging.

  13. 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

  14. Boronic acids for fluorescence imaging of carbohydrates.

    PubMed

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

    2016-02-28

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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. Cancer detection by quantitative fluorescence image analysis.

    PubMed

    Parry, W L; Hemstreet, G P

    1988-02-01

    Quantitative fluorescence image analysis is a rapidly evolving biophysical cytochemical technology with the potential for multiple clinical and basic research applications. We report the application of this technique for bladder cancer detection and discuss its potential usefulness as an adjunct to methods used currently by urologists for the diagnosis and management of bladder cancer. Quantitative fluorescence image analysis is a cytological method that incorporates 2 diagnostic techniques, quantitation of nuclear deoxyribonucleic acid and morphometric analysis, in a single semiautomated system to facilitate the identification of rare events, that is individual cancer cells. When compared to routine cytopathology for detection of bladder cancer in symptomatic patients, quantitative fluorescence image analysis demonstrated greater sensitivity (76 versus 33 per cent) for the detection of low grade transitional cell carcinoma. The specificity of quantitative fluorescence image analysis in a small control group was 94 per cent and with the manual method for quantitation of absolute nuclear fluorescence intensity in the screening of high risk asymptomatic subjects the specificity was 96.7 per cent. The more familiar flow cytometry is another fluorescence technique for measurement of nuclear deoxyribonucleic acid. However, rather than identifying individual cancer cells, flow cytometry identifies cellular pattern distributions, that is the ratio of normal to abnormal cells. Numerous studies by others have shown that flow cytometry is a sensitive method to monitor patients with diagnosed urological disease. Based upon results in separate quantitative fluorescence image analysis and flow cytometry studies, it appears that these 2 fluorescence techniques may be complementary tools for urological screening, diagnosis and management, and that they also may be useful separately or in combination to elucidate the oncogenic process, determine the biological potential of tumors

  1. Advances in optical imaging for pharmacological studies

    PubMed Central

    Arranz, Alicia; Ripoll, Jorge

    2015-01-01

    Imaging approaches are an essential tool for following up over time representative parameters of in vivo models, providing useful information in pharmacological studies. Main advantages of optical imaging approaches compared to other imaging methods are their safety, straight-forward use and cost-effectiveness. A main drawback, however, is having to deal with the presence of high scattering and high absorption in living tissues. Depending on how these issues are addressed, three different modalities can be differentiated: planar imaging (including fluorescence and bioluminescence in vivo imaging), optical tomography, and optoacoustic approaches. In this review we describe the latest advances in optical in vivo imaging with pharmacological applications, with special focus on the development of new optical imaging probes in order to overcome the strong absorption introduced by different tissue components, especially hemoglobin, and the development of multimodal imaging systems in order to overcome the resolution limitations imposed by scattering. PMID:26441646

  2. 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.

  3. 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

  4. Advanced laser image recorder.

    PubMed

    Gramenopoulos, N; Hartfield, E D

    1972-12-01

    A laser image recorder is described, which is unique because of its advanced design and the state-of-the-art components employed to achieve high performance and versatility. The critical components are the pyramidal mirror scanner and the beam focusing lens. The scanner has a six-facet, beryllium mirror accurate to 0.33 sec of arc and rotating at 0-50,000 rpm on air bearings. A rapid change in speed is an important feature of this scanner. The focusing lens is diffraction limited with a flat field of 54 degrees , allowing a 90% duty cycle and the use of photographic film transported by a cylindrical drum. The lens converts the constant angular velocity of the reflected beam to a constant scanning velocity of the focused spot with a linearity of 0.05%. Maximum number of picture elements per line is 36,800 over a format of 228.6 mm. PMID:20119408

  5. 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

  6. Coral monitoring with fluorescence imaging lidar

    NASA Astrophysics Data System (ADS)

    Sasano, Masahiko; Kiriya, Nobuo; Yamanouchi, Hiroshi; Matsumoto, Akira; Hitomi, Kazuo; Tamura, Kenkichi

    2011-06-01

    It has been pointed out that globally hermatypic corals in coral reefs have been seriously damaged in recent years, and it is predicted that such damages will expand in area in the future. It is important to monitor corals globally, in detail, and over long-term periods, for preservation of the marine environment and biodiversity. The spot-check method, one of the major coral monitoring methods, is operated by snorkelers or divers, and therefore, its operation is limited by the seastate, and its monitoring areas are often for specific observation points. On the other hand, the satellite remote sensing, another major coral monitoring methods, can cover composite coral reef areas, but the image resolution is a few meters, and it is not possible to monitor small size coral colonies and deep sea areas. The boat-based fluorescence imaging lidar system has been developed to complement these coral monitoring methods. This system obtains linear coral observation data along the boat track, and makes it possible to build a cooperative coral monitoring network. Since most hermatypic corals have fluorescent proteins, living tissues can be monitored using the blue-to-green fluorescence from UV excitation. It is possible to observe the UV-excited fluorescence images from live coral even in the daytime, by the UV excited fluorescence imaging lidar. Additionally, laser bathymetry is also possible by time-of-flight measurement. We have succeeded in observing the pseudo-coral fluorescent images and depths down to 30 m depth at the testing basin. Secondly, we have succeeded in observing the live coral fluorescent images and their depths by the lidar system using a glass-bottom-boat at Taketomi island, Okinawa, Japan. The system summary and observed data are reported in this paper.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. Supercontinuum Stimulated Emission Depletion Fluorescence Lifetime Imaging

    SciTech Connect

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

    2012-06-13

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

  14. Depth dependence of vascular fluorescence imaging

    PubMed Central

    Davis, Mitchell A.; Shams Kazmi, S. M.; Ponticorvo, Adrien; Dunn, Andrew K.

    2011-01-01

    In vivo surface imaging of fluorescently labeled vasculature has become a widely used tool for functional brain imaging studies. Techniques such as phosphorescence quenching for oxygen tension measurements and indocyanine green fluorescence for vessel perfusion monitoring rely on surface measurements of vascular fluorescence. However, the depth dependence of the measured fluorescence signals has not been modeled in great detail. In this paper, we investigate the depth dependence of the measured signals using a three-dimensional Monte Carlo model combined with high resolution vascular anatomy. We found that a bulk-vascularization assumption to modeling the depth dependence of the signal does not provide an accurate picture of penetration depth of the collected fluorescence signal in most cases. Instead the physical distribution of microvasculature, the degree of absorption difference between extravascular and intravascular space, and the overall difference in absorption at the excitation and emission wavelengths must be taken into account to determine the depth penetration of the fluorescence signal. Additionally, we found that using targeted illumination can provide for superior surface vessel sensitivity over wide-field illumination, with small area detection offering an even greater amount of sensitivity to surface vasculature. Depth sensitivity can be enhanced by either increasing the detector area or increasing the illumination area. Finally, we see that excitation wavelength and vessel size can affect intra-vessel sampling distribution, as well as the amount of signal that originates from inside the vessel under targeted illumination conditions. PMID:22162824

  15. Chemical Approaches for Advanced Optical Imaging

    NASA Astrophysics Data System (ADS)

    Chen, Zhixing

    Advances in optical microscopy have been constantly expanding our knowledge of biological systems. The achievements therein are a result of close collaborations between physicists/engineers who build the imaging instruments and chemists/biochemists who design the corresponding probe molecules. In this work I present a number of chemical approaches for the development of advanced optical imaging methods. Chapter 1 provides an overview of the recent advances of novel imaging approaches taking advantage of chemical tag technologies. Chapter 2 describes the second-generation covalent trimethoprim-tag as a viable tool for live cell protein-specific labeling and imaging. In Chapter 3 we present a fluorescence lifetime imaging approach to map protein-specific micro-environment in live cells using TMP-Cy3 as a chemical probe. In Chapter 4, we present a method harnessing photo-activatable fluorophores to extend the fundamental depth limit in multi-photon microscopy. Chapter 5 describes the development of isotopically edited alkyne palette for multi-color live cell vibrational imaging of cellular small molecules. These studies exemplify the impact of modern chemical approaches in the development of advanced optical microscopies.

  16. Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Quan, Xiangyu; Nitta, Kouichi; Matoba, Osamu; Xia, Peng; Awatsuji, Yasuhiro

    2015-04-01

    Hybrid digital holographic microscopy that combines fluorescence microscopy and digital holographic microscopy into a single system for biological applications is proposed. In the proposed system, a phase image and a fluorescence image can be obtained simultaneously by selecting the different wavelengths of the fluorescent light and the phase measurement. Especially for biological applications, the cell structure can be obtained by the phase imaging based on digital holography and nucleus of the cell can be obtained by the fluorescence imaging. The measurement of fluorescence beads and egera densa presented the feasibility of simultaneous detection of both a phase image and a fluorescence image.

  17. 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.

  18. 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.

  19. Fluorescence lidar imaging of historical monuments.

    PubMed

    Weibring, P; Johansson, T; Edner, H; Svanberg, S; Sundnér, B; Raimondi, V; Cecchi, G; Pantani, L

    2001-11-20

    What is believed to be the first fluorescence imaging of the facades of a historical building, which was accomplished with a scanning fluorescence lidar system, is reported. The mobile system was placed at a distance of ~60 m from the medieval Lund Cathedral (Sweden), and a 355-nm pulsed laser beam was swept over the stone facades row by row while spectrally resolved fluorescence signals of each measurement point were recorded. By multispectral image processing, either by formation of simple spectral-band ratios or by use of multivariate techniques, areas with different spectral signatures were classified. In particular, biological growth was observed and different stone types were distinguished. The technique can yield data for use in facade status assessment and restoration planning. PMID:18364910

  20. Review of Neurosurgical Fluorescence Imaging Methodologies.

    PubMed

    Pogue, Brian W; Gibbs-Strauss, Summer; Valdés, Pablo A; Samkoe, Kimberley; Roberts, David W; Paulsen, Keith D

    2010-05-01

    Fluorescence imaging in neurosurgery has a long historical development, with several different biomarkers and biochemical agents being used, and several technological approaches. This review focuses on the different contrast agents, summarizing endogenous fluorescence, exogenously stimulated fluorescence and exogenous contrast agents, and then on tools used for imaging. It ends with a summary of key clinical trials that lead to consensus studies. The practical utility of protoporphyrin IX (PpIX) as stimulated by administration of δ-aminolevulinic acid (ALA) has had substantial pilot clinical studies and basic science research completed. Recently multi-center clinical trials using PpIx fluorescence to guide resection have shown efficacy for improved short term survival. Exogenous agents are being developed and tested pre-clinically, and hopefully hold the potential for long term survival benefit if they provide additional capabilities for resection of micro-invasive disease or certain tumor sub-types that do not produce PpIX or help delineate low grade tumors. The range of technologies used for measurement and imaging ranges widely, with most clinical trials being carried out with either point probes or modified surgical microscopes. At this point in time, optimized probe approaches are showing efficacy in clinical trials, and fully commercialized imaging systems are emerging, which will clearly help lead to adoption into neurosurgical practice.

  1. Review of Neurosurgical Fluorescence Imaging Methodologies

    PubMed Central

    Pogue, Brian W.; Gibbs-Strauss, Summer; Valdés, Pablo A.; Samkoe, Kimberley; Roberts, David W.; Paulsen, Keith D.

    2010-01-01

    Fluorescence imaging in neurosurgery has a long historical development, with several different biomarkers and biochemical agents being used, and several technological approaches. This review focuses on the different contrast agents, summarizing endogenous fluorescence, exogenously stimulated fluorescence and exogenous contrast agents, and then on tools used for imaging. It ends with a summary of key clinical trials that lead to consensus studies. The practical utility of protoporphyrin IX (PpIX) as stimulated by administration of δ-aminolevulinic acid (ALA) has had substantial pilot clinical studies and basic science research completed. Recently multi-center clinical trials using PpIx fluorescence to guide resection have shown efficacy for improved short term survival. Exogenous agents are being developed and tested pre-clinically, and hopefully hold the potential for long term survival benefit if they provide additional capabilities for resection of micro-invasive disease or certain tumor sub-types that do not produce PpIX or help delineate low grade tumors. The range of technologies used for measurement and imaging ranges widely, with most clinical trials being carried out with either point probes or modified surgical microscopes. At this point in time, optimized probe approaches are showing efficacy in clinical trials, and fully commercialized imaging systems are emerging, which will clearly help lead to adoption into neurosurgical practice. PMID:20671936

  2. Imaging the environment of green fluorescent protein.

    PubMed Central

    Suhling, Klaus; Siegel, Jan; Phillips, David; French, Paul M W; Lévêque-Fort, Sandrine; Webb, Stephen E D; Davis, Daniel M

    2002-01-01

    An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells. PMID:12496126

  3. Multiple frequency fluorescence lifetime imaging microscopy.

    PubMed

    Squire, A; Verveer, P J; Bastiaens, P I

    2000-02-01

    The experimental configuration and the computational algorithms for performing multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) are described. The mfFLIM experimental set-up enables the simultaneous homodyne detection of fluorescence emission modulated at a set of harmonic frequencies. This was achieved in practice by using monochromatic laser light as an excitation source modulated at a harmonic set of frequencies. A minimum of four frequencies were obtained by the use of two standing wave acousto-optic modulators placed in series. Homodyne detection at each of these frequencies was performed simultaneously by mixing with matching harmonics present in the gain characteristics of a microchannel plate (MCP) image intensifier. These harmonics arise as a natural consequence of applying a high frequency sinusoidal voltage to the photocathode of the device, which switches the flow of photoelectrons 'on' and 'off' as the sinus voltage swings from negative to positive. By changing the bias of the sinus it was possible to control the duration of the 'on' state of the intensifier relative to its 'off' state, enabling the amplitude of the higher harmonic content in the gain to be controlled. Relative modulation depths of 400% are theoretically possible from this form of square-pulse modulation. A phase-dependent integrated image is formed by the sum of the mixed frequencies on the phosphor of the MCP. Sampling this signal over a full period of the fundamental harmonic enables each harmonic to be resolved, provided that the Nyquist sampling criterion is satisfied for the highest harmonic component in the signal. At each frequency both the phase and modulation parameters can be estimated from a Fourier analysis of the data. These parameters enable the fractional populations and fluorescence lifetimes of individual components of a complex fluorescence decay to be resolved on a pixel-by-pixel basis using a non-linear fit to the dispersion relationships. The

  4. 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

  5. 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.

  6. Brain tumor resection guided by fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Leblond, Frederic; Fontaine, Kathryn M.; Valdes, Pablo; Ji, Songbai; Pogue, Brian W.; Hartov, Alex; Roberts, David W.; Paulsen, Keith D.

    2009-02-01

    We present the methods that are being used in the scope of an on-going clinical trial designed to assess the usefulness of ALA-PpIX fluorescence imaging when used in conjunction with pre-operative MRI. The overall objective is to develop imaging-based neuronavigation approaches to aid in maximizing the completeness of brain tumor resection, thereby improving patient survival rate. In this paper we present the imaging methods that are used, emphasizing technical aspects relating to the fluorescence optical microscope, including initial validation approaches based on phantom and small-animal experiments. The surgical workflow is then described in detail based on a high-grade glioma resection we performed.

  7. 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

  8. Advanced image memory architecture

    NASA Astrophysics Data System (ADS)

    Vercillo, Richard; McNeill, Kevin M.

    1994-05-01

    A workstation for radiographic images, known as the Arizona Viewing Console (AVC), was developed at the University of Arizona Health Sciences Center in the Department of Radiology. This workstation has been in use as a research tool to aid us in investigating how a radiologist interacts with a workstation, to determine which image processing features are required to aid the radiologist, to develop user interfaces and to support psychophysical and clinical studies. Results from these studies have show a need to increase the current image memory's available storage in order to accommodate high resolution images. The current triple-ported image memory can be allocated to store any number of images up to a combined total of 4 million pixels. Over the past couple of years, higher resolution images have become easier to generate with the advent of laser digitizers and computed radiology systems. As part of our research, a larger 32 million pixel image memory for AVC has been designed to replace the existing image memory.

  9. 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.

  10. 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

  11. 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.

  12. Modern Imaging Technology: Recent Advances

    SciTech Connect

    Welch, Michael J.; Eckelman, William C.

    2004-06-18

    This 2-day conference is designed to bring scientist working in nuclear medicine, as well as nuclear medicine practitioners together to discuss the advances in four selected areas of imaging: Biochemical Parameters using Small Animal Imaging, Developments in Small Animal PET Imaging, Cell Labeling, and Imaging Angiogenesis Using Multiple Modality. The presentations will be on molecular imaging applications at the forefront of research, up to date on the status of molecular imaging in nuclear medicine as well as in related imaging areas. Experts will discuss the basic science of imaging techniques, and scheduled participants will engage in an exciting program that emphasizes the current status of molecular imaging as well as the role of DOE funded research in this area.

  13. Correlation of the gallbladder stone and tissue fluorescent images

    NASA Astrophysics Data System (ADS)

    Kokaj, Jahja O.; Marafi, Mustafa A.; Makdisi, Yacob; Bhatia, Kuldip S.

    2001-11-01

    Fluorescent images of gallbladder stones, tissue and bile are obtained using a streak camera. A Match Spatial Filer (MSF) is made using a stone fluorescent image. The MSF is used to perform correlations with fluorescent tissue and bile image. A method for recognition of the stone and rejection of the tissue during the laser lithotripsy is proposed using the correlation outputs.

  14. 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

  15. Advanced imaging communication system

    NASA Technical Reports Server (NTRS)

    Hilbert, E. E.; Rice, R. F.

    1977-01-01

    Key elements of system are imaging and nonimaging sensors, data compressor/decompressor, interleaved Reed-Solomon block coder, convolutional-encoded/Viterbi-decoded telemetry channel, and Reed-Solomon decoding. Data compression provides efficient representation of sensor data, and channel coding improves reliability of data transmission.

  16. Tissue-Specific Near-Infrared Fluorescence Imaging.

    PubMed

    Owens, Eric A; Henary, Maged; El Fakhri, Georges; Choi, Hak Soo

    2016-09-20

    ) activatable targeting through an internal stimulus, which turns on fluorescence from an off state. Recent advances in nanomedicine and bioimaging offer much needed promise toward fulfilling these stringent requirements as we develop a successful catalog of targeted contrast agents for illuminating both tumors and vital tissues in the same surgical space by employing spectrally distinct fluorophores in real time. These tissue-specific contrast agents can be versatile arsenals to physicians for real-time intraoperative navigation as well as image-guided targeted therapy. There is a versatile library of tissue-specific fluorophores available in the literature, with many discussed herein, which offers clinicians an array of possibilities that will undoubtedly improve intraoperative success and long-term postoperation prognosis. PMID:27564418

  17. Tissue-Specific Near-Infrared Fluorescence Imaging.

    PubMed

    Owens, Eric A; Henary, Maged; El Fakhri, Georges; Choi, Hak Soo

    2016-09-20

    ) activatable targeting through an internal stimulus, which turns on fluorescence from an off state. Recent advances in nanomedicine and bioimaging offer much needed promise toward fulfilling these stringent requirements as we develop a successful catalog of targeted contrast agents for illuminating both tumors and vital tissues in the same surgical space by employing spectrally distinct fluorophores in real time. These tissue-specific contrast agents can be versatile arsenals to physicians for real-time intraoperative navigation as well as image-guided targeted therapy. There is a versatile library of tissue-specific fluorophores available in the literature, with many discussed herein, which offers clinicians an array of possibilities that will undoubtedly improve intraoperative success and long-term postoperation prognosis.

  18. 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

  19. Microscopy imaging device with advanced imaging properties

    SciTech Connect

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2015-11-24

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  20. Microscopy imaging device with advanced imaging properties

    DOEpatents

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2016-10-25

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  1. Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging.

    PubMed

    Streets, Aaron M; Li, Ang; Chen, Tao; Huang, Yanyi

    2014-09-01

    Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.

  2. Advanced Land Imager Assessment System

    NASA Technical Reports Server (NTRS)

    Chander, Gyanesh; Choate, Mike; Christopherson, Jon; Hollaren, Doug; Morfitt, Ron; Nelson, Jim; Nelson, Shar; Storey, James; Helder, Dennis; Ruggles, Tim; Kaita, Ed; Levy, Raviv; Ong, Lawrence; Markham, Brian; Schweiss, Robert

    2008-01-01

    The Advanced Land Imager Assessment System (ALIAS) supports radiometric and geometric image processing for the Advanced Land Imager (ALI) instrument onboard NASA s Earth Observing-1 (EO-1) satellite. ALIAS consists of two processing subsystems for radiometric and geometric processing of the ALI s multispectral imagery. The radiometric processing subsystem characterizes and corrects, where possible, radiometric qualities including: coherent, impulse; and random noise; signal-to-noise ratios (SNRs); detector operability; gain; bias; saturation levels; striping and banding; and the stability of detector performance. The geometric processing subsystem and analysis capabilities support sensor alignment calibrations, sensor chip assembly (SCA)-to-SCA alignments and band-to-band alignment; and perform geodetic accuracy assessments, modulation transfer function (MTF) characterizations, and image-to-image characterizations. ALIAS also characterizes and corrects band-toband registration, and performs systematic precision and terrain correction of ALI images. This system can geometrically correct, and automatically mosaic, the SCA image strips into a seamless, map-projected image. This system provides a large database, which enables bulk trending for all ALI image data and significant instrument telemetry. Bulk trending consists of two functions: Housekeeping Processing and Bulk Radiometric Processing. The Housekeeping function pulls telemetry and temperature information from the instrument housekeeping files and writes this information to a database for trending. The Bulk Radiometric Processing function writes statistical information from the dark data acquired before and after the Earth imagery and the lamp data to the database for trending. This allows for multi-scene statistical analyses.

  3. Neurolight -astonishing advances in brain imaging.

    PubMed

    Rojczyk-Gołębiewska, Ewa; Pałasz, Artur; Worthington, John J; Markowski, Grzegorz; Wiaderkiewicz, Ryszard

    2015-02-01

    In recent years, significant advances in basic neuroanatomical studies have taken place. Moreover, such classical, clinically-oriented human brain imaging methods such as MRI, PET and DTI have been applied to small laboratory animals allowing improvement in current experimental neuroscience. Contemporary structural neurobiology also uses various technologies based on fluorescent proteins. One of these is optogenetics, which integrates physics, genetics and bioengineering to enable temporal precise control of electrical activity of specific neurons. Another important challenge in the field is the accurate imaging of complicated neural networks. To address this problem, three-dimensional reconstruction techniques and retrograde labeling with modified viruses has been developed. However, a revolutionary step was the invention of the "Brainbow" system, utilizing gene constructs including the sequences of fluorescent proteins and the usage of Cre recombinase to create dozens of colour combinations, enabling visualization of neurons and their connections in extremely high resolution. Furthermore, the newly- introduced CLARITY method should make it possible to visualize three-dimensionally the structure of translucent brain tissue using the hydrogel polymeric network. This original technique is a big advance in neuroscience creating novel viewpoints completely different than standard glass slide immunostaining. PMID:24730999

  4. Neurolight -astonishing advances in brain imaging.

    PubMed

    Rojczyk-Gołębiewska, Ewa; Pałasz, Artur; Worthington, John J; Markowski, Grzegorz; Wiaderkiewicz, Ryszard

    2015-02-01

    In recent years, significant advances in basic neuroanatomical studies have taken place. Moreover, such classical, clinically-oriented human brain imaging methods such as MRI, PET and DTI have been applied to small laboratory animals allowing improvement in current experimental neuroscience. Contemporary structural neurobiology also uses various technologies based on fluorescent proteins. One of these is optogenetics, which integrates physics, genetics and bioengineering to enable temporal precise control of electrical activity of specific neurons. Another important challenge in the field is the accurate imaging of complicated neural networks. To address this problem, three-dimensional reconstruction techniques and retrograde labeling with modified viruses has been developed. However, a revolutionary step was the invention of the "Brainbow" system, utilizing gene constructs including the sequences of fluorescent proteins and the usage of Cre recombinase to create dozens of colour combinations, enabling visualization of neurons and their connections in extremely high resolution. Furthermore, the newly- introduced CLARITY method should make it possible to visualize three-dimensionally the structure of translucent brain tissue using the hydrogel polymeric network. This original technique is a big advance in neuroscience creating novel viewpoints completely different than standard glass slide immunostaining.

  5. 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.

  6. 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.

  7. FIND: Fluorescence Imaging in the Nuclear Domain

    SciTech Connect

    Barty, C J

    2005-02-14

    This document examines the potential use of Thomson-Radiated Extreme X-ray (T-REX) sources for Fluorescence Imaging in the Nuclear Domain (FIND) of special nuclear materials. A back-of-the-envelope, relative comparison of T-REX sources vs. Bremsstrahlung sources for this application is presented. It is estimated that use of T-REX for FIND could be as much as 5 x 10{sup 12} more effective than the use of anode based sources. Furthermore it is estimated that illumination of samples of dimension 1 cm on a side could produce up to {approx}10{sup 9} detectable photons per second.

  8. 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.

  9. Advances in high-resolution imaging – techniques for three-dimensional imaging of cellular structures

    PubMed Central

    Lidke, Diane S.; Lidke, Keith A.

    2012-01-01

    A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques. PMID:22685332

  10. 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

  11. 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

  12. 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.

  13. Advances in multimodality molecular imaging

    PubMed Central

    Zaidi, Habib; Prasad, Rameshwar

    2009-01-01

    Multimodality molecular imaging using high resolution positron emission tomography (PET) combined with other modalities is now playing a pivotal role in basic and clinical research. The introduction of combined PET/CT systems in clinical setting has revolutionized the practice of diagnostic imaging. The complementarity between the intrinsically aligned anatomic (CT) and functional or metabolic (PET) information provided in a “one-stop shop” and the possibility to use CT images for attenuation correction of the PET data has been the driving force behind the success of this technology. On the other hand, combining PET with Magnetic Resonance Imaging (MRI) in a single gantry is technically more challenging owing to the strong magnetic fields. Nevertheless, significant progress has been made resulting in the design of few preclinical PET systems and one human prototype dedicated for simultaneous PET/MR brain imaging. This paper discusses recent advances in PET instrumentation and the advantages and challenges of multimodality imaging systems. Future opportunities and the challenges facing the adoption of multimodality imaging instrumentation will also be addressed. PMID:20098557

  14. High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

    PubMed Central

    Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

    2011-01-01

    Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

  15. Live imaging of Tribolium castaneum embryonic development using light-sheet-based fluorescence microscopy.

    PubMed

    Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

    2015-10-01

    Tribolium castaneum has become an important insect model organism for evolutionary developmental biology, genetics and biotechnology. However, few protocols for live fluorescence imaging of Tribolium have been reported, and little image data is available. Here we provide a protocol for recording the development of Tribolium embryos with light-sheet-based fluorescence microscopy. The protocol can be completed in 4-7 d and provides procedural details for: embryo collection, microscope configuration, embryo preparation and mounting, noninvasive live imaging for up to 120 h along multiple directions, retrieval of the live embryo once imaging is completed, and image data processing, for which exemplary data is provided. Stringent quality control criteria for developmental biology studies are also discussed. Light-sheet-based fluorescence microscopy complements existing toolkits used to study Tribolium development, can be adapted to other insect species, and requires no advanced imaging or sample preparation skills.

  16. 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

  17. Recent advances in imaging subcellular processes

    PubMed Central

    Myers, Kenneth A.; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology. PMID:27408708

  18. Recent advances in imaging subcellular processes.

    PubMed

    Myers, Kenneth A; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology. PMID:27408708

  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. 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.

  1. 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

  2. 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.

  3. 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

  4. Ongoing advances in quantitative PpIX fluorescence guided intracranial tumor resection (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Olson, Jonathan D.; Kanick, Stephen C.; Bravo, Jaime J.; Roberts, David W.; Paulsen, Keith D.

    2016-03-01

    Aminolevulinc-acid induced protoporphyrin IX (ALA-PpIX) is being investigated as a biomarker to guide neurosurgical resection of brain tumors. ALA-PpIX fluorescence can be observed visually in the surgical field; however, raw fluorescence emissions can be distorted by factors other than the fluorophore concentration. Specifically, fluorescence emissions are mixed with autofluorescence and attenuated by background absorption and scattering properties of the tissue. Recent work at Dartmouth has developed advanced fluorescence detection approaches that return quantitative assessments of PpIX concentration, which are independent of background optical properties. The quantitative fluorescence imaging (qFI) approach has increased sensitivity to residual disease within the resection cavity at the end of surgery that was not visible to the naked eye through the operating microscope. This presentation outlines clinical observations made during an ongoing investigation of ALA-PpIX based guidance of tumor resection. PpIX fluorescence measurements made in a wide-field hyperspectral imaging approach are co-registered with point-assessment using a fiber optic probe. Data show variations in the measured PpIX accumulation among different clinical tumor grades (i.e. high grade glioma, low grade glioma), types (i.e. primary tumors. metastases) and normal structures of interest (e.g. normal cortex, hippocampus). These results highlight the contrast enhancement and underscore the potential clinical benefit offered from quantitative measurements of PpIX concentration during resection of intracranial tumors.

  5. Mapping intracellular biochemistry with fluorescence anisotropy imaging microscopy

    NASA Astrophysics Data System (ADS)

    Gough, Albert H.; Taylor, D. Lansing

    1994-08-01

    Fluorescence polarization anisotropy can be used to determine the rotational mobility of a fluorescent analog, detect anisotropic orientation distributions, or measure the fluorescence lifetime of a fluorophore. Steady state fluorescence anisotropy can be simply measured in a standard fluorescence microscope equipped with excitation and emission polarizers and therefore, two dimensional maps of fluorescence anisotropy can be easily acquired. We are using steady state fluorescence anisotropy imaging microscopy to study the biochemistry of cell motility. The optimum fluorophore for fluorescence polarization measurements has a fluorescence lifetime that is comparable to the rotational correlation time of the molecule of interest. In order to make imaging measurements with high sensitivity and reasonable time resolution, however, this general rule has to be adjusted, and we have found that FITC-calmodulin has a useful combination of the above features. Calmodulin is a key regulatory protein, that is proposed to be involved in the regulation of the actin-myosin II based force generation in non-muscle cells. The rotational mobility of macromolecules is very sensitive to molecular interactions, yet is relatively insensitive to any surrounding gel matrix. We have taken advantage of this feature to map FITC-calmodulin interactions in the complex cytomatrix in living cells by steady state Fluorescence Anisotropy Imaging Microscopy (FAIM). In addition, we are investigating the use of FAIM for mapping variations in molecular orientation distributions, and fluorescence lifetime distributions.

  6. 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

  7. 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.

  8. Photoactivation and imaging of optical highlighter fluorescent proteins.

    PubMed

    Patterson, George H

    2011-07-01

    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.

  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. Characterization of Fluorescent Polystyrene Microspheres for Advanced Flow Diagnostics

    NASA Technical Reports Server (NTRS)

    Maisto, Pietro M. F.; Lowe, K. Todd; Byun, Guibo; Simpson, Roger; Vercamp, Max; Danley, Jason E.; Koh, Brian; Tiemsin, Pacita; Danehy, Paul M.; Wohl, Christopher J.

    2013-01-01

    Fluorescent dye-doped polystyrene latex microspheres (PSLs) are being developed for velocimetry and scalar measurements in variable property flows. Two organic dyes, Rhodamine B (RhB) and dichlorofluorescence (DCF), are examined to assess laser-induced fluorescence (LIF) properties for flow imaging applications and single-shot temperature measurements. A major interest in the current research is the application of safe dyes, thus DCF is of particular interest, while RhB is used as a benchmark. Success is demonstrated for single-point laser Doppler velocimetry (LDV) and also imaging fluorescence, excited via a continuous wave 2 W laser beam, for exposures down to 10 ms. In contrast, when exciting with a pulsed Nd:YAG laser at 200 mJ/pulse, no fluorescence was detected, even when integrating tens of pulses. We show that this is due to saturation of the LIF signal at relatively low excitation intensities, 4-5 orders of magnitude lower than the pulsed laser intensity. A two-band LIF technique is applied in a heated jet, indicating that the technique effectively removes interfering inputs such as particle diameter variation. Temperature measurement uncertainties are estimated based upon the variance measured for the two-band LIF intensity ratio and the achievable dye temperature sensitivity, indicating that particles developed to date may provide about +/-12.5 C precision, while future improvements in dye temperature sensitivity and signal quality may enable single-shot temperature measurements with sub-degree precision.

  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. CMOS Time-Resolved, Contact, and Multispectral Fluorescence Imaging for DNA Molecular Diagnostics

    PubMed Central

    Guo, Nan; Cheung, Ka Wai; Wong, Hiu Tung; Ho, Derek

    2014-01-01

    Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA) detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS) technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art. PMID:25365460

  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

    2015-11-13

    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.

  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. System and methods for wide-field quantitative fluorescence imaging during neurosurgery.

    PubMed

    Valdes, Pablo A; Jacobs, Valerie L; Wilson, Brian C; Leblond, Frederic; Roberts, David W; Paulsen, Keith D

    2013-08-01

    We report an accurate, precise and sensitive method and system for quantitative fluorescence image-guided neurosurgery. With a low-noise, high-dynamic-range CMOS array, we perform rapid (integration times as low as 50 ms per wavelength) hyperspectral fluorescence and diffuse reflectance detection and apply a correction algorithm to compensate for the distorting effects of tissue absorption and scattering. Using this approach, we generated quantitative wide-field images of fluorescence in tissue-simulating phantoms for the fluorophore PpIX, having concentrations and optical absorption and scattering variations over clinically relevant ranges. The imaging system was tested in a rodent model of glioma, detecting quantitative levels down to 20 ng/ml. The resulting performance is a significant advance on existing wide-field quantitative imaging techniques, and provides performance comparable to a point-spectroscopy probe that has previously demonstrated significant potential for improved detection of malignant brain tumors during surgical resection. PMID:23903142

  16. 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.

  17. Recent advances in fluorescent probes for monitoring of hydrogen sulfide.

    PubMed

    Duan, Cun-Xian; Liu, Yu-Guo

    2013-01-01

    Hydrogen sulfide (H₂S), known for its unpleasant rotten egg smell and its high toxicity, has recently emerged as an important mediator of human physiological and pathological processes, such as the regulation of cell growth, cardiovascular protection, the stimulation of angiogenesis, gastric mucosal injury and Alzheimer's disease. Due to its significant actions in the physiology, H₂S has attracted the abundant concern of numerous researchers in the cutting edge of chemistry, biology and medicine. Recently, several fluorescent probes have been developed for detecting and elucidating the role played by H₂S in biological systems. This review highlights recent advances that have been made on the mechanism and applications of fluorescent probes for the detection of H₂S, demonstrating a new field in which remarkable improvements have been accomplished over the last two years.

  18. Intrinsic fluorescence of selenium nanoparticles for cellular imaging applications.

    PubMed

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

    2016-02-14

    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. PMID:26792107

  19. 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.

  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. Recent advances in imaging technologies in dentistry

    PubMed Central

    Shah, Naseem; Bansal, Nikhil; Logani, Ajay

    2014-01-01

    Dentistry has witnessed tremendous advances in all its branches over the past three decades. With these advances, the need for more precise diagnostic tools, specially imaging methods, have become mandatory. From the simple intra-oral periapical X-rays, advanced imaging techniques like computed tomography, cone beam computed tomography, magnetic resonance imaging and ultrasound have also found place in modern dentistry. Changing from analogue to digital radiography has not only made the process simpler and faster but also made image storage, manipulation (brightness/contrast, image cropping, etc.) and retrieval easier. The three-dimensional imaging has made the complex cranio-facial structures more accessible for examination and early and accurate diagnosis of deep seated lesions. This paper is to review current advances in imaging technology and their uses in different disciplines of dentistry. PMID:25349663

  3. 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.

  4. Fluorescence polarization imaging for delineating nonmelanoma skin cancers

    NASA Astrophysics Data System (ADS)

    Yaroslavsky, A. N.; Neel, V.; Anderson, R. R.

    2004-09-01

    We present a method for detecting nonmelanoma skin cancers using exogenous fluorescence polarization. We built an automated system that permits exogenous fluorescence polarization imaging. It includes a tunable linearly polarized monochromatic light source and a CCD camera equipped with a rotating linear polarizer and a filter to reject excitation light. Two fluorophores that are retained in tumors, toluidine blue and methylene blue, are employed. We demonstrate that fluorescence polarization imaging can be used for accurate delineation of nonmelanoma cancers. The results suggest that this optical technique may be suitable for real-time noninvasive demarcation of epithelial cancers.

  5. 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.

  6. Use of fluorescence lifetime imaging (FLIM) for latent fingerprints detection

    NASA Astrophysics Data System (ADS)

    Wang, Peng; Chao, Zhi Xia; Seah, Leong K.; Murukeshan, Vadakke M.

    2005-04-01

    Fluorescence lifetime imaging (FLIM) in frequency domain enables the mapping of the spatial distribution of fluorescence lifetimes of a specimen. FLIM can provide unique information about fluorophores and hence is widely used in biology and for medical diagnostics. In this paper, a theoretical analysis for the fluorescence lifetime determination of latent fingerprint samples is described, which is followed by the feasibility study of using FLIM in frequency domain for latent fingerprints detection. Experiments are carried out with fingerprint on green paper substrate and postcard substrate treated with certain fluorescent powder. The total phase lag and demodulation factor are calculated to determine the lifetimes pixel by pixel. The resulting fluorescence lifetime image of fingerprint revealed an improvement in the contrast, and was able to detect the latent fingerprint clearly.

  7. Analytical tools for single-molecule fluorescence imaging in cellulo.

    PubMed

    Leake, M C

    2014-07-01

    Recent technological advances in cutting-edge ultrasensitive fluorescence microscopy have allowed single-molecule imaging experiments in living cells across all three domains of life to become commonplace. Single-molecule live-cell data is typically obtained in a low signal-to-noise ratio (SNR) regime sometimes only marginally in excess of 1, in which a combination of detector shot noise, sub-optimal probe photophysics, native cell autofluorescence and intrinsically underlying stochasticity of molecules result in highly noisy datasets for which underlying true molecular behaviour is non-trivial to discern. The ability to elucidate real molecular phenomena is essential in relating experimental single-molecule observations to both the biological system under study as well as offering insight into the fine details of the physical and chemical environments of the living cell. To confront this problem of faithful signal extraction and analysis in a noise-dominated regime, the 'needle in a haystack' challenge, such experiments benefit enormously from a suite of objective, automated, high-throughput analysis tools that can home in on the underlying 'molecular signature' and generate meaningful statistics across a large population of individual cells and molecules. Here, I discuss the development and application of several analytical methods applied to real case studies, including objective methods of segmenting cellular images from light microscopy data, tools to robustly localize and track single fluorescently-labelled molecules, algorithms to objectively interpret molecular mobility, analysis protocols to reliably estimate molecular stoichiometry and turnover, and methods to objectively render distributions of molecular parameters.

  8. A study on a portable fluorescence imaging system

    NASA Astrophysics Data System (ADS)

    Chang, Han-Chao; Wu, Wen-Hong; Chang, Chun-Li; Huang, Kuo-Cheng; Chang, Chung-Hsing; Chiu, Shang-Chen

    2011-09-01

    The fluorescent reaction is that an organism or dye, excited by UV light (200-405 nm), emits a specific frequency of light; the light is usually a visible or near infrared light (405-900 nm). During the UV light irradiation, the photosensitive agent will be induced to start the photochemical reaction. In addition, the fluorescence image can be used for fluorescence diagnosis and then photodynamic therapy can be given to dental diseases and skin cancer, which has become a useful tool to provide scientific evidence in many biomedical researches. However, most of the methods on acquiring fluorescence biology traces are still stay in primitive stage, catching by naked eyes and researcher's subjective judgment. This article presents a portable camera to obtain the fluorescence image and to make up a deficit from observer competence and subjective judgment. Furthermore, the portable camera offers the 375nm UV-LED exciting light source for user to record fluorescence image and makes the recorded image become persuasive scientific evidence. In addition, when the raising the rate between signal and noise, the signal processing module will not only amplify the fluorescence signal up to 70 %, but also decrease the noise significantly from environmental light on bill and nude mouse testing.

  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. 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

  11. 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

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

    NASA Astrophysics Data System (ADS)

    Henderson, Clare A.; Nagul, Edward A.; Cattrall, Robert W.; Kolev, Spas D.; Smith, Trevor A.

    2014-06-01

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

  13. 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

  14. 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

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

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

    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.

  16. 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.

  17. A compact fluorescence and white light imaging system for intraoperative visualization of nerves

    NASA Astrophysics Data System (ADS)

    Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; Tan Hehir, Cristina

    2012-03-01

    Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

  18. Compact fluorescence and white-light imaging system for intraoperative visualization of nerves

    NASA Astrophysics Data System (ADS)

    Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; tan Hehir, Cristina

    2012-02-01

    Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

  19. Imaging tumor hypoxia by near-infrared fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Biswal, Nrusingh C.; Pavlik, Christopher; Smith, Michael B.; Aguirre, Andres; Xu, Yan; Zanganeh, Saeid; Kuhn, Liisa T.; Claffey, Kevin P.; Zhu, Quing

    2011-06-01

    We have developed a novel nitroimidazole indocyanine dye conjugate for tumor-targeted hypoxia fluorescence tomography. The hypoxia probe has been evaluated in vitro using tumor cell lines and in vivo with tumor targeting in mice. The in vitro cell studies were performed to assess fluorescence labeling differences between hypoxia and normoxia conditions. When treated with the hypoxia probe, a fluorescence emission ratio of 2.5-fold was found between the cells incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed by comparing the cells treated with indocyanine dye alone. In vivo tumor targeting in mice showed that the fluorescence signals measured at the tumor site were twice those at the normal site after 150 min post-injection of the hypoxia probe. On the other hand, the fluorescence signals measured after injection of indocyanine dye were the same at tumor and normal sites. In vivo fluorescence tomography images of mice injected with the hypoxia probe showed that the probe remained for more than 5 to 7 h in the tumors, however, the images of mice injected with indocyanine only dye confirmed that the unbound dye washed out in less than 3 h. These findings are supported with fluorescence images of histological sections of tumor samples using a Li-COR scanner and immunohistochemistry technique for tumor hypoxia.

  20. 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.

  1. Advanced optical imaging techniques for neurodevelopment.

    PubMed

    Wu, Yicong; Christensen, Ryan; Colón-Ramos, Daniel; Shroff, Hari

    2013-12-01

    Over the past decade, developmental neuroscience has been transformed by the widespread application of confocal and two-photon fluorescence microscopy. Even greater progress is imminent, as recent innovations in microscopy now enable imaging with increased depth, speed, and spatial resolution; reduced phototoxicity; and in some cases without external fluorescent probes. We discuss these new techniques and emphasize their dramatic impact on neurobiology, including the ability to image neurons at depths exceeding 1mm, to observe neurodevelopment noninvasively throughout embryogenesis, and to visualize neuronal processes or structures that were previously too small or too difficult to target with conventional microscopy.

  2. Advanced Optical Imaging Techniques for Neurodevelopment

    PubMed Central

    Wu, Yicong; Christensen, Ryan; Colón-Ramos, Daniel; Shroff, Hari

    2013-01-01

    Over the past decade, developmental neuroscience has been transformed by the widespread application of confocal and two-photon fluorescence microscopy. Even greater progress is imminent, as recent innovations in microscopy now enable imaging with increased depth, speed, and spatial resolution; reduced phototoxicity; and in some cases without external fluorescent probes. We discuss these new techniques and emphasize their dramatic impact on neurobiology, including the ability to image neurons at depths exceeding 1 mm, to observe neurodevelopment noninvasively throughout embryogenesis, and to visualize neuronal processes or structures that were previously too small or too difficult to target with conventional microscopy. PMID:23831260

  3. 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.

  4. 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.

  5. Fluorescence lidar multi-color imaging of vegetation

    NASA Technical Reports Server (NTRS)

    Johansson, J.; Wallinder, E.; Edner, H.; Svanberg, S.

    1992-01-01

    Multi-color imaging of vegetation fluorescence following laser excitation is reported for distances of 50 m. A mobile laser radar system equipped with a Nd:YAG laser transmitter and a 40 cm diameter telescope was used. Image processing allows extraction of information related to the physiological status of the vegetation and might prove useful in forest decline research.

  6. Diagnosis of colon cancer using frequency domain fluorescence imaging technique

    NASA Astrophysics Data System (ADS)

    Dinish, U. S.; Gulati, P.; Murukeshan, V. M.; Seah, L. K.

    2007-03-01

    Early detection and treatment of colon cancer has been associated with better disease prognosis. Conventional and reported optical techniques have limitations in detecting early stages of colon cancer growth. In this paper, a homodyne signal processing assisted frequency domain (FD) fluorescence imaging methodology is proposed for the early diagnosis of colon cancer. Simulated phantom tissues representing the biopsy samples at different stages of colon cancer growth are prepared and used for the imaging study. Selective imaging of healthy and diseased sites simulated in the samples was achieved even for fluorescence emissions having close lifetimes and wavelength values. Possible extension of the methodology for in vivo investigations is also discussed.

  7. A fluorescent, paramagnetic and PEGylated gold/silica nanoparticle for MRI, CT and fluorescence imaging

    PubMed Central

    van Schooneveld, Matti M.; Cormode, David P.; Koole, Rolf; van Wijngaarden, J. Timon; Calcagno, Claudia; Skajaa, Torjus; Hilhorst, Jan; ’t Hart, Dannis C.; Fayad, Zahi A.; Mulder, Willem J. M.; Meijerink, Andries

    2013-01-01

    An important challenge in medical diagnostics is to design all-in-one contrast agents that can be detected with multiple techniques such as magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), single photon emission tomography (SPECT) or fluorescence imaging (FI). Although many dual labeled agents have been proposed, mainly for combined MRI/FI, constructs for three imaging modalities are scarce. Here gold/silica nanoparticles with a poly(ethylene glycol), paramagnetic and fluorescent lipid coating were synthesized, characterized and applied as trimodal contrast agents to allow for nanoparticle-enhanced imaging of macrophage cells in vitro via MRI, CT and FI, and mice livers in vivo via MRI and CT. This agent can be a useful tool in a multitude of applications, including cell tracking and target-specific molecular imaging, and is a step in the direction of truly multi-modal imaging. PMID:20812290

  8. 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.

  9. Image-guided tumor surgery: will there be a role for fluorescent nanoparticles?

    PubMed Central

    Hill, Tanner K.; Mohs, Aaron M.

    2016-01-01

    Image-guided surgery (IGS) using fluorescent nanoparticles (NPs) has the potential to substantially impact patient treatment. The use of fluorescence imaging provides surgeons with real-time feedback on the location of diseased tissue using safe, low-cost imaging agents and instrumentation. Fluorescent NPs are likely to play a role as they are capable of taking advantage of the enhanced permeability and retention (EPR) effect and can be modified to avoid clearance, increase circulation time, and specifically target tumors. Clinical trials of IGS using the FDA-approved fluorophores indocyanine green and methylene blue have already shown preliminary successes, and incorporation of fluorescent NPs will likely improve detection by providing higher signal to background ratio and reducing false-positive rates through active targeting. Preclinical development of fluorescent NP formulations is advancing rapidly, with strategies ranging from passive targeting to active targeting of cell surface receptors, creating pH-responsive NPs, and increasing cell uptake through cleavable proteins. This collective effort could lead to clinical trials using fluorescent NPs in the near future. PMID:26585556

  10. Thermally activated delayed fluorescence of fluorescein derivative for time-resolved and confocal fluorescence imaging.

    PubMed

    Xiong, Xiaoqing; Song, Fengling; Wang, Jingyun; Zhang, Yukang; Xue, Yingying; Sun, Liangliang; Jiang, Na; Gao, Pan; Tian, Lu; Peng, Xiaojun

    2014-07-01

    Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

  11. Not seeing is not believing: improving the visibility of your fluorescence images

    PubMed Central

    Johnson, Jayme

    2012-01-01

    The digital age has brought both technical advances and ethical quandaries regarding data acquisition and image presentation in the field of cell biology. Image manipulation has drawn considerable attention in the past decade, leading to general guidelines for ethical data processing. However, effective methods of image presentation have been discussed only cursorily and have been largely overlooked. Under standard viewing conditions, the human visual system imposes limitations for readers analyzing fluorescence images. In this paper, I discuss the advantages and limitations of image-manipulation techniques with respect to the human visual system, including contrast stretching, nonlinear grayscale transformations, and pseudocoloring. While online data viewing presents innovative ways to access image information, most images continue to be viewed in static publications, in which image presentation is critical for effective information transmission. PMID:22379119

  12. Segmentation and classification of cell cycle phases in fluorescence imaging.

    PubMed

    Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

    2009-01-01

    Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

  13. A fluorescence imaging lidar for the control of cultural heritage

    NASA Astrophysics Data System (ADS)

    Palombi, Lorenzo; Cecchi, Giovanna; Lognoli, David; Raimondi, Valentina; Masotti, Leonardo

    2007-10-01

    The fluorescence lidar imaging technique turns particularly useful for the control of monuments. The investigated topics range from the detection of biodeteriogens to the characterization of stones and other masonry or restoration materials, such as protective treatments. In addition, the fluorescence lidar imaging is a non-destructive technique offering the possibility of being carried out in situ without the use of scaffolding that, beside being costly, limits the access to the monument and its use. This paper presents the main technical features of a new fluorescence imaging lidar system specifically developed for the diagnostics on the cultural heritage, whose operative conditions include outdoor and indoor environments, and the possibility of monitoring vaults and ceilings. This fluorescence lidar prototype is mainly composed of a Q-switched, tripled frequency Nd:YAG laser (@355 nm), a 1 m focal length Newtonian telescope and a 300 mm focal length spectrometer coupled to an intensified, gated 512 x 512 CCD detector. Imaging is carried out via a scanning system realized with a computer controlled mirror. The lidar prototype includes also a target pointing system for referencing the acquired fluorescence images on the target.

  14. Deep tissue fluorescence imaging and in vivo biological applications

    NASA Astrophysics Data System (ADS)

    Crosignani, Viera; Dvornikov, Alexander; Aguilar, Jose S.; Stringari, Chiara; Edwards, Robert; Mantulin, William W.; Gratton, Enrico

    2012-11-01

    We describe a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.

  15. Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging.

    PubMed

    Hense, Anika; Prunsche, Benedikt; Gao, Peng; Ishitsuka, Yuji; Nienhaus, Karin; Nienhaus, G Ulrich

    2015-12-09

    The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M(-1)cm(-1), mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths.

  16. 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.

  17. Application of fluorescence labeled liposome nanoparticles in the cell imaging

    NASA Astrophysics Data System (ADS)

    Hu, Jianbing; Li, Huimin; He, Xiaoxiao; Gong, Ping; Wang, Kemin; Zhang, Shouchun

    2007-05-01

    Fluorescence labeled liposome nanoparticles were prepared by dispersion of film method. The size of nanoparticles was around 50 nm. DPPE-FITC synthesized in our lab was used to label the liposome nanoparticles. Anti-cytokeratins 19 antibody was connected to the surface of the fluorescence liposome nanoparticles. After incubation with MGC cells and COS-7 cells for 30 min, MGC cells were selectively recognized by anti-cytokeratins 19 antibody modified liposome nanoparticles and well imaged under laser confocal microscope. This fluorescence labeled liposome nanoparticles is expected to have good applications in cell recognition and tumor diagnosis.

  18. Imaging of a targeted PDT drug with fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Muffoletto, Dan; Gupta, Anurag; Xu, Zhiqiang; Mahrer, Chris; Bauer, Gretchen; Galas, Scott; Pandey, Ravindra K.; Sunar, Ulas

    2009-02-01

    We constructed a whole-body fluorescence tomography instrument to monitor novel bifunctional phototherapeutic drugs (e.g., HPPH-Cyanine dye conjugate) in small animals. The instrument allows dense source and detector sampling with a fast galvo scanner and a CCD detector for improved resolution and sensitivity (Patwardhan et al., 2005). Here we report tissue phantom measurements to evaluate the imaging performance with a newly constructed tomography instrument. Phantom measurements showed that strong fluorescence generated by HPPH-Cyanine dye (HPPH-CD), having high fluorescence quantum yield and long wavelength fluorescence emission, allowed deep tissue imaging. We also report in vivo fluorescence measurements of the conjugate in Nude mice bearing A549 human non-small cell lung carcinoma (NSCLC) tumors at 24 hr post injection to evaluate tumor detection ability of the conjugate. Our results indicate that the HPPH-CD shows preferential uptake in tumors compared to surrounding normal tissue at 24 hr post injection. This study demonstrates a potential use of HPPH-CD in detection (fluorescence imaging) and treatment (PDT) of deeply seated tumors.

  19. 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.

  20. Community detection for fluorescent lifetime microscopy image segmentation

    NASA Astrophysics Data System (ADS)

    Hu, Dandan; Sarder, Pinaki; Ronhovde, Peter; Achilefu, Samuel; Nussinov, Zohar

    2014-03-01

    Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes.1 In the current paper, we further explore this method in FLT images of ex vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or "solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1:25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.

  1. 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

  2. Orange/Red Fluorescence of Active Caries by Retrospective Quantitative Light-Induced Fluorescence Image Analysis.

    PubMed

    Felix Gomez, Grace; Eckert, George J; Ferreira Zandona, Andrea

    2016-01-01

    This retrospective clinical study determined the association of caries activity and orange/red fluorescence on quantitative light-induced fluorescence (QLF) images of surfaces that progressed to cavitation, as determined by clinical visual examination. A random sample of QLF images from 565 children (5-13 years) previously enrolled in a longitudinal study was selected. Buccal, lingual and occlusal surface images obtained after professional brushing at baseline and every 4 months over a 4-year period were analyzed for red fluorescence. Surfaces that progressed (n = 224) to cavitation according to the International Caries Detection and Assessment System (ICDAS 0/1/2/3/4 to 5/6 or filling), and surfaces that did not progress (n = 486) were included. QA2 image analysis software outputs the percentage increase of the red/green components as x0394;R and area of x0394;R (areax0394;R) at different thresholds. Mixed-model ANOVA was used to compare progressive and nonprogressive surfaces to account for correlations of red fluorescence (x0394;R and areax0394;R) between surfaces within a subject. The first analysis used the first observation for each surface or the first available visit if the surface was unerupted (baseline), while the second analysis used the last observation prior to cavitation for surfaces that progressed and the last observation for surfaces that did not progress (final). There was a significant (p < 0.05) association between red fluorescence and progression to cavitation at thresholds x0394;R0, x0394;R10, x0394;R20, x0394;R60, x0394;R70, x0394;R80, x0394;R90 and x0394;Rmax at baseline and for x0394;R0 and x0394;R10 at the final observation. Quantification of orange/red fluorescence may help to identify lesions that progress to cavitation. Future studies identifying microbiological factors causing orange/ red fluorescence and its caries activity are indicated. PMID:27160323

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. Advanced Pointing Imaging Camera (APIC) Concept

    NASA Astrophysics Data System (ADS)

    Park, R. S.; Bills, B. G.; Jorgensen, J.; Jun, I.; Maki, J. N.; McEwen, A. S.; Riedel, E.; Walch, M.; Watkins, M. M.

    2016-10-01

    The Advanced Pointing Imaging Camera (APIC) concept is envisioned as an integrated system, with optical bench and flight-proven components, designed for deep-space planetary missions with 2-DOF control capability.

  9. Recent Advancements in Microwave Imaging Plasma Diagnostics

    SciTech Connect

    H. Park; C.C. Chang; B.H. Deng; C.W. Domier; A.J.H. Donni; K. Kawahata; C. Liang; X.P. Liang; H.J. Lu; N.C. Luhmann, Jr.; A. Mase; H. Matsuura; E. Mazzucato; A. Miura; K. Mizuno; T. Munsat; K. and Y. Nagayama; M.J. van de Pol; J. Wang; Z.G. Xia; W-K. Zhang

    2002-03-26

    Significant advances in microwave and millimeter wave technology over the past decade have enabled the development of a new generation of imaging diagnostics for current and envisioned magnetic fusion devices. Prominent among these are revolutionary microwave electron cyclotron emission imaging (ECEI), microwave phase imaging interferometers, imaging microwave scattering and microwave imaging reflectometer (MIR) systems for imaging electron temperature and electron density fluctuations (both turbulent and coherent) and profiles (including transport barriers) on toroidal devices such as tokamaks, spherical tori, and stellarators. The diagnostic technology is reviewed, and typical diagnostic systems are analyzed. Representative experimental results obtained with these novel diagnostic systems are also presented.

  10. 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.

  11. Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

    PubMed

    Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

    2012-11-01

    Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays.

  12. Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast.

    PubMed

    Yao, Junjie; Shcherbakova, Daria M; Li, Chiye; Krumholz, Arie; Lorca, Ramon A; Reinl, Erin; England, Sarah K; Verkhusha, Vladislav V; Wang, Lihong V

    2014-08-01

    Confocal microscopy with optical sectioning has revolutionized biological studies by providing sharper images than conventional optical microscopy. Here, we introduce a fluorescence imaging method with enhanced resolution and imaging contrast, which can be implemented using a commercial confocal microscope setup. This approach, called the reversibly switchable photo-imprint microscopy (rsPIM), is based on the switching dynamics of reversibly switchable fluorophores. When the fluorophores are switched from the bright (ON) state to the dark (OFF) state, their switching rate carries the information about the local excitation light intensity. In rsPIM, a polynomial function is used to fit the fluorescence signal decay during the transition. The extracted high-order coefficient highlights the signal contribution from the center of the excitation volume, and thus sharpens the resolution in all dimensions. In particular, out-of-focus signals are greatly blocked for large targets, and thus the image contrast is considerably enhanced. Notably, since the fluorophores can be cycled between the ON and OFF states, the whole imaging process can be repeated. RsPIM imaging with enhanced image contrast was demonstrated in both fixed and live cells using a reversibly switchable synthetic dye and a genetically encoded red fluorescent protein. Since rsPIM does not require the modification of commercial microscope systems, it may provide a simple and cost-effective solution for subdiffraction imaging of live cells. PMID:25144452

  13. 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

  14. Fast globally optimal segmentation of cells in fluorescence microscopy images.

    PubMed

    Bergeest, Jan-Philip; Rohr, Karl

    2011-01-01

    Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

  15. Color-matched esophagus phantom for fluorescent imaging

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

    We developed a stable, reproducible three-dimensional optical phantom for the evaluation of a wide-field endoscopic molecular imaging system. This phantom mimicked a human esophagus structure with flexibility to demonstrate body movements. At the same time, realistic visual appearance and diffuse spectral reflectance properties of the tissue were simulated by a color matching methodology. A photostable dye-in-polymer technology was applied to represent biomarker probed "hot-spot" locations. Furthermore, fluorescent target quantification of the phantom was demonstrated using a 1.2mm ultrathin scanning fiber endoscope with concurrent fluorescence-reflectance imaging.

  16. Mesh adaptation technique for Fourier-domain fluorescence lifetime imaging

    SciTech Connect

    Soloviev, Vadim Y.

    2006-11-15

    A novel adaptive mesh technique in the Fourier domain is introduced for problems in fluorescence lifetime imaging. A dynamical adaptation of the three-dimensional scheme based on the finite volume formulation reduces computational time and balances the ill-posed nature of the inverse problem. Light propagation in the medium is modeled by the telegraph equation, while the lifetime reconstruction algorithm is derived from the Fredholm integral equation of the first kind. Stability and computational efficiency of the method are demonstrated by image reconstruction of two spherical fluorescent objects embedded in a tissue phantom.

  17. Desktop supercomputers. Advance medical imaging.

    PubMed

    Frisiello, R S

    1991-02-01

    Medical imaging tools that radiologists as well as a wide range of clinicians and healthcare professionals have come to depend upon are emerging into the next phase of functionality. The strides being made in supercomputing technologies--including reduction of size and price--are pushing medical imaging to a new level of accuracy and functionality.

  18. 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.

  19. A Patch-Based Method for Repetitive and Transient Event Detection in Fluorescence Imaging

    PubMed Central

    Boulanger, Jérôme; Gidon, Alexandre; Kervran, Charles; Salamero, Jean

    2010-01-01

    Automatic detection and characterization of molecular behavior in large data sets obtained by fast imaging in advanced light microscopy become key issues to decipher the dynamic architectures and their coordination in the living cell. Automatic quantification of the number of sudden and transient events observed in fluorescence microscopy is discussed in this paper. We propose a calibrated method based on the comparison of image patches expected to distinguish sudden appearing/vanishing fluorescent spots from other motion behaviors such as lateral movements. We analyze the performances of two statistical control procedures and compare the proposed approach to a frame difference approach using the same controls on a benchmark of synthetic image sequences. We have then selected a molecular model related to membrane trafficking and considered real image sequences obtained in cells stably expressing an endocytic-recycling trans-membrane protein, the Langerin-YFP, for validation. With this model, we targeted the efficient detection of fast and transient local fluorescence concentration arising in image sequences from a data base provided by two different microscopy modalities, wide field (WF) video microscopy using maximum intensity projection along the axial direction and total internal reflection fluorescence microscopy. Finally, the proposed detection method is briefly used to statistically explore the effect of several perturbations on the rate of transient events detected on the pilot biological model. PMID:20976222

  20. Optofluidic fluorescent imaging cytometry on a cell phone.

    PubMed

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

    2011-09-01

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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. Diagnostic imaging advances in murine models of colitis

    PubMed Central

    Brückner, Markus; Lenz, Philipp; Mücke, Marcus M; Gohar, Faekah; Willeke, Peter; Domagk, Dirk; Bettenworth, Dominik

    2016-01-01

    Inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis are chronic-remittent inflammatory disorders of the gastrointestinal tract still evoking challenging clinical diagnostic and therapeutic situations. Murine models of experimental colitis are a vital component of research into human IBD concerning questions of its complex pathogenesis or the evaluation of potential new drugs. To monitor the course of colitis, to the present day, classical parameters like histological tissue alterations or analysis of mucosal cytokine/chemokine expression often require euthanasia of animals. Recent advances mean revolutionary non-invasive imaging techniques for in vivo murine colitis diagnostics are increasingly available. These novel and emerging imaging techniques not only allow direct visualization of intestinal inflammation, but also enable molecular imaging and targeting of specific alterations of the inflamed murine mucosa. For the first time, in vivo imaging techniques allow for longitudinal examinations and evaluation of intra-individual therapeutic response. This review discusses the latest developments in the different fields of ultrasound, molecularly targeted contrast agent ultrasound, fluorescence endoscopy, confocal laser endomicroscopy as well as tomographic imaging with magnetic resonance imaging, computed tomography and fluorescence-mediated tomography, discussing their individual limitations and potential future diagnostic applications in the management of human patients with IBD. PMID:26811642

  6. Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

    PubMed Central

    Changou, Chun A.; Wolfson, Deanna L.; Ahluwalia, Balpreet Singh; Bold, Richard J.; Kung, Hsing-Jien; Chuang, Frank Y.S.

    2013-01-01

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12. Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of

  7. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-01-01

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  8. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-05-03

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  9. Advanced Imaging Algorithms for Radiation Imaging Systems

    SciTech Connect

    Marleau, Peter

    2015-10-01

    The intent of the proposed work, in collaboration with University of Michigan, is to develop the algorithms that will bring the analysis from qualitative images to quantitative attributes of objects containing SNM. The first step to achieving this is to develop an indepth understanding of the intrinsic errors associated with the deconvolution and MLEM algorithms. A significant new effort will be undertaken to relate the image data to a posited three-dimensional model of geometric primitives that can be adjusted to get the best fit. In this way, parameters of the model such as sizes, shapes, and masses can be extracted for both radioactive and non-radioactive materials. This model-based algorithm will need the integrated response of a hypothesized configuration of material to be calculated many times. As such, both the MLEM and the model-based algorithm require significant increases in calculation speed in order to converge to solutions in practical amounts of time.

  10. Five-color fluorescent imaging in living tumor cells

    NASA Astrophysics Data System (ADS)

    Wang, Liang; Yang, Jie; Chu, Jun; Luo, Qingming; Zhang, Zhihong

    2008-12-01

    The fluorescent probes based on fluorescent proteins (FP) have been widely used to investigate the molecules of interest in living cells. It is well-known that the molecular events in the living cells are very complicate and all of the cell activities are involved by multi-molecular interaction. With the development of novel fluorescent protein mutants and imaging technology, the molecular signal in living cells could be detected accurately. In this study, with the appropriate targeting signals, the fluorescent proteins were localized to plasma membrane (Rac1-mCerulean), Golgi membrane (EYFP-go), ER membrane (RFP2-er), mitochondrial membrane (RFP1-mt). Cultured Hela cells were cotransfected with these four plasmids, and 36 h later, labeled with Hoechst33258 which located in the nucleus of a living cell. Using a confocal microscopy, with 405 nm, 458 nm and 514 nm laser lines employed respectively, a five-color fluorescent image was obtained in which five subcellular structures were clearly shown in living cells. The technique of multi-color imaging in a single cell provides a powerful tool to simultaneously study the multi-molecular events in living cells.

  11. 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.

  12. 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.

  13. 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.

  14. Real-time absorption reduced surface fluorescence imaging

    PubMed Central

    Yang, Bin; Tunnell, James W.

    2014-01-01

    Abstract. We introduce a technique that limits absorption effects in fluorescence imaging and does not require extensive imaging processing, thus allowing for video rate imaging. The absorption minimization is achieved using spatial frequency domain imaging at a single high spatial frequency with standard three-phase demodulation. At a spatial frequency f=0.5  mm−1, we demonstrated in both in-vitro phantoms and ex-vivo tissue that the absorption can be significantly reduced. In the real-time implementation, we achieved a video rate of 19  frames/s. This technique has potential in cancer visualization and tumor margin detection. PMID:25250826

  15. Real-time absorption reduced surface fluorescence imaging.

    PubMed

    Yang, Bin; Tunnell, James W

    2014-09-01

    We introduce a technique that limits absorption effects in fluorescence imaging and does not require extensive imaging processing, thus allowing for video rate imaging. The absorption minimization is achieved using spatial frequency domain imaging at a single high spatial frequency with standard three-phase demodulation. At a spatial frequency f ¼ 0.5 mm−1, we demonstrated in both in-vitro phantoms and ex-vivo tissue that the absorption can be significantly reduced. In the real-time implementation, we achieved a video rate of 19 frames∕s. This technique has potential in cancer visualization and tumor margin detection. PMID:25250826

  16. 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.

  17. Doped semiconductor nanocrystal based fluorescent cellular imaging probes

    NASA Astrophysics Data System (ADS)

    Maity, Amit Ranjan; Palmal, Sharbari; Basiruddin, Sk; Karan, Niladri Sekhar; Sarkar, Suresh; Pradhan, Narayan; Jana, Nikhil R.

    2013-05-01

    Doped semiconductor nanocrystals such as Mn doped ZnS, Mn doped ZnSe and Cu doped InZnS, are considered as new classes of fluorescent biological probes with low toxicity. Although the synthesis in high quality of such nanomaterials is now well established, transforming them into functional fluorescent probes remains a challenge. Here we report a fluorescent cellular imaging probe made of high quality doped semiconductor nanocrystals. We have identified two different coating approaches suitable for transforming the as synthesized hydrophobic doped semiconductor nanocrystals into water-soluble functional nanoparticles. Following these approaches we have synthesized TAT-peptide- and folate-functionalized nanoparticles of 10-80 nm hydrodynamic diameter and used them as a fluorescent cell label. The results shows that doped semiconductor nanocrystals can be an attractive alternative for conventional cadmium based quantum dots with low toxicity.Doped semiconductor nanocrystals such as Mn doped ZnS, Mn doped ZnSe and Cu doped InZnS, are considered as new classes of fluorescent biological probes with low toxicity. Although the synthesis in high quality of such nanomaterials is now well established, transforming them into functional fluorescent probes remains a challenge. Here we report a fluorescent cellular imaging probe made of high quality doped semiconductor nanocrystals. We have identified two different coating approaches suitable for transforming the as synthesized hydrophobic doped semiconductor nanocrystals into water-soluble functional nanoparticles. Following these approaches we have synthesized TAT-peptide- and folate-functionalized nanoparticles of 10-80 nm hydrodynamic diameter and used them as a fluorescent cell label. The results shows that doped semiconductor nanocrystals can be an attractive alternative for conventional cadmium based quantum dots with low toxicity. Electronic supplementary information available: Characterization details of coating and

  18. 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.

  19. 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.

  20. 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

  1. Polyacrylamide based ICG nanocarriers for enhanced fluorescence and photoacoustic imaging

    NASA Astrophysics Data System (ADS)

    Ray, Aniruddha; Yoon, Hyung Ki; Ryu, HeeJu; Koo Lee, Yong-Eun; Kim, Gwangseong; Wang, Xueding; Kopelman, Raoul

    2013-02-01

    Indocyanine green (ICG) is an FDA approved tricarbocyanine dye. This dye, with a strong absorbance in the near infrared (NIR) region, has been extensively used for fluorescence and photoacoustic imaging in vivo. ICG in its free form, however, has a few drawbacks that limit its in vivo applications, such as non-targetability, tendency to form aggregates which changes its optical properties, fast degradation, short plasma lifetime and reduced fluorescence at body temperature. In order to bypass these inherent drawbacks, we demonstrate a polyacrylamide based nanocarrier that was particularly designed to carry the negatively charged ICG molecules. These nanocarriers are biodegradable, biocompatible and can be specifically targeted to any cell or tissue. Using these nanocarriers we avoid all the problems associated with free ICG, such as degradation, aggregation and short plasma lifetime, and also enhance demonstrate its ability towards photoacoustics and fluorescence imaging.

  2. Imaging of the pancreas: Recent advances

    PubMed Central

    Chaudhary, Vikas; Bano, Shahina

    2011-01-01

    A wide spectrum of anomalies of pancreas and the pancreatic duct system are commonly encountered at radiological evaluation. Diagnosing pancreatic lesions generally requires a multimodality approach. This review highlights the new advances in pancreatic imaging and their applications in the diagnosis and management of pancreatic pathologies. The mainstay techniques include computed tomography (CT), magnetic resonance imaging (MRI), endoscopic ultrasound (EUS), radionuclide imaging (RNI) and optical coherence tomography (OCT). PMID:21847450

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

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

    PubMed

    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/.

  9. 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.

  10. 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.

  11. Photoacoustic tomography of human hepatic malignancies using intraoperative indocyanine green fluorescence imaging.

    PubMed

    Miyata, Akinori; Ishizawa, Takeaki; Kamiya, Mako; Shimizu, Atsushi; Kaneko, Junichi; Ijichi, Hideaki; Shibahara, Junji; Fukayama, Masashi; Midorikawa, Yutaka; Urano, Yasuteru; Kokudo, Norihiro

    2014-01-01

    Recently, fluorescence imaging following the preoperative intravenous injection of indocyanine green has been used in clinical settings to identify hepatic malignancies during surgery. The aim of this study was to evaluate the ability of photoacoustic tomography using indocyanine green as a contrast agent to produce representative fluorescence images of hepatic tumors by visualizing the spatial distribution of indocyanine green on ultrasonographic images. Indocyanine green (0.5 mg/kg, intravenous) was preoperatively administered to 9 patients undergoing hepatectomy. Intraoperatively, photoacoustic tomography was performed on the surface of the resected hepatic specimens (n = 10) under excitation with an 800 nm pulse laser. In 4 hepatocellular carcinoma nodules, photoacoustic imaging identified indocyanine green accumulation in the cancerous tissue. In contrast, in one hepatocellular carcinoma nodule and five adenocarcinoma foci (one intrahepatic cholangiocarcinoma and 4 colorectal liver metastases), photoacoustic imaging delineated indocyanine green accumulation not in the cancerous tissue but rather in the peri-cancerous hepatic parenchyma. Although photoacoustic tomography enabled to visualize spatial distribution of ICG on ultrasonographic images, which was consistent with fluorescence images on cut surfaces of the resected specimens, photoacoustic signals of ICG-containing tissues decreased approximately by 40% even at 4 mm depth from liver surfaces. Photoacoustic tomography using indocyanine green also failed to identify any hepatocellular carcinoma nodules from the body surface of model mice with non-alcoholic steatohepatitis. In conclusion, photoacoustic tomography has a potential to enhance cancer detectability and differential diagnosis by ultrasonographic examinations and intraoperative fluorescence imaging through visualization of stasis of bile-excreting imaging agents in and/or around hepatic tumors. However, further technical advances are needed

  12. Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Periasamy, Ammasi; Kay, Steve A.; Day, Richard N.

    1997-05-01

    Fluorescence resonance energy transfer (FRET) imaging microscopy is a unique tool to visualize the spatiotemporal dynamics of protein interactions in living cells. Genetic vectors that encode protein fusions with green fluorescent protein (GFP) provide a method for imaging protein localization in living cells. We used FRET to study dimerization of the pituitary-specific transcription factor Pit-1 fused to GFP and BFP. A fusion protein containing GFP separated from BFP by 29 amino acids served as a positive control for FRET. Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. Using optimized excitation and emission filters, cells expressing the fluorescently-tagged Pit-1 proteins were imaged with a back- thinned, back-illuminated CCD chip that has about 50% quantum efficiency in the blue range. 2D FRET images acquired at the focal plane demonstrated Pit-1 protein associations in the nucleus of living cells. The significance of 2- and 3-D energy transfer imaging from these living cells is discussed.

  13. Dual-color fluorescence imaging of tumor/host interaction with green and red fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Amoh, Yasuyuki; Li, Lingna; Baranov, Eugene; Wang, Jin Wei; Jiang, Ping; Moossa, A. R.; Hoffman, Robert M.

    2004-06-01

    Dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP) expressing transgenic mice has been shown to be a powerful technology to study tumor-host interaction. Host animals include mice which express the GFP transgene in essentially all cells as well as animals in which the regulatory elements of the stem cell marker nestin drive GFP. The general GFP-transgenic mouse is available in both the normal and athymic nude (nu/nu) background. These models show with great clarity the details of the tumor-stroma interaction especially tumor induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. GFP-expressing tumor vasculature could be visualized interacting with the RFP-expressing tumor cells transplanted to the nestin-driven GFP transgenic mice which expressed nestin-GFP in nascent blood vessels was shown as a marker of nascent tumor angiogenesis. Dual-color fluorescence imaging, which visualizes the tumor-host interaction by whole-body imaging and at the cellular level in fresh tissues, dramatically expanding previous studies in fixed and stained preparations (1).

  14. Targeted thiazole orange derivative with folate: synthesis, fluorescence and in vivo fluorescence imaging.

    PubMed

    Fei, Xuening; Gu, Yingchun; Wang, Yiqi; Meng, Qingyang; Zhang, Baolian

    2010-10-11

    A Thiazole Orange conjugated with folate derivative was synthesized in two steps. Firstly, folate was coupled with 1-(3-aminopropyl)-4-methylquinolinium bromide to afford folate-methylquinolinium bromide, which then reacted with benzothiazolium to obtain the title folate-conjugated compound. The compound was evaluated by ¹H-NMR MS, TG/DTA and fluorescence spectroscopic methods. The title compound could selectively target folate receptor expressing tumors according to the in vivo fluorescence imaging preliminarily performed on nude mice with breast tumors.

  15. 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.

  16. Experimental validation of L-shell x-ray fluorescence computed tomography imaging: phantom study.

    PubMed

    Bazalova-Carter, Magdalena; Ahmad, Moiz; Xing, Lei; Fahrig, Rebecca

    2015-10-01

    Thanks to the current advances in nanoscience, molecular biochemistry, and x-ray detector technology, x-ray fluorescence computed tomography (XFCT) has been considered for molecular imaging of probes containing high atomic number elements, such as gold nanoparticles. The commonly used XFCT imaging performed with K-shell x rays appears to have insufficient imaging sensitivity to detect the low gold concentrations observed in small animal studies. Low energy fluorescence L-shell x rays have exhibited higher signal-to-background ratio and appeared as a promising XFCT mode with greatly enhanced sensitivity. The aim of this work was to experimentally demonstrate the feasibility of L-shell XFCT imaging and to assess its achievable sensitivity. We built an experimental L-shell XFCT imaging system consisting of a miniature x-ray tube and two spectrometers, a silicon drift detector (SDD), and a CdTe detector placed at [Formula: see text] with respect to the excitation beam. We imaged a 28-mm-diameter water phantom with 4-mm-diameter Eppendorf tubes containing gold solutions with concentrations of 0.06 to 0.1% Au. While all Au vials were detectable in the SDD L-shell XFCT image, none of the vials were visible in the CdTe L-shell XFCT image. The detectability limit of the presented L-shell XFCT SDD imaging setup was 0.007% Au, a concentration observed in small animal studies.

  17. Experimental validation of L-shell x-ray fluorescence computed tomography imaging: phantom study.

    PubMed

    Bazalova-Carter, Magdalena; Ahmad, Moiz; Xing, Lei; Fahrig, Rebecca

    2015-10-01

    Thanks to the current advances in nanoscience, molecular biochemistry, and x-ray detector technology, x-ray fluorescence computed tomography (XFCT) has been considered for molecular imaging of probes containing high atomic number elements, such as gold nanoparticles. The commonly used XFCT imaging performed with K-shell x rays appears to have insufficient imaging sensitivity to detect the low gold concentrations observed in small animal studies. Low energy fluorescence L-shell x rays have exhibited higher signal-to-background ratio and appeared as a promising XFCT mode with greatly enhanced sensitivity. The aim of this work was to experimentally demonstrate the feasibility of L-shell XFCT imaging and to assess its achievable sensitivity. We built an experimental L-shell XFCT imaging system consisting of a miniature x-ray tube and two spectrometers, a silicon drift detector (SDD), and a CdTe detector placed at [Formula: see text] with respect to the excitation beam. We imaged a 28-mm-diameter water phantom with 4-mm-diameter Eppendorf tubes containing gold solutions with concentrations of 0.06 to 0.1% Au. While all Au vials were detectable in the SDD L-shell XFCT image, none of the vials were visible in the CdTe L-shell XFCT image. The detectability limit of the presented L-shell XFCT SDD imaging setup was 0.007% Au, a concentration observed in small animal studies. PMID:26839910

  18. Experimental validation of L-shell x-ray fluorescence computed tomography imaging: phantom study

    PubMed Central

    Bazalova-Carter, Magdalena; Ahmad, Moiz; Xing, Lei; Fahrig, Rebecca

    2015-01-01

    Abstract. Thanks to the current advances in nanoscience, molecular biochemistry, and x-ray detector technology, x-ray fluorescence computed tomography (XFCT) has been considered for molecular imaging of probes containing high atomic number elements, such as gold nanoparticles. The commonly used XFCT imaging performed with K-shell x rays appears to have insufficient imaging sensitivity to detect the low gold concentrations observed in small animal studies. Low energy fluorescence L-shell x rays have exhibited higher signal-to-background ratio and appeared as a promising XFCT mode with greatly enhanced sensitivity. The aim of this work was to experimentally demonstrate the feasibility of L-shell XFCT imaging and to assess its achievable sensitivity. We built an experimental L-shell XFCT imaging system consisting of a miniature x-ray tube and two spectrometers, a silicon drift detector (SDD), and a CdTe detector placed at ±120  deg with respect to the excitation beam. We imaged a 28-mm-diameter water phantom with 4-mm-diameter Eppendorf tubes containing gold solutions with concentrations of 0.06 to 0.1% Au. While all Au vials were detectable in the SDD L-shell XFCT image, none of the vials were visible in the CdTe L-shell XFCT image. The detectability limit of the presented L-shell XFCT SDD imaging setup was 0.007% Au, a concentration observed in small animal studies. PMID:26839910

  19. Advanced noninvasive imaging of spinal vascular malformations

    PubMed Central

    Eddleman, Christopher S.; Jeong, Hyun; Cashen, Ty A.; Walker, Matthew; Bendok, Bernard R.; Batjer, H. Hunt; Carroll, Timothy J.

    2010-01-01

    Spinal vascular malformations (SVMs) are an uncommon, heterogeneous group of vascular anomalies that can render devastating neurological consequences if they are not diagnosed and treated in a timely fashion. Imaging SVMs has always presented a formidable challenge because their clinical and imaging presentations resemble those of neoplasms, demyelination diseases, and infection. Advancements in noninvasive imaging modalities (MR and CT angiography) have increased during the last decade and have improved the ability to accurately diagnose spinal vascular anomalies. In addition, intraoperative imaging techniques have been developed that aid in the intraoperative assessment before, during, and after resection of these lesions with minimal and/or optimal use of spinal digital subtraction angiography. In this report, the authors review recent advancements in the imaging of SVMs that will likely lead to more timely diagnoses and treatment while reducing procedural risk exposure to the patients who harbor these uncommon spinal lesions. PMID:19119895

  20. Fluorescent rhenium-naphthalimide conjugates as cellular imaging agents.

    PubMed

    Langdon-Jones, Emily E; Symonds, Nadine O; Yates, Sara E; Hayes, Anthony J; Lloyd, David; Williams, Rebecca; Coles, Simon J; Horton, Peter N; Pope, Simon J A

    2014-04-01

    A range of biologically compatible, fluorescent rhenium-naphthalimide conjugates, based upon the rhenium fac-tricarbonyl core, has been synthesized. The fluorescent ligands are based upon a N-functionalized, 4-amino-derived 1,8-naphthalimide core and incorporate a dipicolyl amine binding unit to chelate Re(I); the structural variations accord to the nature of the alkylated imide with ethyl ester glycine (L(1)), 3-propanol (L(2)), diethylene glycol (L(3)), and benzyl alcohol (L(4)) variants. The species are fluorescent in the visible region between 505 and 537 nm through a naphthalimide-localized intramolecular charge transfer, with corresponding fluorescent lifetimes of up to 9.8 ns. The ligands and complexes were investigated for their potential as imaging agents for human osteoarthritic cells and protistan fish parasite Spironucleus vortens using confocal fluorescence microscopy. The results show that the specific nature of the naphthalimide structure serves to control the uptake and intracellular localization of these imaging agents. Significant differences were noted between the free ligands and complexes, with the Re(I) complex of L(2) showing hydrogenosomal localization in S. vortens.

  1. 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.

  2. 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

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

    PubMed Central

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

    2013-01-01

    Paradigm shifts in surgery arise when surgeons are empowered to perform surgery faster, better, and/or less expensively. Optical imaging that exploits invisible near-infrared fluorescent light has the potential to improve cancer surgery outcomes while minimizing anesthesia time and lowering healthcare costs. Because of this, the last few years have witnessed an explosion of proof-of-concept clinical trials in the field. In this review, we introduce the concept of near-infrared fluorescence imaging for cancer surgery, review the clinical trial literature to date, outline the key issues pertaining to imaging system and contrast agent optimization, discuss limitations and leverage, and provide a framework for making the technology available for the routine care of cancer patients in the near future. PMID:23881033

  4. 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.

  5. Visualizing photosynthesis through processing of chlorophyll fluorescence images

    SciTech Connect

    Daley, P.F. ); Ball, J.T. . Desert Research Inst.); Berry, J.A. . Dept. of Plant Biology); Patzke, J.; Raschke, K. )

    1990-01-01

    Measurements of terrestrial plant photosynthesis frequently exploit sensing of gas exchange from leaves enclosed in gas-tight, climate controlled chambers. 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 mechanism (q{sub N}) that dissipate excess excitation energy, otherwise damaging to the light harvesting apparatus. 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. 15 refs., 4 figs.

  6. Trends in Fluorescence Image-guided Surgery for Gliomas

    PubMed Central

    Liu, Jonathan T.C.; Meza, Daphne; Sanai, Nader

    2014-01-01

    Mounting evidence suggests that a more extensive surgical resection is associated with an improved life expectancy for both low-grade and high-grade glioma patients. However, radiographically complete resections are not often achieved in many cases due to the lack of sensitivity and specificity of current neurosurgical guidance techniques at the margins of diffuse infiltrative gliomas. Intraoperative fluorescence imaging offers the potential to improve the extent of resection and to investigate the possible benefits of resecting beyond the radiographic margins. Here, we provide a review of wide-field and high-resolution fluorescence-imaging strategies that are being developed for neurosurgical guidance, with a focus on emerging imaging technologies and clinically viable contrast agents. The strengths and weaknesses of these approaches will be discussed, as well as issues that are being addressed to translate these technologies into the standard of care. PMID:24618801

  7. Trends in fluorescence image-guided surgery for gliomas.

    PubMed

    Liu, Jonathan T C; Meza, Daphne; Sanai, Nader

    2014-07-01

    Mounting evidence suggests that a more extensive surgical resection is associated with an improved life expectancy for both low-grade and high-grade glioma patients. However, radiographically complete resections are not often achieved in many cases because of the lack of sensitivity and specificity of current neurosurgical guidance techniques at the margins of diffuse infiltrative gliomas. Intraoperative fluorescence imaging offers the potential to improve the extent of resection and to investigate the possible benefits of resecting beyond the radiographic margins. Here, we provide a review of wide-field and high-resolution fluorescence-imaging strategies that are being developed for neurosurgical guidance, with a focus on emerging imaging technologies and clinically viable contrast agents. The strengths and weaknesses of these approaches will be discussed, as well as issues that are being addressed to translate these technologies into the standard of care.

  8. 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

  9. 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

  10. 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.

  11. Fluorescence lifetime imaging by asynchronous pump-probe microscopy.

    PubMed Central

    Dong, C Y; So, P T; French, T; Gratton, E

    1995-01-01

    We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE. Images FIGURE 2 FIGURE 6 FIGURE 7 FIGURE 8 PMID:8599631

  12. 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.

  13. 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

  14. Dynamic fluorescence lifetime imaging based on acousto-optic deflectors

    NASA Astrophysics Data System (ADS)

    Yan, Wei; Peng, Xiao; Qi, Jing; Gao, Jian; Fan, Shunping; Wang, Qi; Qu, Junle; Niu, Hanben

    2014-11-01

    We report a dynamic fluorescence lifetime imaging (D-FLIM) system that is based on a pair of acousto-optic deflectors for the random regions of interest (ROI) study in the sample. The two-dimensional acousto-optic deflector devices are used to rapidly scan the femtosecond excitation laser beam across the sample, providing specific random access to the ROI. Our experimental results using standard fluorescent dyes in live cancer cells demonstrate that the D-FLIM system can dynamically monitor the changing process of the microenvironment in the ROI in live biological samples.

  15. 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

  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 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.

  18. 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

  19. Advanced Microwave/Millimeter-Wave Imaging Technology

    NASA Astrophysics Data System (ADS)

    Shen, Zuowei; Yang, Lu; Luhmann, N. C., Jr.; Domier, C. W.; Ito, N.; Kogi, Y.; Liang, Y.; Mase, A.; Park, H.; Sakata, E.; Tsai, W.; Xia, Z. G.; Zhang, P.

    Millimeter wave technology advances have made possible active and passive millimeter wave imaging for a variety of applications including advanced plasma diagnostics, radio astronomy, atmospheric radiometry, concealed weapon detection, all-weather aircraft landing, contraband goods detection, harbor navigation/surveillance in fog, highway traffic monitoring in fog, helicopter and automotive collision avoidance in fog, and environmental remote sensing data associated with weather, pollution, soil moisture, oil spill detection, and monitoring of forest fires, to name but a few. The primary focus of this paper is on technology advances which have made possible advanced imaging and visualization of magnetohydrodynamic (MHD) fluctuations and microturbulence in fusion plasmas. Topics of particular emphasis include frequency selective surfaces, planar Schottky diode mixer arrays, electronically controlled beam shaping/steering arrays, and high power millimeter wave local oscillator and probe sources.

  20. Advances in endoscopic imaging in ulcerative colitis.

    PubMed

    Tontini, Gian Eugenio; Pastorelli, Luca; Ishaq, Sauid; Neumann, Helmut

    2015-01-01

    Modern strategies for the treatment of ulcerative colitis require more accurate tools for gastrointestinal imaging to better assess mucosal disease activity and long-term prognostic clinical outcomes. Recent advances in gastrointestinal luminal endoscopy are radically changing the role of endoscopy in every-day clinical practice and research trials. Advanced endoscopic imaging techniques including high-definition endoscopes, optical magnification endoscopy, and various chromoendoscopy techniques have remarkably improved endoscopic assessment of ulcerative colitis. More recently, optical biopsy techniques with either endocytoscopy or confocal laser endomicroscopy have shown great potential in predicting several histological changes in real time during ongoing endoscopy. Here, we review current applications of advanced endoscopic imaging techniques in ulcerative colitis and present the most promising upcoming headways in this field. PMID:26365308

  1. Fluorescence Lifetime Imaging and Spectroscopy as Tools for Nondestructive Analysis of Works of Art

    NASA Astrophysics Data System (ADS)

    Comelli, Daniela; D'Andrea, Cosimo; Valentini, Gianluca; Cubeddu, Rinaldo; Colombo, Chiara; Toniolo, Lucia

    2004-04-01

    A system for advanced fluorescence investigation of works of art has been assembled and integrated in a characterization procedure that allows one to localize and identify organic compounds that are present in artworks. At the beginning of the investigation, fluorescence lifetime imaging and spectroscopy address a selective microsampling of the artwork. Then analytical measurements of microsamples identify the chemical composition of the materials under investigation. Finally, on the basis of fluorescence lifetime and amplitude maps, analytical data are extended to the whole artwork. In such a way, information on the spatial distribution of organic materials can be inferred. These concepts have been successfully applied in an extensive campaign for analysis of Renaissance fresco paintings in Castiglione Olona, Italy. Residue of various types of glue and stucco left from a restoration carried out in the early 1970s was localized and classified. Insight into the technique used by the painter to make gilded reliefs was also obtained.

  2. Two-Photon Fluorescence Anisotropy Imaging to Elucidate the Dynamics and the Stability of Immobilized Proteins.

    PubMed

    Orrego, Alejandro H; García, Carolina; Mancheño, José M; Guisán, Jose M; Lillo, M Pilar; López-Gallego, Fernando

    2016-01-28

    Time/spatial-resolved fluorescence determines anisotropy values of supported-fluorescent proteins through different immobilization chemistries, evidencing some of the molecular mechanisms that drive the stabilization of proteins at the interfaces with solid surfaces. Fluorescence anisotropy imaging provides a normalized protein mobility parameter that serves as a guide to study the effect of different immobilization parameters (length and flexibility of the spacer arm and multivalency of the protein-support interaction) on the final stability of the supported proteins. Proteins in a more constrained environment correspond to the most thermostable ones, as was shown by thermal inactivation studies. This work contributes to explain the experimental evidence found with conventional methods based on observable measurements; thus this advanced characterization technique provides reliable molecular information about the immobilized proteins with sub-micrometer spatial resolution. Such information has been very useful for fabricating highly stable heterogeneous biocatalysts with high interest in industrial developments.

  3. 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.

  4. 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.

  5. Advances in electromagnetic brain imaging

    NASA Astrophysics Data System (ADS)

    Nagarajan, Srikantan S.

    2010-02-01

    Non-invasive and dynamic imaging of brain activity in the sub-millisecond time-scale is enabled by measurements on or near the scalp surface using an array of sensors that measure magnetic fields (magnetoencephalography (MEG)) or electric potentials (electroencephalography (EEG)). Algorithmic reconstruction of brain activity from MEG and EEG data is referred to as electromagnetic brain imaging (EBI). Reconstructing the actual brain response to external events and distinguishing unrelated brain activity has been a challenge for many existing algorithms in this field. Furthermore, even under conditions where there is very little interference, accurately determining the spatial locations and timing of brain sources from MEG and EEG data is challenging problem because it involves solving for unknown brain activity across thousands of voxels from just a few sensors (~300). In recent years, my research group has developed a suite of novel and powerful algorithms for EBI that we have shown to be considerably superior to existing benchmark algorithms. Specifically, these algorithms can solve for many brain sources, including sources located far from the sensors, in the presence of large interference from unrelated brain sources. Our algorithms efficiently model interference contributions to sensors, accurately estimate sparse brain source activity using fast and robust probabilistic inference techniques. Here, we review some of these algorithms and illustrate their performance in simulations and real MEG/EEG data.

  6. 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

  7. Personnel screening with advanced multistatic imaging technology

    NASA Astrophysics Data System (ADS)

    Ahmed, Sherif S.

    2013-05-01

    Personnel screening is demanded nowadays for securing air traffic as well as critical infrastructures. The millimeter-waves are able to penetrate clothes and detect concealed objects, making them an attractive choice for security screening. Imaging methods based on multistatic architecture can ensure high quality imagery in terms of resolution and dynamic range. Following the advances in semiconductor technology, fully electronic solutions delivering real-time imaging are becoming feasible. Furthermore, the continuously increasing capabilities of digital signal processing units allow for the utilization of digital-beamforming techniques for image reconstruction, thus offering new opportunities for imaging systems to use sophisticated operation modes. Based on these modern technologies, an advanced realization addressing personnel screening in E-band with planar multistatic sparse array design is demonstrated.

  8. Advances in Molecular Imaging with Ultrasound

    PubMed Central

    Gessner, Ryan; Dayton, Paul A.

    2010-01-01

    Ultrasound imaging has long demonstrated utility in the study and measurement of anatomic features and noninvasive observation of blood flow. Within the last decade, advances in molecular biology and contrast agents have allowed researchers to use ultrasound to detect changes in the expression of molecular markers on the vascular endothelium and other intravascular targets. This new technology, referred to as ultrasonic molecular imaging, is still in its infancy. However, in preclinical studies, ultrasonic molecular imaging has shown promise in assessing angiogenesis, inflammation, and thrombus. In this review, we discuss recent advances in microbubble-type contrast agent development, ultrasound technology, and signal processing strategies that have the potential to substantially improve the capabilities and utility of ultrasonic molecular imaging. PMID:20487678

  9. Biochip Image Grid Normalization Absolute Signal Fluorescence Measurement Using

    SciTech Connect

    Alferov, Oleg

    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 to 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.

  10. 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

  11. Biochip Image Grid Normalization Absolute Signal Fluorescence Measurement Using

    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

  12. Extended focus optical coherence microscopy and fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Villiger, Martin; Blatter, Cedric; Bachmann, Adrian; Lasser, Theo; Leitgeb, Rainer A.

    2008-02-01

    Fourier domain Optical coherence microscopy (FDOCM) offers excellent sensitivity and high axial resolution to image the structure of biological tissue. The depth information is extracted in parallel and allows very high volume acquisition rates. The present system uses a diffractionless beam, produced with an axicon lens, to achieve high lateral resolution all while maintaining an extended depth of field (xf). The xfOCM signal reveals the spatial distribution of changes of the refractive index in the sample that scatter the incident light. To identify and validate the functionality of the observed structures can proof difficult. In this work the xfOCM setup was interfaced with a fluorescent lifetime imaging (FLIM) system, working in the Fourier domain and measuring the phase offset between the modulated excitation signal and the returned fluorescence intensity. Both the fluorescence amplitude and lifetime are retrieved. The amplitude contains important information due to the selective labeling of the tissue. The lifetime is very sensitive to the surrounding environment and varies for different fluorophores, adding further contrast. The xfOCM tomograms and FLIM images are acquired in parallel. A complementary view of the sample is obtained that helps to understand and interpret the xfOCM signal. The lifetime measurement provides further contrast to perform functional imaging on biological samples such as the rat hair follicle.

  13. 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.

  14. 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.

  15. 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.

  16. [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

  17. [Intraoperative graft assessment using fluorescent imaging system (SPY)].

    PubMed

    Kawashima, T; Naraoka, S; Kakizaki, T

    2009-07-01

    We investigated the efficacy of intraoperative fluorescent imaging system for the assessment of coronary artery bypass grafting (CABG). We used SPY imaging system in 100 CABG (57 off-pump and 43 on-pump CABG), totalling 287 distal anastomoses. The total graft patency rate on postoperative angiography in this series was 96.2% (276/287). Graft revision was done in 10 cases (10.0%) and 13 anastomoses (4.5%) by SPY imaging, which all resulted in good patency at postoperative angiography. On the other hand, 7 distal anastomoses and 1 mammary graft (2.8%) appeared to be successful on intraoperative SPY imaging, but were revealed to be occluded by postoperative angiography. SPY imaging system is useful for graft validation, and may contribute to improvement of coronary bypass graft patency.

  18. 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.

  19. Standard Fluorescent Imaging of Live Cells is Highly Genotoxic

    PubMed Central

    Ge, Jing; Wood, David K.; Weingeist, David M.; Prasongtanakij, Somsak; Navasumrit, Panida; Ruchirawat, Mathuros; Engelward, Bevin P.

    2013-01-01

    Fluorescence microscopy is commonly used for imaging live mammalian cells. Here, we describe studies aimed at revealing the potential genotoxic effects of standard fluorescence microscopy. To assess DNA damage, a high throughput platform for single cell gel electrophoresis is used (e.g., the CometChip). Light emitted by three standard filters was studied: a) violet light [340–380 nm], used to excite DAPI and other blue fluorophores, b) blue light [460–500 nm] commonly used to image GFP and Calcein AM, and c) green light [528–553], useful for imaging red fluorophores. Results show that exposure of samples to light during imaging is indeed genotoxic even when the selected wavelengths are outside the range known to induce significant levels. Shorter excitation wavelengths and longer irradiation times lead to higher levels of DNA damage. We have also measured DNA damage in cells expressing enhanced green fluorescent protein (GFP) or stained with Calcein AM, a widely used green fluorophore. Data show that Calcein AM leads to a synergistic increase in the levels of DNA damage and that even cells that are not being directly imaged sustain significant DNA damage from exposure to indirect light. The nature of light-induced DNA damage during imaging was assessed using the Fpg glycosylase, an enzyme that enables quantification of oxidative DNA damage. Oxidative damage was evident in cells exposed to violet light. Furthermore the Fpg glycosylase revealed the presence of oxidative DNA damage in blue-light exposed cells for which DNA damage was not detected using standard analysis conditions. Taken together, the results of these studies call attention to the potential confounding effects of DNA damage induced by standard imaging conditions, and identify wavelength, exposure time and fluorophore as parameters that can be modulated to reduce light-induced DNA damage. PMID:23650257

  20. Fluorescence-enhanced gadolinium-doped zinc oxide quantum dots for magnetic resonance and fluorescence imaging.

    PubMed

    Liu, Yanlan; Ai, Kelong; Yuan, Qinghai; Lu, Lehui

    2011-02-01

    We report here the development of Gd-doped ZnO quantum dots (QDs) as dual modal fluorescence and magnetic resonance imaging nanoprobes. They are fabricated in a simple, versatile and environmentally friendly method, not only decreasing the difficulty and complexity, but also avoiding the increase of particle's size brought about by silica coating procedure in the synthesis of nanoprobes reported previously. These nanoprobes, with exceptionally small size and enhanced fluorescence resulting from the Gd doping, can label successfully the HeLa cells in short time and present no evidence of toxicity or adverse affect on cell growth even at the concentration up to 1 mm. These results show that such nanoprobes have low toxicity, especially in comparison with the traditional PEGylated CdSe/ZnS or CdSe/CdS QDs. In MRI studies, they exert strong positive contrast effect with a large longitudinal relaxivity (r(1)) of water proton of 16 mm(-1) s(-1). Their capability of imaging HeLa cells with MRI implies that they have great potential as MRI contrast agents. Combining the high sensitivity of fluorescence imaging with high spatial resolution of MRI, We expect that the as-prepared Gd-doped Zno QDs can provide a better reliability of the collected data and find promising applications in biological, medical and other fields.

  1. 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

  2. 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

  3. 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

  4. 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

  5. Flexible peritoneal windows for quantitative fluorescence and bioluminescence preclinical imaging.

    PubMed

    Souris, Jeffrey S; Hickson, Jonathan A; Msezane, Lambda; Rinker-Schaeffer, Carrie W; Chen, Chin-Tu

    2013-01-01

    At present, there is considerable interest in the use of in vivo fluorescence and bioluminescence imaging to track the onset and progression of pathologic processes in preclinical models of human disease. Optical quantitation of such phenomena, however, is often problematic, frequently complicated by the overlying tissue's scattering and absorption of light, as well as the presence of endogenous cutaneous and subcutaneous fluorophores. To partially circumvent this information loss, we report here the development of flexible, surgically implanted, transparent windows that enhance quantitative in vivo fluorescence and bioluminescence imaging of optical reporters. These windows are metal and glass free and thus compatible with computed tomography, magnetic resonance imaging, positron emission tomography, and single-photon emission computed tomography; they also permit visualization of much larger areas with fewer impediments to animal locomotion and grooming than those previously described. To evaluate their utility in preclinical imaging, we surgically implanted these windows in the abdominal walls of female athymic nude mice and subsequently inoculated each animal with 1 × 10(4) to 1 × 10(6) bioluminescent human ovarian cancer cells (SKOV3ip.1-luc). Longitudinal imaging studies of fenestrated animals revealed up to 48-fold gains in imaging sensitivity relative to nonfenestrated animals, with relatively few complications, allowing wide-field in vivo visualization of nascent metastatic ovarian cancer colonization.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

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

    PubMed

    Zhao, Qiaole; Schelen, Ben; Schouten, Raymond; van den Oever, Rein; Leenen, René; van Kuijk, Harry; Peters, Inge; Polderdijk, Frank; Bosiers, Jan; Raspe, Marcel; Jalink, Kees; Geert Sander de Jong, Jan; van Geest, Bert; Stoop, Karel; Young, Ian Ted

    2012-12-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

  11. 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.

  12. High spectral resolution imager for solar induced fluorescence observation

    NASA Astrophysics Data System (ADS)

    Barducci, A.; Guzzi, D.; Lastri, C.; Marcoionni, P.; Nardino, V.; Pippi, I.; Raimondi, V.; Sandri, P.

    2011-11-01

    The use of high-resolution imagers for determination of solar-induced fluorescence of natural bodies by observing the infilling of Fraunhofer lines has been frequently adopted as a tool for vegetation characterization. The option to perform those measurements from airborne platforms was addressed in the past. In-field observations gave evidence of the main requirements for an imaging spectrometer to be used for Sun-induced fluorescence measurements such as high spectral resolution and fine radiometric accuracy needed to resolve the shape of observed Fraunhofer lines with a high level of accuracy. In this paper, some solutions for the design of a high spectral resolution push-broom imaging spectrometer for Sun-induced fluorescence measurements are analysed. The main constraints for the optical design are a spectral resolution better than 0.01 nm and a wide field of view. Due to the fine instrumental spectral resolution, bidimensional focal plane arrays characterized by high quantum efficiency, low read-out noise, and high sensitivity are requested. The development of a lightweight instrument is a benefit for aerospace implementations of this technology. First results coming from laboratory measurements and optical simulations are presented and discussed taking into account their feasibility.

  13. Fluorescence and image guided resection in high grade glioma.

    PubMed

    Panciani, Pier Paolo; Fontanella, Marco; Schatlo, Bawarjan; Garbossa, Diego; Agnoletti, Alessandro; Ducati, Alessandro; Lanotte, Michele

    2012-01-01

    The extent of resection in high grade glioma is increasingly been shown to positively effect survival. Nevertheless, heterogeneity and migratory behavior of glioma cells make gross total resection very challenging. Several techniques were used in order to improve the detection of residual tumor. Aim of this study was to analyze advantages and limitations of fluorescence and image guided resection. A multicentric prospective study was designed to evaluate the accuracy of each method. Furthermore, the role of 5-aminolevulinc acid and neuronavigation were reviewed. Twenty-three patients harboring suspected high grade glioma, amenable to complete resection, were enrolled. Fluorescence and image guides were used to perform surgery. Multiple samples were obtained from the resection cavity of each lesion according to 5-ALA staining positivity and boundaries as delineated by neuronavigation. All samples were analyzed by a pathologist blinded to the intra-operative labeling. Decision-making based on fluorescence showed a sensitivity of 91.1% and a specificity of 89.4% (p<0.001). On the other hand, the image-guided resection accuracy was low (sensitivity: 57.8%; specificity: 57.4%; p=0.346). We observed that the sensitivity of 5-ALA can be improved by the combined use of neuronavigation, but this leads to a significant reduction in specificity. Thus, the use of auxiliary techniques should always be subject to critical skills of the surgeon. We advocate a large-scale study to further improve the assessment of multimodal approaches.

  14. 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

  15. Validation of image processing tools for 3-D fluorescence microscopy.

    PubMed

    Dieterlen, Alain; Xu, Chengqi; Gramain, Marie-Pierre; Haeberlé, Olivier; Colicchio, Bruno; Cudel, Christophe; Jacquey, Serge; Ginglinger, Emanuelle; Jung, Georges; Jeandidier, Eric

    2002-04-01

    3-D optical fluorescent microscopy becomes nowadays an efficient tool for volumic investigation of living biological samples. Using optical sectioning technique, a stack of 2-D images is obtained. However, due to the nature of the system optical transfer function and non-optimal experimental conditions, acquired raw data usually suffer from some distortions. In order to carry out biological analysis, raw data have to be restored by deconvolution. The system identification by the point-spread function is useful to obtain the knowledge of the actual system and experimental parameters, which is necessary to restore raw data. It is furthermore helpful to precise the experimental protocol. In order to facilitate the use of image processing techniques, a multi-platform-compatible software package called VIEW3D has been developed. It integrates a set of tools for the analysis of fluorescence images from 3-D wide-field or confocal microscopy. A number of regularisation parameters for data restoration are determined automatically. Common geometrical measurements and morphological descriptors of fluorescent sites are also implemented to facilitate the characterisation of biological samples. An example of this method concerning cytogenetics is presented.

  16. Implantable CMOS imaging device with absorption filters for green fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Sunaga, Yoshinori; Haruta, Makito; Takehara, Hironari; Ohta, Yasumi; Motoyama, Mayumi; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Ohta, Jun

    2014-03-01

    Green fluorescent materials such as Green Fluorescence Protein (GFP) and fluorescein are often used for observing neural activities. Thus, it is important to observe the fluorescence in a freely moving state in order to understand neural activities corresponding to behaviors. In this work, we developed an implantable CMOS imaging device for in-vivo green fluorescence imaging with efficient excitation light rejection using a combination of absorption filters. An interference filter is usually used for a fluorescence microscope in order to achieve high fluorescence imaging sensitivity. However, in the case of the implantable device, interference filters are not suitable because their transmission spectra depend on incident angle. To solve this problem we used two kinds of absorption filters that do not have angle dependence. An absorption filter consisting of yellow dye (VARYFAST YELLOW 3150) was coated on the pixel array of an image sensor. The rejection ratio of ideal excitation light (490 nm) against green fluorescence (510 nm) was 99.66%. However, the blue LED as an excitation light source has a broad emission spectrum and its intensity at 510 nm is 2.2 x 10-2 times the emission peak intensity. By coating LEDs with the emission absorption filters, the intensity of the unwanted component of the excitation light was reduced to 1.4 x 10-4. Using the combination of absorption filters, we achieved excitation light transmittance of 10-5 onto the image sensor. It is expected that high-sensitivity green fluorescence imaging of neural activities in a freely moving mouse will be possible by using this technology.

  17. Fluorescence imaging efficiency of cold atoms in free fall.

    PubMed

    Serre, I; Pruvost, L; Duong, H T

    1998-02-20

    A fluorescence detection scheme coupled to a highly sensitive nitrogen-cooled CCD camera is used to image the spatial distribution of a low-density falling rubidium atomic cloud released from an optical trap. The falling cloud passes through a thin probe laser beam tuned to resonance. The performance of the scheme is illustrated in the analysis of cold atomic clouds collimated by pinholes during their free fall under the influence of gravity. Clouds of approximately 10(4) atoms and with typically 10(6) at./cm(3) density are analyzed spatially with 24-mum resolution. This method is compared with different atomic cloud imaging techniques.

  18. 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.

  19. Advanced endoscopic imaging to improve adenoma detection

    PubMed Central

    Neumann, Helmut; Nägel, Andreas; Buda, Andrea

    2015-01-01

    Advanced endoscopic imaging is revolutionizing our way on how to diagnose and treat colorectal lesions. Within recent years a variety of modern endoscopic imaging techniques was introduced to improve adenoma detection rates. Those include high-definition imaging, dye-less chromoendoscopy techniques and novel, highly flexible endoscopes, some of them equipped with balloons or multiple lenses in order to improve adenoma detection rates. In this review we will focus on the newest developments in the field of colonoscopic imaging to improve adenoma detection rates. Described techniques include high-definition imaging, optical chromoendoscopy techniques, virtual chromoendoscopy techniques, the Third Eye Retroscope and other retroviewing devices, the G-EYE endoscope and the Full Spectrum Endoscopy-system. PMID:25789092

  20. Noninvasive fluorescence imaging in animal models of stroke.

    PubMed

    Stemmer, N; Mehnert, J; Steinbrink, J; Wunder, A

    2012-01-01

    Noninvasive fluorescence imaging (NFI) is a powerful tool to study physiology and pathophysiology in animal disease models. NFI has been successfully applied in a number of animal disease models including cancer, arthritis, and stroke. Furthermore, several applications in humans have been described. NFI is widely available in research laboratories because it has a number of advantages: It uses non-ionizing radiation and requires comparably simple, inexpensive instrumentation, and easy to handle. Fluorochromes can be detected with high sensitivity, and image acquisition time is relatively short. Furthermore, a plethora of fluorescent imaging agents is available including unspecific, target-specific, and activatable imaging probes. With these probes, biological processes such as inflammation, cell death or enzyme activity, and many others can be visualized in living animals. This review offers an overview of current approaches in NFI of stroke pathophysiology in animal models of cerebral ischemia. First, the instrumentation and the different types of imaging agents for NFI are described. Second, a short introduction to animal models of stroke is provided. Third, examples for NFI in animal models of stroke are given. Finally, the use of NFI in human stroke is critically discussed.

  1. 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.

  2. Advanced technologies for remote sensing imaging applications

    SciTech Connect

    Wood, L.L.

    1993-06-07

    Generating and returning imagery from great distances has been generally associated with national security activities, with emphasis on reliability of system operation. (While the introduction of such capabilities was usually characterized by high levels of innovation, the evolution of such systems has followed the classical track of proliferation of ``standardized items`` expressing ever more incremental technological advances.) Recent focusing of interest on the use of remote imaging systems for commercial and scientific purposes can be expected to induce comparatively rapid advances along the axes of efficiency and technological sophistication, respectively. This paper reviews the most basic reasons for expecting the next decade of advances to dwarf the impressive accomplishments of the past ten years. The impact of these advances clearly will be felt in all major areas of large-scale human endeavor, commercial, military and scientific.

  3. 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

    2016-01-01

    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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Advances in retinal ganglion cell imaging

    PubMed Central

    Balendra, S I; Normando, E M; Bloom, P A; Cordeiro, M F

    2015-01-01

    Glaucoma is one of the leading causes of blindness worldwide and will affect 79.6 million people worldwide by 2020. It is caused by the progressive loss of retinal ganglion cells (RGCs), predominantly via apoptosis, within the retinal nerve fibre layer and the corresponding loss of axons of the optic nerve head. One of its most devastating features is its late diagnosis and the resulting irreversible visual loss that is often predictable. Current diagnostic tools require significant RGC or functional visual field loss before the threshold for detection of glaucoma may be reached. To propel the efficacy of therapeutics in glaucoma, an earlier diagnostic tool is required. Recent advances in retinal imaging, including optical coherence tomography, confocal scanning laser ophthalmoscopy, and adaptive optics, have propelled both glaucoma research and clinical diagnostics and therapeutics. However, an ideal imaging technique to diagnose and monitor glaucoma would image RGCs non-invasively with high specificity and sensitivity in vivo. It may confirm the presence of healthy RGCs, such as in transgenic models or retrograde labelling, or detect subtle changes in the number of unhealthy or apoptotic RGCs, such as detection of apoptosing retinal cells (DARC). Although many of these advances have not yet been introduced to the clinical arena, their successes in animal studies are enthralling. This review will illustrate the challenges of imaging RGCs, the main retinal imaging modalities, the in vivo techniques to augment these as specific RGC-imaging tools and their potential for translation to the glaucoma clinic. PMID:26293138

  9. Advanced laser systems for photoacoustic imaging

    NASA Astrophysics Data System (ADS)

    Klosner, Marc; Sampathkumar, Ashwin; Chan, Gary; Wu, Chunbai; Gross, Daniel; Heller, Donald F.

    2015-03-01

    We describe the ongoing development of laser systems for advanced photoacoustic imaging (PAI). We discuss the characteristics of these laser systems and their particular benefits for soft tissue imaging and next-generation breast cancer diagnostics. We provide an overview of laser performance and compare this with other laser systems that have been used for early-stage development of PAI. These advanced systems feature higher pulse energy output at clinically relevant repetition rates, as well as a novel wavelength-cycling output pulse format. Wavelength cycling provides pulse sequences for which the output repeatedly alternates between two wavelengths that provide differential imaging. This capability improves co-registration of captured differential images. We present imaging results of phantoms obtained with a commercial ultrasound detector system and a wavelength-cycling laser source providing ~500 mJ/pulse at 755 and 797 nm, operating at 25 Hz. The results include photoacoustic images and corresponding pulse-echo data from a tissue mimicking phantom containing inclusions, simulating tumors in the breast. We discuss the application of these systems to the contrast-enhanced detection of various tissue types and tumors.

  10. Emerging in vivo analyses of cell function using fluorescence imaging (*).

    PubMed

    Lippincott-Schwartz, Jennifer

    2011-01-01

    Understanding how cells of all types sense external and internal signals and how these signals are processed to yield particular responses is a major goal of biology. Genetically encoded fluorescent proteins (FPs) and fluorescent sensors are playing an important role in achieving this comprehensive knowledge base of cell function. Providing high sensitivity and immense versatility while being minimally perturbing to a biological specimen, the probes can be used in different microscopy techniques to visualize cellular processes on many spatial scales. Three review articles in this volume discuss recent advances in probe design and applications. These developments help expand the range of biochemical processes in living systems suitable for study. They provide researchers with exciting new tools to explore how cellular processes are organized and their activity regulated in vivo.

  11. 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.

  12. Wide-field multispectral super-resolution imaging using spin-dependent fluorescence in nanodiamonds.

    PubMed

    Chen, Edward H; Gaathon, Ophir; Trusheim, Matthew E; Englund, Dirk

    2013-05-01

    Recent advances in fluorescence microscopy have enabled spatial resolution below the diffraction limit by localizing multiple temporally or spectrally distinguishable fluorophores. Here, we introduce a super-resolution technique that deterministically controls the brightness of uniquely addressable, photostable emitters. We modulate the fluorescence brightness of negatively charged nitrogen-vacancy (NV(-)) centers in nanodiamonds through magnetic resonance techniques. Using a CCD camera, this "deterministic emitter switch microscopy" (DESM) technique enables super-resolution imaging with localization down to 12 nm across a 35 × 35 μm(2) area. DESM is particularly well suited for biological applications such as multispectral particle tracking since fluorescent nanodiamonds are not only cytocompatible but also nonbleaching and bright. We observe fluorescence count rates exceeding 1.5 × 10(6) photons per second from single NV(-) centers at saturation. When combined with emerging NV(-)-based techniques for sensing magnetic and electric fields, DESM opens the door to rapid, super-resolution imaging for tracking and sensing applications in the life and physical sciences.

  13. 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

  14. Image reconstruction enables high resolution imaging at large penetration depths in fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Dilipkumar, Shilpa; Montalescot, Sandra; Mondal, Partha Pratim

    2013-10-01

    Imaging thick specimen at a large penetration depth is a challenge in biophysics and material science. Refractive index mismatch results in spherical aberration that is responsible for streaking artifacts, while Poissonian nature of photon emission and scattering introduces noise in the acquired three-dimensional image. To overcome these unwanted artifacts, we introduced a two-fold approach: first, point-spread function modeling with correction for spherical aberration and second, employing maximum-likelihood reconstruction technique to eliminate noise. Experimental results on fluorescent nano-beads and fluorescently coated yeast cells (encaged in Agarose gel) shows substantial minimization of artifacts. The noise is substantially suppressed, whereas the side-lobes (generated by streaking effect) drops by 48.6% as compared to raw data at a depth of 150 μm. Proposed imaging technique can be integrated to sophisticated fluorescence imaging techniques for rendering high resolution beyond 150 μm mark.

  15. Near-infrared fluorescent probes in cancer imaging and therapy: an emerging field

    PubMed Central

    Yi, Xiaomin; Wang, Fuli; Qin, Weijun; Yang, Xiaojian; Yuan, Jianlin

    2014-01-01

    Near-infrared fluorescence (NIRF) imaging is an attractive modality for early cancer detection with high sensitivity and multi-detection capability. Due to convenient modification by conjugating with moieties of interests, NIRF probes are ideal candidates for cancer targeted imaging. Additionally, the combinatory application of NIRF imaging and other imaging modalities that can delineate anatomical structures extends fluorometric determination of biomedical information. Moreover, nanoparticles loaded with NIRF dyes and anticancer agents contribute to the synergistic management of cancer, which integrates the advantage of imaging and therapeutic functions to achieve the ultimate goal of simultaneous diagnosis and treatment. Appropriate probe design with targeting moieties can retain the original properties of NIRF and pharmacokinetics. In recent years, great efforts have been made to develop new NIRF probes with better photostability and strong fluorescence emission, leading to the discovery of numerous novel NIRF probes with fine photophysical properties. Some of these probes exhibit tumoricidal activities upon light radiation, which holds great promise in photothermal therapy, photodynamic therapy, and photoimmunotherapy. This review aims to provide a timely and concise update on emerging NIRF dyes and multifunctional agents. Their potential uses as agents for cancer specific imaging, lymph node mapping, and therapeutics are included. Recent advances of NIRF dyes in clinical use are also summarized. PMID:24648733

  16. Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy.

    PubMed

    Okabe, Kohki; Inada, Noriko; Gota, Chie; Harada, Yoshie; Funatsu, Takashi; Uchiyama, Seiichi

    2012-02-28

    Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18-0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.

  17. Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy

    PubMed Central

    Okabe, Kohki; Inada, Noriko; Gota, Chie; Harada, Yoshie; Funatsu, Takashi; Uchiyama, Seiichi

    2012-01-01

    Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18–0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function. PMID:22426226

  18. 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

  19. 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.

  20. Foundations of Advanced Magnetic Resonance Imaging

    PubMed Central

    Bammer, Roland; Skare, Stefan; Newbould, Rexford; Liu, Chunlei; Thijs, Vincent; Ropele, Stefan; Clayton, David B.; Krueger, Gunnar; Moseley, Michael E.; Glover, Gary H.

    2005-01-01

    Summary: During the past decade, major breakthroughs in magnetic resonance imaging (MRI) quality were made by means of quantum leaps in scanner hardware and pulse sequences. Some advanced MRI techniques have truly revolutionized the detection of disease states and MRI can now—within a few minutes—acquire important quantitative information noninvasively from an individual in any plane or volume at comparatively high resolution. This article provides an overview of the most common advanced MRI methods including diffusion MRI, perfusion MRI, functional MRI, and the strengths and weaknesses of MRI at high magnetic field strengths. PMID:15897944

  1. Foundations of advanced magnetic resonance imaging.

    PubMed

    Bammer, Roland; Skare, Stefan; Newbould, Rexford; Liu, Chunlei; Thijs, Vincent; Ropele, Stefan; Clayton, David B; Krueger, Gunnar; Moseley, Michael E; Glover, Gary H

    2005-04-01

    During the past decade, major breakthroughs in magnetic resonance imaging (MRI) quality were made by means of quantum leaps in scanner hardware and pulse sequences. Some advanced MRI techniques have truly revolutionized the detection of disease states and MRI can now-within a few minutes-acquire important quantitative information noninvasively from an individual in any plane or volume at comparatively high resolution. This article provides an overview of the most common advanced MRI methods including diffusion MRI, perfusion MRI, functional MRI, and the strengths and weaknesses of MRI at high magnetic field strengths.

  2. Magnetic field mapping using an image-intensifying fluorescent probe

    NASA Astrophysics Data System (ADS)

    Tou, T. Y.; Blackwell, B. D.; Sharp, L. E.

    1991-05-01

    A simple, cost-effective image-intensifying fluorescent probe designed for mapping the magnetic surfaces in the heliac sheila is described. It consists of a phosphor-coated metal plate which is enclosed in a grounded U-channel that provides electrostatic shielding. An adjustable accelerating voltage is applied to the metal plate to greatly increase the cathodoluminescence produced by the directed electron beam from an electron gun, and the visible electron-beam image is recorded by a CCD camera. The gain in the image brightness allows significant reduction of the electron-beam energy to minimize the deviation of the measured drift surfaces from the true magnetic surfaces, and to improve resolution for detailed studies of surfaces in the newer stellarator experiments. This technique is particularly suited to electron energies below the phosphor activation threshold, when external image intensifying systems are likely to be very inefficient. Up to 36 toroidal rotations have been observed, limited mainly by the effective cross sections of the fluorescent probe and the electron gun. Mapping at low magnetic field strengths allows detection of small fixed amplitude field errors. Measurements of the gain characteristics and resolution are presented, with an example of the electrically variable resolution achievable with this design. The effect of electron energy on drift surfaces of a heliac is demonstrated.

  3. 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.

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

    PubMed

    Hayashi, Shinichi; Okada, Yasushi

    2015-05-01

    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 micro-tubules, 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.

  5. 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

  6. Real-time Fluorescence Image-Guided Oncologic Surgery

    PubMed Central

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

    2014-01-01

    Medical imaging plays a critical role in cancer diagnosis and planning. Many of these patients rely on surgical intervention for curative outcomes. This requires a careful identification of the primary and microscopic tumors, and the complete removal of cancer. Although there have been efforts to adapt traditional imaging modalities for intraoperative image guidance, they suffer from several constraints such as large hardware footprint, high operation cost, and disruption of the surgical workflow. Because of the ease of image acquisition, relatively low cost devices and intuitive operation, optical imaging methods have received tremendous interests for use in real-time image-guided surgery. To improve imaging depth under low interference by tissue autofluorescence, many of these applications utilize light in the near-infra red (NIR) wavelengths, which is invisible to human eyes. With the availability of a wide selection of tumor-avid contrast agents, advancements in imaging sensors, electronic and optical designs, surgeons are able to combine different attributes of NIR optical imaging techniques to improve treatment outcomes. The emergence of diverse commercial and experimental image guidance systems, which are in various stages of clinical translation, attests to the potential high impact of intraoperative optical imaging methods to improve speed of oncologic surgery with high accuracy and minimal margin positivity. PMID:25287689

  7. 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)].

  8. 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

  9. 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

  10. 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

  11. 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.

  12. Portable multispectral fluorescence imaging system for food safety applications

    NASA Astrophysics Data System (ADS)

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

    2004-03-01

    Fluorescence can be a sensitive method for detecting food contaminants. Of particular interest is detection of fecal contamination as feces is the source of many pathogenic organisms. Feces generally contain chlorophyll a and related compounds due to ingestion of plant materials, and these compounds can readily be detected using fluorescence techniques. Described is a fluorescence-imaging system consisting primarily of a UV light source, an intensified camera with a six-position filter wheel, and software for controlling the system and automatically analyzing the resulting images. To validate the system, orchard apples artificially contaminated with dairy feces were used in a "hands-on" public demonstration. The contamination sites were easily identified using automated edge detection and threshold detection algorithms. In addition, by applying feces to apples and then washing sets of apples at hourly intervals, it was determined that five h was the minimum contact time that allowed identification of the contamination site after the apples were washed. There are many potential uses for this system, including studying the efficacy of apple washing systems.

  13. Brain tumor resection guided by fluorescence imaging and MRI image guidance

    NASA Astrophysics Data System (ADS)

    Valdes, Pablo; Harris, Brent T.; Leblond, Frederic; Fontaine, Kathryn M.; Ji, Songbai; Pogue, Brian W.; Hartov, Alex; Roberts, David W.; Paulsen, Keith D.

    2009-02-01

    Recent evidence suggests a correlation between extent of tumor resection and patient prognosis, making maximal tumor resection a clinical ideal for neurosurgeons. Our group is currently undertaking a clinical study using fluorescence-based detection of tumor coupled with a standard 3-D image guidance system to study the effectiveness of fluorescence-based detection in the neurosurgical operating room. For fluorescence-based detection, we used 5-aminolevulinic acid to induce accumulation of protoporphyrin IX in malignant tissues. In this paper, we chose one prototypical, highly fluorescent case of glioblastoma multiforme, a high-grade glioma, to highlight some of the key findings and methodology used in our study of fluorescence-based detection and resection of brain tumors.

  14. Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging.

    PubMed

    Ulrich, Volker; Fischer, Peter; Riemann, Iris; Königt, Karsten

    2004-01-01

    An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution. PMID:15536977

  15. Image formation in fluorescence coherence-gated imaging through scattering media

    NASA Astrophysics Data System (ADS)

    Bilenca, A.; Lasser, T.; Ozcan, A.; Leitgeb, R. A.; Bouma, B. E.; Tearney, G. J.

    2007-03-01

    Recently, we have experimentally demonstrated a new form of cross-sectional, coherence-gated fluorescence imaging referred to as SD-FCT (‘spectral-domain fluorescence coherence tomography’). Imaging in SD-FCT is accomplished by spectrally detecting self-interference of the spontaneous emission of fluorophores, thereby providing depth-resolved information on the axial positions of fluorescent probes. Here, we present a theoretical investigation of the factors affecting the detected SD-FCT signal through scattering media. An imaging equation for SD-FCT is derived that includes the effects of defocusing, numerical-aperture, and the optical properties of the medium. A comparison between the optical sectioning capabilities of SD-FCT and confocal microscopy is also presented. Our results suggest that coherence gating in fluorescence imaging may provide an improved approach for depth-resolved imaging of fluorescently labeled samples; high axial resolution (a few microns) can be achieved with low numerical apertures (NA<0.09) while maintaining a large depth of field (a few hundreds of microns) in a relatively low scattering medium (6 mean free paths), whereas moderate NA’s can be used to enhance depth selectivity in more highly scattering biological samples.

  16. 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

  17. 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

  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. Total variation versus wavelet-based methods for image denoising in fluorescence lifetime imaging microscopy

    PubMed Central

    Chang, Ching-Wei; Mycek, Mary-Ann

    2014-01-01

    We report the first application of wavelet-based denoising (noise removal) methods to time-domain box-car fluorescence lifetime imaging microscopy (FLIM) images and compare the results to novel total variation (TV) denoising methods. Methods were tested first on artificial images and then applied to low-light live-cell images. Relative to undenoised images, TV methods could improve lifetime precision up to 10-fold in artificial images, while preserving the overall accuracy of lifetime and amplitude values of a single-exponential decay model and improving local lifetime fitting in live-cell images. Wavelet-based methods were at least 4-fold faster than TV methods, but could introduce significant inaccuracies in recovered lifetime values. The denoising methods discussed can potentially enhance a variety of FLIM applications, including live-cell, in vivo animal, or endoscopic imaging studies, especially under challenging imaging conditions such as low-light or fast video-rate imaging. PMID:22415891

  20. Terahertz Tools Advance Imaging for Security, Industry

    NASA Technical Reports Server (NTRS)

    2010-01-01

    Picometrix, a wholly owned subsidiary of Advanced Photonix Inc. (API), of Ann Arbor, Michigan, invented the world s first commercial terahertz system. The company improved the portability and capabilities of their systems through Small Business Innovation Research (SBIR) agreements with Langley Research Center to provide terahertz imaging capabilities for inspecting the space shuttle external tanks and orbiters. Now API s systems make use of the unique imaging capacity of terahertz radiation on manufacturing floors, for thickness measurements of coatings, pharmaceutical tablet production, and even art conservation.

  1. Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging.

    PubMed

    Wei, Lu; Chen, Zhixing; Min, Wei

    2012-06-01

    Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.

  2. Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging

    PubMed Central

    Wei, Lu; Chen, Zhixing; Min, Wei

    2012-01-01

    Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8. PMID:22741091

  3. 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.

  4. 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.

  5. 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

  6. 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.

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

    PubMed Central

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

    2015-01-01

    Abstract. 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

  8. 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...

  9. 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

  10. Real-time intraoperative fluorescence imaging system using light-absorption correction

    NASA Astrophysics Data System (ADS)

    Themelis, George; Yoo, Jung Sun; Soh, Kwang-Sup; Schulz, Ralf; Ntziachristos, Vasilis

    2009-11-01

    We present a novel fluorescence imaging system developed for real-time interventional imaging applications. The system implements a correction scheme that improves the accuracy of epi-illumination fluorescence images for light intensity variation in tissues. The implementation is based on the use of three cameras operating in parallel, utilizing a common lens, which allows for the concurrent collection of color, fluorescence, and light attenuation images at the excitation wavelength from the same field of view. The correction is based on a ratio approach of fluorescence over light attenuation images. Color images and video is used for surgical guidance and for registration with the corrected fluorescence images. We showcase the performance metrics of this system on phantoms and animals, and discuss the advantages over conventional epi-illumination systems developed for real-time applications and the limits of validity of corrected epi-illumination fluorescence imaging.

  11. 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

  12. Imaging Tumor Hypoxia to Advance Radiation Oncology

    PubMed Central

    Lee, Chen-Ting; Boss, Mary-Keara

    2014-01-01

    Abstract Significance: Most solid tumors contain regions of low oxygenation or hypoxia. Tumor hypoxia has been associated with a poor clinical outcome and plays a critical role in tumor radioresistance. Recent Advances: Two main types of hypoxia exist in the tumor microenvironment: chronic and cycling hypoxia. Chronic hypoxia results from the limited diffusion distance of oxygen, and cycling hypoxia primarily results from the variation in microvessel red blood cell flux and temporary disturbances in perfusion. Chronic hypoxia may cause either tumor progression or regressive effects depending on the tumor model. However, there is a general trend toward the development of a more aggressive phenotype after cycling hypoxia. With advanced hypoxia imaging techniques, spatiotemporal characteristics of tumor hypoxia and the changes to the tumor microenvironment can be analyzed. Critical Issues: In this review, we focus on the biological and clinical consequences of chronic and cycling hypoxia on radiation treatment. We also discuss the advanced non-invasive imaging techniques that have been developed to detect and monitor tumor hypoxia in preclinical and clinical studies. Future Directions: A better understanding of the mechanisms of tumor hypoxia with non-invasive imaging will provide a basis for improved radiation therapeutic practices. Antioxid. Redox Signal. 21, 313–337. PMID:24329000

  13. 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.

  14. Optical fibers for high-resolution in vivo microendoscopic fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Oh, Gyungseok; Chung, Euiheon; Yun, Seok H.

    2013-12-01

    Optical fiber-based high-resolution fluorescence imaging techniques have promising applications in clinical practice and preclinical research using animals. Here we review the instrumentation and applications of microendoscopy based on various types of optical fibers. Single-mode fibers and double-clad fibers have been widely used for delivering light from light sources to tissues and collecting light from tissues to photodetectors. Coherent fiber bundles, cylindrical graded-index lenses, and multi-mode fibers have been employed in both beam-scanning and non-scanning microscopy. With continuing advances of optical fiber technologies, further innovations in optical microendoscopy are expected.

  15. Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging.

    PubMed

    Battisti, Antonella; Digman, Michelle A; Gratton, Enrico; Storti, Barbara; Beltram, Fabio; Bizzarri, Ranieri

    2012-05-25

    A versatile pH-dependent fluorescent protein was applied to intracellular pH measurements by means of the phasor approach to fluorescence lifetime imaging. By this fit-less method we obtain intracellular pH maps under resting or altered physiological conditions by single-photon confocal or two-photon microscopy.

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

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

    PubMed

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

    2014-01-01

    Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo. PMID:24487319

  18. FIZICS: fluorescent imaging zone identification system, a novel macro imaging system.

    PubMed

    Skwish, Stephen; Asensio, Francisco; King, Greg; Clarke, Glenn; Kath, Gary; Salvatore, Michael J; Dufresne, Claude

    2004-12-01

    Constantly improving biological assay development continues to drive technological requirements. Recently, a specification was defined for capturing white light and fluorescent images of agar plates ranging in size from the NUNC Omni tray (96-well footprint, 128 x 85 mm) to the NUNC Bio Assay Dish (245 x 245 mm). An evaluation of commercially available products failed to identify any system capable of fluorescent macroimaging with discrete wavelength selection. To address the lack of a commercially available system, a custom imaging system was designed and constructed. This system provides the same capabilities of many commercially available systems with the added ability to fluorescently image up to a 245 x 245 mm area using wavelengths in the visible light spectrum.

  19. 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.

  20. X-Ray Fluorescence Imaging of Ancient Artifacts

    NASA Astrophysics Data System (ADS)

    Thorne, Robert; Geil, Ethan; Hudson, Kathryn; Crowther, Charles

    2011-03-01

    Many archaeological artifacts feature inscribed and/or painted text or figures which, through erosion and aging, have become difficult or impossible to read with conventional methods. Often, however, the pigments in paints contain metallic elements, and traces may remain even after visible markings are gone. A promising non-destructive technique for revealing these remnants is X-ray fluorescence (XRF) imaging, in which a tightly focused beam of monochromatic synchrotron radiation is raster scanned across a sample. At each pixel, an energy-dispersive detector records a fluorescence spectrum, which is then analyzed to determine element concentrations. In this way, a map of various elements is made across a region of interest. We have succesfully XRF imaged ancient Greek, Roman, and Mayan artifacts, and in many cases, the element maps have revealed significant new information, including previously invisible painted lines and traces of iron from tools used to carve stone tablets. X-ray imaging can be used to determine an object's provenance, including the region where it was produced and whether it is authentic or a copy.

  1. 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.

  2. 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

  3. Image stabilization for SWIR advanced optoelectronic device

    NASA Astrophysics Data System (ADS)

    Schiopu, Paul; Manea, Adrian; Cristea, Ionica; Grosu, Neculai; Craciun, Anca-Ileana; Craciun, Alexandru; Granciu, Dana

    2015-02-01

    At long ranges and under low visibility conditions, Advanced Optoelectronic Device provides the signal-to-noise ratio and image quality in the Short-wave Infra-red - SWIR (wavelengths between 1,1 ÷2,5 μm), significantly better than in the near wave infrared - NWIR and visible spectral bands [1,2]. The quality of image is nearly independent of the polarization in the incoming light, but it is influenced by the relative movement between the optical system and the observer (the operators' handshake), and the movement towards the support system (land and air vehicles). All these make it difficult to detect objectives observation in real time. This paper presents some systems enhance which the ability of observation and sighting through the optical systems without the use of the stands, tripods or other means. We have to eliminate the effect of "tremors of the hands" and the vibration in order to allow the use of optical devices by operators on the moving vehicles on land, on aircraft, or on boats, and to provide additional comfort for the user to track the moving object through the optical system, without losing the control in the process of detection and tracking. The practical applications of stabilization image process, in SWIR, are the most advanced part of the optical observation systems available worldwide [3,4,5]. This application has a didactic nature, because it ensures understanding by the students about image stabilization and their participation in research.

  4. Comparative studies of X-ray images and fluorescence images of the same specimens

    NASA Astrophysics Data System (ADS)

    Majima, T.; Tomie, T.; Shimizu, H.

    2003-03-01

    A flash contact soft x-ray microscope using laser-induced plasma as a flash x-ray source is a practical instrument for observation of living organisms in water [1-4]. As previously reported we developed a tabletop flash contact soft x-ray microscope System [3]. In this System, x-ray images are given as whole projection of the specimens on the PMMA membrane. This causes us some complexity for understanding the x-ray images. It is necessary to attribute features in the x-ray images to sub-cellular structures of the specimen. For this purpose we have developed a new sample holder, where specimens are observable with a fluorescence microscope just before x-ray exposure. Fluorescence images of onion epidermal cells stained by DAPI and x-ray images of the same specimens are compared.

  5. Red fluorescent chitosan nanoparticles grafted with poly(2-methacryloyloxyethyl phosphorylcholine) for live cell imaging.

    PubMed

    Wang, Ke; Fan, Xingliang; Zhang, Xiaoyong; Zhang, Xiqi; Chen, Yi; Wei, Yen

    2016-08-01

    Poly(2-methacryloyloxyethyl phosphorylcholine) conjugated red fluorescent chitosan nanoparticles (GCC-pMPC) were facilely fabricated by "grafting from" method via surface initiated atom transfer radical polymerization (ATRP). Firstly, glutaraldehyde crosslinked red fluorescent chitosan nanoparticles (GCC NPs) with many amino groups and hydroxyl groups on their surface were prepared, which were then reacted with 2-bromoisobutyryl bromide to form GCC-Br; subsequently, poly(MPC) (pMPC) brushes were grafted onto GCC NPs surface using GCC-Br as initiator via ATRP. Compared with PEGylated nanoparticles, zwitterionic polymers modified nanoparticles demonstrated better performance in their cellular uptake. Moreover, the obtained GCC-pMPC demonstrated excellent water-dispersibility, biocompatibility, and photostability, which made them highly potential for long-term tracing applications. Importantly, the successful live cell imaging of GCC-pMPC would remarkably advance the research of their further bioapplications.

  6. Performance evaluation of integrating detectors for near-infrared fluorescence molecular imaging

    NASA Astrophysics Data System (ADS)

    Zhu, Banghe; Rasmussen, John C.; Sevick-Muraca, Eva M.

    2014-05-01

    Although there has been a plethora of devices advanced for clinical translation, there has been no standards to compare and determine the optical device for fluorescence molecular imaging. In this work, we compare different CCD configurations using a solid phantom developed to mimic pM - fM concentrations of near-infrared fluorescent dyes in tissues. Our results show that intensified CCD systems (ICCDs) offer greater contrast at larger signal-tonoise ratios (SNRs) in comparison to their un-intensified CCD systems operated at clinically reasonable, sub-second acquisition times. Furthermore, we compared our investigational ICCD device to the commercial NOVADAQ SPY system, demonstrating different performance in both SNR and contrast.

  7. 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.

  8. 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,...

  9. Understanding Protein Folding from Advances of Fluorescence Energy Transfer.

    NASA Astrophysics Data System (ADS)

    Tcherkasskaya, Olga; Gronenborn, Angela M.

    2001-03-01

    A multi-site fluorescence energy transfer method was developed for the study of protein folding. Technique uses "tyrosine-phenylalanine" substitution mutagenesis to place the "tyrosine-guest" into positions of interest into the protein structure. Tetranitromethane modification of the tyrosine-guest renders this amino acid an acceptor of the tryptophan fluorescence. This approach can be applied to any protein system, and, most importantly, does not require single- or double-labeling of the protein molecule by a donor and/or an acceptor fluorophore. It is equally suited for equilibrium as well as kinetic studies of folding. We tested the methodology to monitor the equilibrium (un)folding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) induced by guanidine hydrochloride. Wild-type GB1 contains three tyrosines located at positions 3, 33, 45 and a single tryptophan residue at position 43. Two of the three tyrosines were replaced in turn, thereby allowing us to measure the energy transfer from Trp43 to each particular tyrosine. Overall, multi-parametrical experiments on GB1 including circular dichroism, steady state and time-resolved fluorescence, as well as fluorescence energy transfer revealed the existence of highly stable unfolded intermediates, which precede the formation of the rigid (native) secondary structure.

  10. Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities.

    PubMed

    Heuff, Romey F; Swift, Jody L; Cramb, David T

    2007-04-28

    Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.

  11. 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

  12. 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

  13. 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.

  14. Red emitting neutral fluorescent glycoconjugates for membrane optical imaging.

    PubMed

    Redon, Sébastien; Massin, Julien; Pouvreau, Sandrine; De Meulenaere, Evelien; Clays, Koen; Queneau, Yves; Andraud, Chantal; Girard-Egrot, Agnès; Bretonnière, Yann; Chambert, Stéphane

    2014-04-16

    A family of neutral fluorescent probes was developed, mimicking the overall structure of natural glycolipids in order to optimize their membrane affinity. Nonreducing commercially available di- or trisaccharidic structures were connected to a push-pull chromophore based on dicyanoisophorone electron-accepting group, which proved to fluoresce in the red region with a very large Stokes shift. This straightforward synthetic strategy brought structural variations to a series of probes, which were studied for their optical, biophysical, and biological properties. The insertion properties of the different probes into membranes were evaluated on a model system using the Langmuir monolayer balance technique. Confocal fluorescence microscopy performed on muscle cells showed completely different localizations and loading efficiencies depending on the structure of the probes. When compared to the commercially available ANEPPS, a family of commonly used membrane imaging dyes, the most efficient probes showed a similar brightness, but a sharper pattern was observed. According to this study, compounds bearing one chromophore, a limited size of the carbohydrate moiety, and an overall rod-like shape gave the best results.

  15. 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.

  16. 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.

  17. Sentinel lymph node imaging by a fluorescently labeled DNA tetrahedron.

    PubMed

    Kim, Kyoung-Ran; Lee, Yong-Deok; Lee, Taemin; Kim, Byeong-Su; Kim, Sehoon; Ahn, Dae-Ro

    2013-07-01

    Sentinel lymph nodes (SLNs) are the first lymph nodes which cancer cells reach after traveling through lymphatic vessels from the primary tumor. Evaluating the nodal status is crucial in accurate staging of human cancers and accordingly determines prognosis and the most appropriate treatment. The commonly used methods for SLN identification in clinics are based on employment of a colloid of radionuclide or injection of a small dye. Although these methods have certainly contributed to improve surgical practice, new imaging materials are still required to overcome drawbacks of the techniques such as inconvenience of handling radioactive materials and short retention time of small dyes in SLNs. Here, we prepare a fluorescence-labeled DNA tetrahedron and perform SLN imaging by using the DNA nanoconstruct. With a successful identification of SLNs by the DNA nanoconstruct, we suggest that DNA tetrahedron hold great promises for clinical applications.

  18. 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

  19. 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

  20. Fluorescence imaging of chromosomal DNA using click chemistry

    PubMed Central

    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

  1. Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging

    PubMed Central

    Hense, Anika; Prunsche, Benedikt; Gao, Peng; Ishitsuka, Yuji; Nienhaus, Karin; Ulrich Nienhaus, G.

    2015-01-01

    The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M−1cm−1, mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths. PMID:26648024

  2. Co-registered optical coherence tomography and fluorescence molecular imaging for simultaneous morphological and molecular imaging

    NASA Astrophysics Data System (ADS)

    Yuan, Shuai; Roney, Celeste A.; Wierwille, Jeremiah; Chen, Chao-Wei; Xu, Biying; Griffiths, Gary; 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 is a great deal of interest 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 mm2 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 the APCmin mouse model is presented as an example of biological applications of this co-registered OCT/FMI system.

  3. Fluorescence imaging to localize head and neck squamous cell carcinoma for enhanced pathological assessment.

    PubMed

    Warram, Jason M; de Boer, Esther; van Dam, Gooitzen M; Moore, Lindsay S; Bevans, Stephanie L; Walsh, Erika M; Young, Erik S; Carroll, William R; Stevens, Todd M; Rosenthal, Eben L

    2016-04-01

    Accurately identifying close or positive margins in real-time permits re-excision during surgical procedures. Intraoperative assessment of margins via gross examination and frozen section is a widely used tool to assist the surgeon in achieving complete resection. While this methodology permits diagnosis of freshly resected tissue, the process is fraught with misinterpretation and sampling errors. During fluorescence-guided surgery, an exogenous fluorescent agent specific for the target disease is imaged in order to navigate the surgical excision. As this technique quickly advances into the clinic, we hypothesize that the disease-specific fluorescence inherently contained within the resected tissues can be used to guide histopathological assessment. To evaluate the feasibility of fluorescence-guided pathology, we evaluated head and neck squamous cell carcinoma tumour specimens and margins resected from animals and patients after systemic injection of cetuximab-IRDye800CW. In a preclinical model of luciferase-positive tumour resection using bioluminescence as the gold standard, fluorescence assessment determined by closed-field fluorescence imaging of fresh resected margins accurately predicted the presence of disease in 33/39 positive margins yielding an overall sensitivity of 85%, specificity of 95%, positive predictive value (PPV) of 94%, and a negative predictive value (NPV) of 87%, which was superior to both surgical assessment (54%, 61%, 57%, and 58%) and pathological assessment (49%, 95%, 91%, and 66%), respectively. When the power of the technique was evaluated using human-derived tumour tissues, as little as 0.5mg (1mm(3)) of tumour tissue was identified (tumour-to-background-ratio:5.2). When the sensitivity/specificity of fluorescence-guided pathology was determined using traditional histological assessment as the gold standard in human tissues obtained during fluorescence-guided surgery, the technique was highly accurate with a sensitivity of 91

  4. Experimental demonstration of novel imaging geometries for x-ray fluorescence computed tomography

    PubMed Central

    Fu, Geng; Meng, Ling-Jian; Eng, Peter; Newville, Matt; Vargas, Phillip; Riviere, Patrick La

    2013-01-01

    Purpose: X-ray fluorescence computed tomography (XFCT) is an emerging imaging modality that maps the three-dimensional distribution of elements, generally metals, in ex vivo specimens and potentially in living animals and humans. At present, it is generally performed at synchrotrons, taking advantage of the high flux of monochromatic x rays, but recent work has demonstrated the feasibility of using laboratory-based x-ray tube sources. In this paper, the authors report the development and experimental implementation of two novel imaging geometries for mapping of trace metals in biological samples with ∼50–500 μm spatial resolution. Methods: One of the new imaging approaches involves illuminating and scanning a single slice of the object and imaging each slice's x-ray fluorescent emissions using a position-sensitive detector and a pinhole collimator. The other involves illuminating a single line through the object and imaging the emissions using a position-sensitive detector and a slit collimator. They have implemented both of these using synchrotron radiation at the Advanced Photon Source. Results: The authors show that it is possible to achieve 250 eV energy resolution using an electron multiplying CCD operating in a quasiphoton-counting mode. Doing so allowed them to generate elemental images using both of the novel geometries for imaging of phantoms and, for the second geometry, an osmium-stained zebrafish. Conclusions: The authors have demonstrated the feasibility of these two novel approaches to XFCT imaging. While they use synchrotron radiation in this demonstration, the geometries could readily be translated to laboratory systems based on tube sources. PMID:23718594

  5. 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.

  6. Imaging mitochondrial reactive oxygen species with fluorescent probes: current applications and challenges.

    PubMed

    Zhang, X; Gao, F

    2015-04-01

    Mitochondrial reactive oxygen species (ROS) is a key element in the regulation of several physiological functions and in the development or progression of multiple pathological events. A key task in the study of mitochondrial ROS is to establish reliable methods for measuring the ROS level in mitochondria with high selectivity, sensitivity, and spatiotemporal resolution. Over the last decade, imaging tools with fluorescent indicators from either small-molecule dyes or genetically encoded probes that can be targeted to mitochondria have been developed, which provide a powerful method to visualize and even quantify mitochondrial ROS level not only in live cells, but also in live animals. These innovative tools that have bestowed exciting new insights in mitochondrial ROS biology have been further promoted with the invention of new techniques in indicator design and fluorescent detection. However, these probes present some limitations in terms of specificity, sensitivity, and kinetics; failure to recognize these limitations often results in inappropriate interpretations of data. This review evaluates the recent advances in mitochondrial ROS imaging approaches with emphasis on their proper application and limitations, and highlights the future perspectives in the development of novel fluorescent probes for visualizing all species of ROS.

  7. Fluorescence lifetime imaging of membrane lipid order with a ratiometric fluorescent probe.

    PubMed

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

    2015-05-19

    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

  8. 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

  9. 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

  10. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  12. Recent advances in computer image generation simulation.

    PubMed

    Geltmacher, H E

    1988-11-01

    An explosion in flight simulator technology over the past 10 years is revolutionizing U.S. Air Force (USAF) operational training. The single, most important development has been in computer image generation. However, other significant advances are being made in simulator handling qualities, real-time computation systems, and electro-optical displays. These developments hold great promise for achieving high fidelity combat mission simulation. This article reviews the progress to date and predicts its impact, along with that of new computer science advances such as very high speed integrated circuits (VHSIC), on future USAF aircrew simulator training. Some exciting possibilities are multiship, full-mission simulators at replacement training units, miniaturized unit level mission rehearsal training simulators, onboard embedded training capability, and national scale simulator networking.

  13. Rapid analysis and exploration of fluorescence microscopy images.

    PubMed

    Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason M; Steininger, Robert J; Wu, Lani F; Altschuler, Steven J

    2014-03-19

    Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard. Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

  14. 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

  15. Nonnegative matrix factorization: a blind spectra separation method for in vivo fluorescent optical imaging.

    PubMed

    Montcuquet, Anne-Sophie; Hervé, Lionel; Navarro, Fabrice; Dinten, Jean-Marc; Mars, Jérôme I

    2010-01-01

    Fluorescence imaging in diffusive media is an emerging imaging modality for medical applications that uses injected fluorescent markers that bind to specific targets, e.g., carcinoma. The region of interest is illuminated with near-IR light and the emitted back fluorescence is analyzed to localize the fluorescence sources. To investigate a thick medium, as the fluorescence signal decreases with the light travel distance, any disturbing signal, such as biological tissues intrinsic fluorescence (called autofluorescence) is a limiting factor. Several specific markers may also be simultaneously injected to bind to different molecules, and one may want to isolate each specific fluorescent signal from the others. To remove the unwanted fluorescence contributions or separate different specific markers, a spectroscopic approach is explored. The nonnegative matrix factorization (NMF) is the blind positive source separation method we chose. We run an original regularized NMF algorithm we developed on experimental data, and successfully obtain separated in vivo fluorescence spectra.

  16. Advances in computed tomography imaging technology.

    PubMed

    Ginat, Daniel Thomas; Gupta, Rajiv

    2014-07-11

    Computed tomography (CT) is an essential tool in diagnostic imaging for evaluating many clinical conditions. In recent years, there have been several notable advances in CT technology that already have had or are expected to have a significant clinical impact, including extreme multidetector CT, iterative reconstruction algorithms, dual-energy CT, cone-beam CT, portable CT, and phase-contrast CT. These techniques and their clinical applications are reviewed and illustrated in this article. In addition, emerging technologies that address deficiencies in these modalities are discussed.

  17. Fluorescence imaging and chlorophyll fluorescence to evaluate the role of EDU in UV-B protection in cucumber

    NASA Astrophysics Data System (ADS)

    Sandhu, Ravinder K.; Kim, Moon S.; Krizek, Donald T.; Middleton, Elizabeth M.

    1997-07-01

    A fluorescence imaging system and chlorophyll fluorescence emissions were used to evaluate whether EDU, N-[2-(2-oxo-1- imidazolidinyl) ethyl]-N'-phenylurea, provided protection against ultraviolet-B (UV-B) irradiation (290 - 320 nm) in cucumber (Cucumis sativus L.) leaves. Plants were grown in growth chambers illuminated for 14 h per day with 400 W high pressure sodium and metal halide lamps. Photosynthetically active radiation (PAR) for 1 hr at the beginning and end of each cycle was provided at 270 micrometers ol m-2 s-1 PAR; during the other 12 hr of the photoperiod, the plants received 840 micrometers ol m-2 s-1 PAR. Beginning on the twelfth day, the plants were exposed to UV-B radiation (0.2 & 18.0 kJ m-2d-1) for 2 days at 8 h per day centered in the photoperiod. Rapidly acquired (less than 1 s), high spatial resolution (less than 1 mm2) images were obtained for whole adaxial leaf surfaces using a fluorescence imaging system. The steady-state fluorescence images were acquired in four spectral regions: blue (F450 nm), green (F550 nm), red (F680 nm), and far-red (F740 nm). Fluorescence emission spectra for leaf pigments extracted in dimethyl sulfoxide (DMSO) were obtained by excitation at 280 and 380 nm (280EX 300 - 530 nm; 380EX 400 - 800 nm). Both UV-B and EDU induced stress responses in cucumber leaves that altered the fluorescence emissions obtained from extracts. In the fluorescence images only UV-B induced stress responses were observed but this damage was detected before it was visually apparent. There was no evidence that EDU afforded protection against UV-B irradiation. Use of fluorescence imaging may provide an early stress detection capability for helping to assess damage to the photosynthetic apparatus of plants.

  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.

  19. 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

  20. Detecting fluorescence hot-spots using mosaic maps generated from multimodal endoscope imaging

    NASA Astrophysics Data System (ADS)

    Yang, Chenying; Soper, Timothy D.; Seibel, Eric J.

    2013-03-01

    Fluorescence labeled biomarkers can be detected during endoscopy to guide early cancer biopsies, such as high-grade dysplasia in Barrett's Esophagus. To enhance intraoperative visualization of the fluorescence hot-spots, a mosaicking technique was developed to create full anatomical maps of the lower esophagus and associated fluorescent hot-spots. The resultant mosaic map contains overlaid reflectance and fluorescence images. It can be used to assist biopsy and document findings. The mosaicking algorithm uses reflectance images to calculate image registration between successive frames, and apply this registration to simultaneously acquired fluorescence images. During this mosaicking process, the fluorescence signal is enhanced through multi-frame averaging. Preliminary results showed that the technique promises to enhance the detectability of the hot-spots due to enhanced fluorescence signal.

  1. 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.

  2. Advanced imaging of the scapholunate ligamentous complex.

    PubMed

    Shahabpour, Maryam; Staelens, Barbara; Van Overstraeten, Luc; De Maeseneer, Michel; Boulet, Cedric; De Mey, Johan; Scheerlinck, Thierry

    2015-12-01

    The scapholunate joint is one of the most involved in wrist injuries. Its stability depends on primary and secondary stabilisers forming together the scapholunate complex. This ligamentous complex is often evaluated by wrist arthroscopy. To avoid surgery as diagnostic procedure, optimization of MR imaging parameters as use of three-dimensional (3D) sequences with very thin slices and high spatial resolution, is needed to detect lesions of the intrinsic and extrinsic ligaments of the scapholunate complex. The paper reviews the literature on imaging of radial-sided carpal ligaments with advanced computed tomographic arthrography (CTA) and magnetic resonance arthrography (MRA) to evaluate the scapholunate complex. Anatomy and pathology of the ligamentous complex are described and illustrated with CTA, MRA and corresponding arthroscopy. Sprains, mid-substance tears, avulsions and fibrous infiltrations of carpal ligaments could be identified on CTA and MRA images using 3D fat-saturated PD and 3D DESS (dual echo with steady-state precession) sequences with 0.5-mm-thick slices. Imaging signs of scapholunate complex pathology include: discontinuity, nonvisualization, changes in signal intensity, contrast extravasation (MRA), contour irregularity and waviness and periligamentous infiltration by edema, granulation tissue or fibrosis. Based on this preliminary experience, we believe that 3 T MRA using 3D sequences with 0.5-mm-thick slices and multiplanar reconstructions is capable to evaluate the scapholunate complex and could help to reduce the number of diagnostic arthroscopies.

  3. Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning

    PubMed Central

    de Groot, Mattijs; Evans, Conor L.; de Boer, Johannes F.

    2012-01-01

    We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 µm over a 500 µm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a three-dimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan. PMID:22772223

  4. Robust overlay schemes for the fusion of fluorescence and color channels in biological imaging.

    PubMed

    Glatz, Jürgen; Symvoulidis, Panagiotis; Garcia-Allende, P Beatriz; Ntziachristos, Vasilis

    2014-04-01

    Molecular fluorescence imaging is a commonly used method in various biomedical fields and is undergoing rapid translation toward clinical applications. Color images are commonly superimposed with fluorescence measurements to provide orientation, anatomical information, and molecular tissue properties in a single image. New adaptive methods that produce a more robust composite image than conventional lime green alpha blending are presented and demonstrated herein. Moreover, visualization through temporal changes is showcased as an alternative for real-time imaging systems.

  5. Method for single illumination source combined optical coherence tomography and fluorescence imaging of fluorescently labeled ocular structures in transgenic mice.

    PubMed

    McNabb, Ryan P; Blanco, Tomas; Bomze, Howard M; Tseng, Henry C; Saban, Daniel R; Izatt, Joseph A; Kuo, Anthony N

    2016-10-01

    In vivo imaging permits longitudinal study of ocular disease processes in the same animal over time. Two different in vivo optical imaging modalities - optical coherence tomography (OCT) and fluorescence - provide important structural and cellular data respectively about disease processes. In this Methods in Eye Research article, we describe and demonstrate the combination of these two modalities producing a truly simultaneous OCT and fluorescence imaging system for imaging of fluorescently labeled animal models. This system uses only a single light source to illuminate both modalities, and both share the same field of view. This allows simultaneous acquisition of OCT and fluorescence images, and the benefits of both techniques are realized without incurring increased costs in variability, light exposure, time, and post-processing effort as would occur when the modalities are used separately. We then utilized this system to demonstrate multi-modal imaging in a progression of samples exhibiting both fluorescence and OCT scattering beginning with resolution targets, ex vivo thy1-YFP labeled neurons in mouse eyes, and finally an in vivo longitudinal time course of GFP labeled myeloid cells in a mouse model of ocular allergy. PMID:27519152

  6. 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.

  7. 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}.

  8. Recent advances in fluorescent arylboronic acids for glucose sensing.

    PubMed

    Hansen, Jon Stefan; Christensen, Jørn Bolstad

    2013-01-01

    Continuous glucose monitoring (CGM) is crucial in order to avoid complications caused by change in blood glucose for patients suffering from diabetes mellitus. The long-term consequences of high blood glucose levels include damage to the heart, eyes, kidneys, nerves and other organs, among others, caused by malign glycation of vital protein structures. Fluorescent monitors based on arylboronic acids are promising candidates for optical CGM, since arylboronic acids are capable of forming arylboronate esters with 1,2-cis-diols or 1,3-diols fast and reversibly, even in aqueous solution. These properties enable arylboronic acid dyes to provide immediate information of glucose concentrations. Thus, the replacement of the commonly applied semi-invasive and non-invasive techniques relying on glucose binding proteins, such as concanavalin A, or enzymes, such as glucose oxidase, glucose dehydrogenase and hexokinases/glucokinases, might be possible. The recent progress in the development of fluorescent arylboronic acid dyes will be emphasized in this review. PMID:25586415

  9. Recent Advances in Fluorescent Arylboronic Acids for Glucose Sensing

    PubMed Central

    Hansen, Jon Stefan; Christensen, Jørn Bolstad

    2013-01-01

    Continuous glucose monitoring (CGM) is crucial in order to avoid complications caused by change in blood glucose for patients suffering from diabetes mellitus. The long-term consequences of high blood glucose levels include damage to the heart, eyes, kidneys, nerves and other organs, among others, caused by malign glycation of vital protein structures. Fluorescent monitors based on arylboronic acids are promising candidates for optical CGM, since arylboronic acids are capable of forming arylboronate esters with 1,2-cis-diols or 1,3-diols fast and reversibly, even in aqueous solution. These properties enable arylboronic acid dyes to provide immediate information of glucose concentrations. Thus, the replacement of the commonly applied semi-invasive and non-invasive techniques relying on glucose binding proteins, such as concanavalin A, or enzymes, such as glucose oxidase, glucose dehydrogenase and hexokinases/glucokinases, might be possible. The recent progress in the development of fluorescent arylboronic acid dyes will be emphasized in this review. PMID:25586415

  10. 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.

  11. A novel approach for phytotoxicity assessment by CCD fluorescence imaging.

    PubMed

    Gavel, Alan; Marsálek, Blahoslav

    2004-08-01

    Rapidity, cost effectiveness, ecological reliability, and the possibility of the direct interpretation of bioassay results are the main motivations for the development of new approaches in ecotoxicity testing. Color, turbidity, and nutrient content are factors of great importance in phytotoxicity testing of natural samples. Some algal bioassay end points are markedly influenced by such factors or are impossible to estimate in their presence. An algal toxicity test applicable as an early-warning system has to be able to give a signal in the shortest time possible (hours). We used CCD fluorescence imaging to evaluate toxicity effects in algae, cyanobacteria, and vascular plants, and the data were compared with standard end points. Plant physiologists use this device mainly for photosynthesis research, but common photosynthetic parameters used to characterize chlorophyll fluorescence (F(v)/F(m), F(0), and F(m)) or its quenching (NPQ) have only limited ecotoxicological applicability. Previously published estimations based on the geometrical complement to measured data (complementary area) in the fast kinetics (Kautsky effect) respond especially to pollutants affecting electron transport in the PSII. We recommend using a definite integral value combined with relative fluorescence decay (Rfd) to obtain a sensitive and fast toxicity response. Our approach integrates more mechanisms of toxicity (effects on membranes, proton pump, ATP synthesis, etc.) and improves the toxicity signal, which is more ecotoxicologically relevant. This method can give results after 2-6 h of exposure and is especially useful as an early-warning system and for the toxicity assessment of environmental samples with unknown nutrient status. Results with our approach after 4-6 h are comparable with those obtained with a 96-h standard algal assay. A similar methodical approach can be applied for toxicity evaluation of plants or lichens, or for in situ ecotoxicological studies of microphytobenthos

  12. 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.

  13. Tumor Endothelial Marker Imaging in Melanomas Using Dual-Tracer Fluorescence Molecular Imaging

    PubMed Central

    Tichauer, Kenneth M.; Deharvengt, Sophie J.; Samkoe, Kimberley S.; Gunn, Jason R.; Bosenberg, Marcus W.; Turk, Mary-Jo; Hasan, Tayyaba; Stan, Radu V.; Pogue, Brian W.

    2014-01-01

    Purpose Cancer-specific endothelial markers available for intravascular binding are promising targets for new molecular therapies. In this study, a molecular imaging approach of quantifying endothelial marker concentrations (EMCI) is developed and tested in highly light-absorbing melanomas. The approach involves injection of targeted imaging tracer in conjunction with an untargeted tracer, which is used to account for nonspecific uptake and tissue optical property effects on measured targeted tracer concentrations. Procedures Theoretical simulations and a mouse melanoma model experiment were used to test out the EMCI approach. The tracers used in the melanoma experiments were fluorescently labeled anti-Plvap/PV1 antibody (plasmalemma vesicle associated protein Plvap/PV1 is a transmembrane protein marker exposed on the luminal surface of endothelial cells in tumor vasculature) and a fluorescent isotype control antibody, the uptakes of which were measured on a planar fluorescence imaging system. Results The EMCI model was found to be robust to experimental noise under reversible and irreversible binding conditions and was capable of predicting expected overexpression of PV1 in melanomas compared to healthy skin despite a 5-time higher measured fluorescence in healthy skin compared to melanoma: attributable to substantial light attenuation from melanin in the tumors. Conclusions This study demonstrates the potential of EMCI to quantify endothelial marker concentrations in vivo, an accomplishment that is currently unavailable through any other methods, either in vivo or ex vivo. PMID:24217944

  14. Fluorescence Imaging with One-nanometer Accuracy (FIONA)

    PubMed Central

    Sheung, Janet; Lee, Sang Hak; Teng, Kai Wen; Selvin, Paul R.

    2014-01-01

    Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved. PMID:25286081

  15. 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.

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

    PubMed Central

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

    2016-01-01

    Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide–mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions. PMID:27386579

  17. Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging

    NASA Astrophysics Data System (ADS)

    Peter, Sébastien; Elgass, Kirstin; Sackrow, Marcus; Caesar, Katharina; Born, Anne-Kathrin; Maniura, Katharina; Harter, Klaus; Meixner, Alfred J.; Schleifenbaum, Frank

    2010-02-01

    Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.

  18. 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.

  19. 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.

  20. 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.

  1. Fluorescent magnetic nanoprobes: design and application for cell imaging.

    PubMed

    Zhang, Guo; Feng, Jianghua; Lu, Lehui; Zhang, Baohua; Cao, Linyuan

    2010-11-01

    Multifunctional nanoprobes combining magnetic nanoparticles with organic dyes have attracted tremendous interest due to their promising applications in biomedical field. Here we demonstrate a facile and general strategy for the fabrication of robust fluorescent magnetic nanoprobes with high payloads of dye molecules and their use as multimodal nanoprobes for cell imaging. These nanoprobes not only effectively keep photochemical stability of dyes, but also provide a platform for grafting other functional or targeted moieties into silica surface via primary amines. Moreover, the nanoprobes are uniformly spherical morphology and can be dispersed well in aqueous solution, which are very desirable for biomedical applications. Importantly, this method can be extended to synthesize other bifunctional nanoprobes by using the dyes with isothiocyanate group.

  2. Elemental mapping with an X-ray fluorescence imaging microscope

    NASA Astrophysics Data System (ADS)

    Yamamoto, Kimitake; Watanabe, Norio; Takeuchi, Akihisa; Takano, Hidekazu; Aota, Tatuya; Kumegawa, Masaru; Ohigashi, Takuji; Tanoue, Ryuichi; Yokosuka, Hiroki; Aoki, Sadao

    2000-05-01

    An X-ray fluorescence (XRF) imaging microscope with a Wolter-type grazing-incidence mirror as an objective was constructed at the beamline 39XU of SPring-8 (8 GeV, 70 mA) at Japan Synchrotron Radiation Institute. The monochromatic undulator X-rays in the energy range of 6-10 keV were used to produce XRF of a specimen. The microscope system was set normal to the incident beam to reduce elastic scattering from a specimen and to improve signal/background ratio. The two-dimensional elemental mappings of a test specimen (Cu, Ni, Fe wires) and inclusions (Fe, Co, Ni) in a synthesized diamond could be obtained by utilizing the absorption edges of the corresponding elements.

  3. Ultra-sensitive fluorescent proteins for imaging neuronal activity

    PubMed Central

    Chen, Tsai-Wen; Wardill, Trevor J.; Sun, Yi; Pulver, Stefan R.; Renninger, Sabine L.; Baohan, Amy; Schreiter, Eric R.; Kerr, Rex A.; Orger, Michael B.; Jayaraman, Vivek; Looger, Loren L.; Svoboda, Karel; Kim, Douglas S.

    2013-01-01

    Summary Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultra-sensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies, and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5 - 40 micrometers long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales. PMID:23868258

  4. Multimodal optical coherence tomography and fluorescence lifetime imaging with interleaved excitation sources for simultaneous endogenous and exogenous fluorescence

    PubMed Central

    Shrestha, Sebina; Serafino, Michael J.; Rico-Jimenez, Jesus; Park, Jesung; Chen, Xi; Zhaorigetu, Siqin; Walton, Brian L.; Jo, Javier A.; Applegate, Brian E.

    2016-01-01

    Multimodal imaging probes a variety of tissue properties in a single image acquisition by merging complimentary imaging technologies. Exploiting synergies amongst the data, algorithms can be developed that lead to better tissue characterization than could be accomplished by the constituent imaging modalities taken alone. The combination of optical coherence tomography (OCT) with fluorescence lifetime imaging microscopy (FLIM) provides access to detailed tissue morphology and local biochemistry. The optical system described here merges 1310 nm swept-source OCT with time-domain FLIM having excitation at 355 and 532 nm. The pulses from 355 and 532 nm lasers have been interleaved to enable simultaneous acquisition of endogenous and exogenous fluorescence signals, respectively. The multimodal imaging system was validated using tissue phantoms. Nonspecific tagging with Alexa Flour 532 in a Watanbe rabbit aorta and active tagging of the LOX-1 receptor in human coronary artery, demonstrate the capacity of the system for simultaneous acquisition of OCT, endogenous FLIM, and exogenous FLIM in tissues.

  5. 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

  6. Segmentation of fluorescence microscopy images for quantitative analysis of cell nuclear architecture.

    PubMed

    Russell, Richard A; Adams, Niall M; Stephens, David A; Batty, Elizabeth; Jensen, Kirsten; Freemont, Paul S

    2009-04-22

    Considerable advances in microscopy, biophysics, and cell biology have provided a wealth of imaging data describing the functional organization of the cell nucleus. Until recently, cell nuclear architecture has largely been assessed by subjective visual inspection of fluorescently labeled components imaged by the optical microscope. This approach is inadequate to fully quantify spatial associations, especially when the patterns are indistinct, irregular, or highly punctate. Accurate image processing techniques as well as statistical and computational tools are thus necessary to interpret this data if meaningful spatial-function relationships are to be established. Here, we have developed a thresholding algorithm, stable count thresholding (SCT), to segment nuclear compartments in confocal laser scanning microscopy image stacks to facilitate objective and quantitative analysis of the three-dimensional organization of these objects using formal statistical methods. We validate the efficacy and performance of the SCT algorithm using real images of immunofluorescently stained nuclear compartments and fluorescent beads as well as simulated images. In all three cases, the SCT algorithm delivers a segmentation that is far better than standard thresholding methods, and more importantly, is comparable to manual thresholding results. By applying the SCT algorithm and statistical analysis, we quantify the spatial configuration of promyelocytic leukemia nuclear bodies with respect to irregular-shaped SC35 domains. We show that the compartments are closer than expected under a null model for their spatial point distribution, and furthermore that their spatial association varies according to cell state. The methods reported are general and can readily be applied to quantify the spatial interactions of other nuclear compartments.

  7. Advances in Fluorescence Sensing Systems for the Remote Assessment of Nitrogen Supply in Field Corn

    NASA Technical Reports Server (NTRS)

    Corp, L. A.; Chappelle, E. W.; McMurtrey, J. E.; Daughtry, C. S. T.; Kim, M. S.

    2000-01-01

    The studies described herein were conducted to better define changes in fluorescence properties of leaves from field grown corn (Zea mays L.) as they relate to varying levels of nitrogen (N) fertilization. This research was directed toward: 1) providing a remote non-destructive sensing technique to aid in the determination of optimal rates of N fertilization in corn crops and, 2) defining parameters for further development of fluorescence instrumentation to be operated remotely at field canopy levels. Fluorescence imaging bands centered in the blue (450 nm), green (525 nm), red (680 nm), and far-red (740 nm) and ratios of these bands were compared with the following plant parameters: rates of photosynthesis, N:C ratio, pigment concentrations, and grain yields. Both the fluorescence and physiological measures exhibited similar curvilinear responses to N fertilization level while significant linear correlations were obtained among fluorescence bands and band ratios to certain physiological measures of plant productivity. The red / blue, red / green, far-red / blue, far-red /green fluorescence ratios are well suited for remote observation and provided high correlations to grain yield, LAI, N:C, and chlorophyll contents. The results from this investigation indicate that fluorescence technology could aid in the determination of N fertilization requirements for corn. This discussion will also address design concepts and preliminary field trials of a mobile field-based Laser Induced Fluorescence Imaging System (LIFIS) capable of simultaneously acquiring images of four fluorescence emission bands from areas of plant canopies equaling 1 sq m and greater without interference of ambient solar radiation.

  8. Combining optical coherence tomography with fluorescence molecular imaging: towards simultaneous morphology and molecular imaging

    NASA Astrophysics Data System (ADS)

    Yuan, Shuai; Lai, Michael; Roney, Celeste A.; Jiang, James; Li, Qian; Ma, Hongzhou; Cable, Alex; Summers, Ronald M.; Chen, Yu

    2009-02-01

    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, a new 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 x 2.4 mm field of view. The transverse resolutions of OCT and FMI of the system are both 10 μm. Capillary tubes filled with Cy 5.5 fluorescent dye in different concentrations (750nM to 24μM) under a scattering medium (1% - 2% intralipid) 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. In conclusion, a co-registering OCT and FMI imaging system has been demonstrated. The system enables simultaneous visualization of tissue morphology and molecular information at high resolutions over a 2-3 mm field-of-view.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25248645

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

    PubMed

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

    2014-11-01

    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.

  11. 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

  12. Laser-induced fluorescence imaging of plants using a liquid crystal tunable filter and charge coupled device imaging camera

    NASA Astrophysics Data System (ADS)

    Saito, Yasunori; Matsubara, Tomohiro; Koga, Tomoya; Kobayashi, Fumitoshi; Kawahara, Takuya D.; Nomura, Akio

    2005-10-01

    We developed a laser-induced fluorescence imaging system for plant monitoring use, with which it was possible to make an image at any wavelength between 430 and 750nm. The excitation source for the fluorescence was a cw ultraviolet laser diode with 398nm, and the detector was an image-intensified charge coupled device. A liquid crystal tunable filter was used as the fluorescence wavelength selection device. All of the system performance including the wavelength tuning was electrically controlled, so that it could be operated with no mechanical vibration noise. The fluorescence images of a coffee tree leaf obtained at 440, 530, 685, and 740nm clearly showed a distribution pattern of the fluorescence intensity over the leaf. The pattern reflected the different physiological statuses of the plant. Advantages of the imaging system were experimentally discussed on a point of detection of inhomogeneous physiological activities over a plant leaf.

  13. Imaging of Surfaces by Concurrent Surface Plasmon Resonance and Surface Plasmon Resonance-Enhanced Fluorescence

    PubMed Central

    Thariani, Rahber; Yager, Paul

    2010-01-01

    Surface plasmon resonance imaging and surface plasmon induced fluorescent are sensitive tools for surface analysis. However, existing instruments in this area have provided limited capability for concurrent detection, and may be large and expensive. We demonstrate a highly cost-effective system capable of concurrent surface plasmon resonance microscopy (SPRM) and surface plasmon resonance-enhanced fluorescence (SPRF) imaging, allowing for simultaneous monitoring of reflectivity and fluorescence from discrete spatial regions. The instrument allows for high performance imaging and quantitative measurements with surface plasmon resonance, and surface plasmon induced fluorescence, with inexpensive off-the-shelf components. PMID:20360841

  14. Digital aberration correction of fluorescent images with coherent holographic image reconstruction by phase transfer (CHIRPT)

    NASA Astrophysics Data System (ADS)

    Field, Jeffrey J.; Bartels, Randy A.

    2016-03-01

    Coherent holographic image reconstruction by phase transfer (CHIRPT) is an imaging method that permits digital holographic propagation of fluorescent light. The image formation process in CHIRPT is based on illuminating the specimen with a precisely controlled spatio-temporally varying intensity pattern. This pattern is formed by focusing a spatially coherent illumination beam to a line focus on a spinning modulation mask, and image relaying the mask plane to the focal plane of an objective lens. Deviations from the designed spatio-temporal illumination pattern due to imperfect mounting of the circular modulation mask onto the rotation motor induce aberrations in the recovered image. Here we show that these aberrations can be measured and removed non-iteratively by measuring the disk aberration phase externally. We also demonstrate measurement and correction of systematic optical aberrations in the CHIRPT microscope.

  15. 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.

  16. Noninvasive multimodal evaluation of bioengineered cartilage constructs combining time-resolved fluorescence and ultrasound imaging.

    PubMed

    Fite, Brett Z; Decaris, Martin; Sun, Yinghua; Sun, Yang; Lam, Adrian; Ho, Clark K L; Leach, J Kent; Marcu, Laura

    2011-04-01

    A multimodal diagnostic system that integrates time-resolved fluorescence spectroscopy, fluorescence lifetime imaging microscopy, and ultrasound backscatter microscopy is evaluated here as a potential tool for assessing changes in engineered tissue composition and microstructure nondestructively and noninvasively. The development of techniques capable of monitoring the quality of engineered tissue, determined by extracellular matrix (ECM) content, before implantation would alleviate the need for destructive assays over multiple time points and advance the widespread development and clinical application of engineered tissues. Using a prototype system combining time-resolved fluorescence spectroscopy, FLIM, and UBM, we measured changes in ECM content occurring during chondrogenic differentiation of equine adipose stem cells on 3D biodegradable matrices. The optical and ultrasound results were validated against those acquired via conventional techniques, including collagen II immunohistochemistry, picrosirius red staining, and measurement of construct stiffness. Current results confirm the ability of this multimodal approach to follow the progression of tissue maturation along the chondrogenic lineage by monitoring ECM production (namely, collagen type II) and by detecting resulting changes in mechanical properties of tissue constructs. Although this study was directed toward monitoring chondrogenic tissue maturation, these data demonstrate the feasibility of this approach for multiple applications toward engineering other tissues, including bone and vascular grafts. PMID:21303258

  17. A multi-threaded mosaicking algorithm for fast image composition of fluorescence bladder images

    NASA Astrophysics Data System (ADS)

    Behrens, Alexander; Bommes, Michael; Stehle, Thomas; Gross, Sebastian; Leonhardt, Steffen; Aach, Til

    2010-02-01

    The treatment of urinary bladder cancer is usually carried out using fluorescence endoscopy. A narrow-band bluish illumination activates a tumor marker resulting in a red fluorescence. Because of low illumination power the distance between endoscope and bladder wall is kept low during the whole bladder scan, which is carried out before treatment. Thus, only a small field of view (FOV) of the operation field is provided, which impedes navigation and relocating of multi-focal tumors. Although off-line calculated panorama images can assist surgery planning, the immediate display of successively growing overview images composed from single video frames in real-time during the bladder scan, is well suited to ease navigation and reduce the risk of missing tumors. Therefore we developed an image mosaicking algorithm for fluorescence endoscopy. Due to fast computation requirements a flexible multi-threaded software architecture based on our RealTimeFrame platform is developed. Different algorithm tasks, like image feature extraction, matching and stitching are separated and applied by independent processing threads. Thus, different implementation of single tasks can be easily evaluated. In an optimization step we evaluate the trade-off between feature repeatability and total processing time, consider the thread synchronization, and achieve a constant workload of each thread. Thus, a fast computation of panoramic images is performed on a standard hardware platform, preserving full input image resolution (780x576) at the same time. Displayed on a second clinical monitor, the extended FOV of the image composition promises high potential for surgery assistance.

  18. Hyperspectral image projector for advanced sensor characterization

    NASA Astrophysics Data System (ADS)

    Brown, S. W.; Rice, J. P.; Neira, J. E.; Bousquet, R.; Johnson, B. C.

    2006-08-01

    In this work, we describe radiometric platforms able to produce realistic spectral distributions and spatial scenes for the development of application-specific metrics to quantify the performance of sensors and systems. Using these platforms, sensor and system performance may be quantified in terms of the accuracy of measurements of standardized sets of complex source distributions. The same platforms can also serve as a basis for algorithm testing and instrument comparison. The platforms consist of spectrally tunable light sources (STS's) coupled with spatially programmable projection systems. The resultant hyperspectral image projectors (HIP) can generate complex spectral distributions with high spectral fidelity; that is, scenes with realistic spectral content. Using the same fundamental technology, platforms can be developed for the ultraviolet, visible, and infrared regions. These radiometric platforms will facilitate advanced sensor characterization testing, enabling a pre-flight validation of the pre-flight calibration.

  19. Fluorescence Behavioral Imaging (FBI) Tracks Identity in Heterogeneous Groups of Drosophila

    PubMed Central

    Ramdya, Pavan; Schaffter, Thomas; Floreano, Dario; Benton, Richard

    2012-01-01

    Distinguishing subpopulations in group behavioral experiments can reveal the impact of differences in genetic, pharmacological and life-histories on social interactions and decision-making. Here we describe Fluorescence Behavioral Imaging (FBI), a toolkit that uses transgenic fluorescence to discriminate subpopulations, imaging hardware that simultaneously records behavior and fluorescence expression, and open-source software for automated, high-accuracy determination of genetic identity. Using FBI, we measure courtship partner choice in genetically mixed groups of Drosophila. PMID:23144871

  20. Fluorescence behavioral imaging (FBI) tracks identity in heterogeneous groups of Drosophila.

    PubMed

    Ramdya, Pavan; Schaffter, Thomas; Floreano, Dario; Benton, Richard

    2012-01-01

    Distinguishing subpopulations in group behavioral experiments can reveal the impact of differences in genetic, pharmacological and life-histories on social interactions and decision-making. Here we describe Fluorescence Behavioral Imaging (FBI), a toolkit that uses transgenic fluorescence to discriminate subpopulations, imaging hardware that simultaneously records behavior and fluorescence expression, and open-source software for automated, high-accuracy determination of genetic identity. Using FBI, we measure courtship partner choice in genetically mixed groups of Drosophila.

  1. Complementary Metal-Oxide-Semiconductor Image Sensor with Microchamber Array for Fluorescent Bead Counting

    NASA Astrophysics Data System (ADS)

    Sasagawa, Kiyotaka; Ando, Keisuke; Kobayashi, Takuma; Noda, Toshihiko; Tokuda, Takashi; Kim, Soo Hyeon; Iino, Ryota; Noji, Hiroyuki; Ohta, Jun

    2012-02-01

    We fabricated a complementary metal-oxide-semiconductor image sensor with a femtoliter microchamber array. The microchamber array plate is used for trapping microbeads and limiting the incident angle of light detected by the sensor. The sensor has an interference filter for fluorescent microbeads imaging. We detected fluorescent and nonfluorescent microbead with this sensor and showed its capability for counting the number of fluorescent chambers.

  2. Noninvasive two-photon fluorescence microscopy imaging of mouse retina and RPE through the pupil of the eye

    PubMed Central

    Palczewska, Grazyna; Dong, Zhiqian; Golczak, Marcin; Hunter, Jennifer J.; Williams, David R.; Alexander, Nathan S.; Palczewski, Krzysztof

    2014-01-01

    Two-photon excitation microscopy (TPM) can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in sub-cellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all–trans–retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here we report repetitive, dynamic imaging of these compounds in live mice, through the pupil of the eye. Leveraging advanced adaptive optics we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium (RPE) by their characteristic localization, spectral properties, and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions. PMID:24952647

  3. Attenuation-corrected fluorescence extraction for image-guided surgery in spatial frequency domain

    PubMed Central

    Yang, Bin; Sharma, Manu

    2013-01-01

    Abstract. A new approach to retrieve the attenuation-corrected fluorescence using spatial frequency-domain imaging is demonstrated. Both in vitro and ex vivo experiments showed the technique can compensate for the fluorescence attenuation from tissue absorption and scattering. This approach has potential in molecular image-guided surgery. PMID:23955392

  4. Attenuation-corrected fluorescence extraction for image-guided surgery in spatial frequency domain.

    PubMed

    Yang, Bin; Sharma, Manu; Tunnell, James W

    2013-08-01

    A new approach to retrieve the attenuation-corrected fluorescence using spatial frequency-domain imaging is demonstrated. Both in vitro and ex vivo experiments showed the technique can compensate for the fluorescence attenuation from tissue absorption and scattering. This approach has potential in molecular image-guided surgery. PMID:23955392

  5. 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.

  6. Probing peroxisome dynamics and biogenesis by fluorescence imaging.

    PubMed

    Jauregui, Miluska; Kim, Peter K

    2014-03-03

    Peroxisomes are the most recently discovered classical organelles, and only lately have their diverse functions been truly recognized. Peroxisomes are highly dynamic structures, changing both morphologically and in number in response to both extracellular and intracellular signals. This metabolic organelle came to prominence due to the many genetic disorders caused by defects in its biogenesis or enzymatic functions. There is now growing evidence that suggests peroxisomes are involved in lipid biosynthesis, innate immunity, redox homeostasis, and metabolite scavenging, among other functions. Therefore, it is important to have available suitable methods and techniques to visualize and quantify peroxisomes in response to various cellular signals. This unit includes a number of protocols that will enable researchers to image, qualify, and quantify peroxisome numbers and morphology-with both steady-state and time-lapse imaging using mammalian cells. The use of photoactivatable fluorescent proteins to detect and measure peroxisome biogenesis is also described. Altogether, the protocols described here will facilitate understanding of the dynamic changes that peroxisomes undergo in response to various cellular signals.

  7. Spectrally-Resolved Response Properties of the Three Most Advanced FRET Based Fluorescent Protein Voltage Probes

    PubMed Central

    Dimitrov, Dimitar; Iwamoto, Yuka; Akemann, Walther; Chudakov, Dmitriy M.; Knöpfel, Thomas

    2009-01-01

    Genetically-encoded optical probes for membrane potential hold the promise of monitoring electrical signaling of electrically active cells such as specific neuronal populations in intact brain tissue. The most advanced class of these probes was generated by molecular fusion of the voltage sensing domain (VSD) of Ci-VSP with a fluorescent protein (FP) pair. We quantitatively compared the three most advanced versions of these probes (two previously reported and one new variant), each involving a spectrally distinct tandem of FPs. Despite these different FP tandems and dissimilarities within the amino acid sequence linking the VSD to the FPs, the amplitude and kinetics of voltage dependent fluorescence changes were surprisingly similar. However, each of these fluorescent probes has specific merits when considering different potential applications. PMID:19234605

  8. Wavelength dispersive X-ray fluorescence imaging using a high-sensitivity imaging sensor

    NASA Astrophysics Data System (ADS)

    Ohmori, Takashi; Kato, Shuichi; Doi, Makoto; Shoji, Takashi; Tsuji, Kouichi

    2013-05-01

    A new wavelength-dispersive X-ray fluorescence (WD-XRF) imaging spectrometer equipped with a high-sensitivity imaging sensor was developed in our laboratory. In this instrument, a straight polycapillary optic was applied instead of a Soller slit as well as a 2D imaging X-ray detector instead of X-ray counters, which are used in conventional WD-XRF spectrometers. Therefore, images of elemental distribution were available after a short exposure time. Ni Kα images and Cu Kα images were clearly obtained at corresponding diffraction angles for a short exposure time of 10 s. By optimizing the spectrometer, the time required for imaging is reduced, leading to XRF image movies. It is difficult to distinguish two peaks (Ti Kα (4.508 keV) and Ba Lα (4.465 keV)) due to the poor energy resolution of EDXRS. However, Ti and Ba images could be successfully observed by the WD-XRF imaging spectrometer. The energy resolution of the developed spectrometer was 25 eV at the Ti Kα peak.

  9. Eco-friendly carbon-nanodot-based fluorescent paints for advanced photocatalytic systems.

    PubMed

    Park, So Young; Lee, Hyun Uk; Lee, Young-Chul; Choi, Saehae; Cho, Dae Hyun; Kim, Hee Sik; Bang, Sunghee; Seo, Soonjoo; Lee, Soon Chang; Won, Jonghan; Son, Byung-Chul; Yang, Mino; Lee, Jouhahn

    2015-01-01

    Fluorescent carbon nanomaterials, especially zero-dimensional (0D) carbon nanodots (CDs), are widely used in broad biological and optoelectronic applications. CDs have unique characteristics such as strong fluorescence, biocompatibility, sun-light response, and capability of mass-production. Beyond the previous green CD obtained from harmful natural substances, we report a new type of fluid-based fluorescent CD paints (C-paints) derived from polyethylene glycol (PEG; via simple ultrasound irradiation at room temperatures) and produced in quantum yields of up to ~14%. Additionally, C-paints possess a strong, UV- and visible-light-responsive photoluminescent (PL) property. Most especially, C-paints, by incorporation into a photocatalytic system, show additional roles in the emission of fluorescent light for activation of TiO2 nanoparticles (NPs) and the resultant detoxification of most organic dyes, thus further enabling embarkation in advanced water purification.

  10. Eco-friendly carbon-nanodot-based fluorescent paints for advanced photocatalytic systems.

    PubMed

    Park, So Young; Lee, Hyun Uk; Lee, Young-Chul; Choi, Saehae; Cho, Dae Hyun; Kim, Hee Sik; Bang, Sunghee; Seo, Soonjoo; Lee, Soon Chang; Won, Jonghan; Son, Byung-Chul; Yang, Mino; Lee, Jouhahn

    2015-01-01

    Fluorescent carbon nanomaterials, especially zero-dimensional (0D) carbon nanodots (CDs), are widely used in broad biological and optoelectronic applications. CDs have unique characteristics such as strong fluorescence, biocompatibility, sun-light response, and capability of mass-production. Beyond the previous green CD obtained from harmful natural substances, we report a new type of fluid-based fluorescent CD paints (C-paints) derived from polyethylene glycol (PEG; via simple ultrasound irradiation at room temperatures) and produced in quantum yields of up to ~14%. Additionally, C-paints possess a strong, UV- and visible-light-responsive photoluminescent (PL) property. Most especially, C-paints, by incorporation into a photocatalytic system, show additional roles in the emission of fluorescent light for activation of TiO2 nanoparticles (NPs) and the resultant detoxification of most organic dyes, thus further enabling embarkation in advanced water purification. PMID:26201431

  11. Eco-friendly carbon-nanodot-based fluorescent paints for advanced photocatalytic systems

    NASA Astrophysics Data System (ADS)

    Young Park, So; Uk Lee, Hyun; Lee, Young-Chul; Choi, Saehae; Hyun Cho, Dae; Sik Kim, Hee; Bang, Sunghee; Seo, Soonjoo; Chang Lee, Soon; Won, Jonghan; Son, Byung-Chul; Yang, Mino; Lee, Jouhahn

    2015-07-01

    Fluorescent carbon nanomaterials, especially zero-dimensional (0D) carbon nanodots (CDs), are widely used in broad biological and optoelectronic applications. CDs have unique characteristics such as strong fluorescence, biocompatibility, sun-light response, and capability of mass-production. Beyond the previous green CD obtained from harmful natural substances, we report a new type of fluid-based fluorescent CD paints (C-paints) derived from polyethylene glycol (PEG; via simple ultrasound irradiation at room temperatures) and produced in quantum yields of up to ~14%. Additionally, C-paints possess a strong, UV- and visible-light-responsive photoluminescent (PL) property. Most especially, C-paints, by incorporation into a photocatalytic system, show additional roles in the emission of fluorescent light for activation of TiO2 nanoparticles (NPs) and the resultant detoxification of most organic dyes, thus further enabling embarkation in advanced water purification.

  12. Precision Automation of Cell Type Classification and Sub-Cellular Fluorescence Quantification from Laser Scanning Confocal Images.

    PubMed

    Hall, Hardy C; Fakhrzadeh, Azadeh; Luengo Hendriks, Cris L; Fischer, Urs

    2016-01-01

    While novel whole-plant phenotyping technologies have been successfully implemented into functional genomics and breeding programs, the potential of automated phenotyping with cellular resolution is largely unexploited. Laser scanning confocal microscopy has the potential to close this gap by providing spatially highly resolved images containing anatomic as well as chemical information on a subcellular basis. However, in the absence of automated methods, the assessment of the spatial patterns and abundance of fluorescent markers with subcellular resolution is still largely qualitative and time-consuming. Recent advances in image acquisition and analysis, coupled with improvements in microprocessor performance, have brought such automated methods within reach, so that information from thousands of cells per image for hundreds of images may be derived in an experimentally convenient time-frame. Here, we present a MATLAB-based analytical pipeline to (1) segment radial plant organs into individual cells, (2) classify cells into cell type categories based upon Random Forest classification, (3) divide each cell into sub-regions, and (4) quantify fluorescence intensity to a subcellular degree of precision for a separate fluorescence channel. In this research advance, we demonstrate the precision of this analytical process for the relatively complex tissues of Arabidopsis hypocotyls at various stages of development. High speed and robustness make our approach suitable for phenotyping of large collections of stem-like material and other tissue types. PMID:26904081

  13. Understanding Fluorescence Measurements through a Guided-Inquiry and Discovery Experiment in Advanced Analytical Laboratory

    ERIC Educational Resources Information Center

    Wilczek-Vera, Grazyna; Salin, Eric Dunbar

    2011-01-01

    An experiment on fluorescence spectroscopy suitable for an advanced analytical laboratory is presented. Its conceptual development used a combination of the expository and discovery styles. The "learn-as-you-go" and direct "hands-on" methodology applied ensures an active role for a student in the process of visualization and discovery of concepts.…

  14. 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

  15. Imaging HIV-1 Tat Trafficking and Interactions by Engineered Green-Fluorescent-Protein Tagging

    NASA Astrophysics Data System (ADS)

    Beltram, Fabio

    2002-03-01

    The direct monitoring of protein function in live cells under physiologically relevant conditions is one of the most powerful and innovative methodologies for proteomics. Efficient florescent probes fully compatible with human-cell expression are the fundamental tools for these studies and their optimization opens the way to resolution at the single-protein level. Biological events involving protein pairs are also directly accessible thanks to tuning of protein-tag spectral properties and production of complementary pairs. Such pairs are characterized by overlapping absorption (for the acceptor tag) and emission (for the donor tag) spectra. By tagging the proteins of interest with acceptor and donor molecules, protein interaction can be directly visualized by FRET, fluorescent resonant energy transfer. In this talk we shall present the design by molecular dynamics calculations and the application of optimized green fluorescent proteins to the study of the human immunodeficiency virus HIV-1 proteomics. In particular trafficking and cellular interactions of HIV-1 transactivator protein Tat in live human cells will be presented. Tat localization and complex internalization pathways of exogenous molecules will be presented thanks to the peculiar optical properties of mutated GFPs. Cellular protein partners and subcellular interaction sites will be identified and directly visualized. The relevance of such results and of advanced spectroscopic and imaging techniques for a new level of understanding of biological processes and its significance for advancement in molecular biology will be underlined. A. Marcello et al., J. Biol. Chem. 276, 39220 (2001). R. Cinelli et al., Appl. Phys. Lett. 79, 3353 (2001).

  16. Depth-resolved imaging of colon tumor using optical coherence tomography and fluorescence laminar optical tomography (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Tang, Qinggong; Frank, Aaron; Wang, Jianting; Chen, Chao-wei; Jin, Lily; Lin, Jon; Chan, Joanne M.; Chen, Yu

    2016-03-01

    Early detection of neoplastic changes remains a critical challenge in clinical cancer diagnosis and treatment. Many cancers arise from epithelial layers such as those of the gastrointestinal (GI) tract. Current standard endoscopic technology is unable to detect those subsurface lesions. Since cancer development is associated with both morphological and molecular alterations, imaging technologies that can quantitative image tissue's morphological and molecular biomarkers and assess the depth extent of a lesion in real time, without the need for tissue excision, would be a major advance in GI cancer diagnostics and therapy. In this research, we investigated the feasibility of multi-modal optical imaging including high-resolution optical coherence tomography (OCT) and depth-resolved high-sensitivity fluorescence laminar optical tomography (FLOT) for structural and molecular imaging. APC (adenomatous polyposis coli) mice model were imaged using OCT and FLOT and the correlated histopathological diagnosis was obtained. Quantitative structural (the scattering coefficient) and molecular imaging parameters (fluorescence intensity) from OCT and FLOT images were developed for multi-parametric analysis. This multi-modal imaging method has demonstrated the feasibility for more accurate diagnosis with 87.4% (87.3%) for sensitivity (specificity) which gives the most optimal diagnosis (the largest area under receiver operating characteristic (ROC) curve). This project results in a new non-invasive multi-modal imaging platform for improved GI cancer detection, which is expected to have a major impact on detection, diagnosis, and characterization of GI cancers, as well as a wide range of epithelial cancers.

  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. 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.

  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. 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.

  2. Laser induced fluorescence imaging of thermal damage in polymer matrix composites

    SciTech Connect

    Fisher, W.G.; Meyer, K.E.; Wachter, E.A.; Perl, D.R.; Kulowitch, P.J.

    1997-06-01

    A simple, fluorescence based imaging system has been developed that is capable of identifying regions of thermal damage in polymer matrix composites (PMCs). These materials are playing an increasingly important role in the production of high performance vehicles and aircraft, where their low weight and high mechanical strength, combined with advancements in manufacturing technology, ensure increased use for a variety of applications. Of particular concern in the aerospace industry is the tendency of some PMC materials to become irreversibly damaged when exposed to elevated temperatures. Traditional nondestructive testing (NDT) techniques are capable of detecting physical anomalies such as cracks and delaminations but cannot detect initial heat damage, which occurs on a molecular scale. Spectroscopic techniques such as laser induced fluorescence provide an attractive means for detecting this type of damage and are amenable to imaging large, irregularly shaped surfaces. In this report the authors describe instrumentation capable of rapidly detecting thermal damage in graphite epoxy components and suggest improvements which will enable this technology to make quantitative judgments concerning the mechanical strength properties of heat damaged specimens.

  3. Simultaneous fluorescence and phase imaging with extensions toward sub-diffraction resolution via structured-illumination (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Chowdhury, Shwetadwip; Izatt, Joseph A.

    2016-03-01

    In the biological sciences, there is much emphasis on elucidating the functions of various biological components and processes. To do so, advances in general microscopy have yielded various imaging modalities to probe such processes under specific visualization and contrast requirements. Examples of modalities that are popularly integrated into conventional biological studies include fluorescent, dark-field, phase-contrast, and polarization-sensitive microscopies, with each modality offering unique insights into the biological function of the sample. Often times, however, a comprehensive understanding of biological phenomena requires the integration of the unique and separate visualizations of various modalities. Unfortunately, conventional microscopes typically support only one modality and rarely allow multiple modalities to be used in conjunction. Though high-end microscopes may support multimodal visualization, they often require either mechanical (and often manual) toggling, which obstruct real-time multimodal imaging, or simultaneous detection via multiple cameras, which dramatically increases the microscope's cost. Here, we present a one-shot technique that allows multiple imaging channels, of potentially different modalities, to be simultaneously detected by a single camera. We experimentally demonstrate this method on transparent cells that have been tagged for F-actin and nuclear fluorescence. Our multimodal system consists of 2-channel fluorescence and 1-channel quantitative-phase (QP) imaging, and clearly demonstrates ability for simultaneous fluorescent and QP visualization. Though we experimentally verify our framework using dual fluorescent/QP imaging, we emphasize that our framework for single-shot, simultaneous single-camera detection is applicable to an arbitrary number of widefield imaging modalities so long as they fulfill criteria for Fourier spectra separation, SNR, and detector dynamic range

  4. Lifetime estimation of moving subcellular objects in frequency-domain fluorescence lifetime imaging microscopy.

    PubMed

    Roudot, Philippe; Kervrann, Charles; Blouin, Cedric M; Waharte, Francois

    2015-10-01

    Fluorescence lifetime is usually defined as the average nanosecond-scale delay between excitation and emission of fluorescence. It has been established that lifetime measurements yield numerous indications on cellular processes such as interprotein and intraprotein mechanisms through fluorescent tagging and Förster resonance energy transfer. In this area, frequency-domain fluorescence lifetime imaging microscopy is particularly appropriate to probe a sample noninvasively and quantify these interactions in living cells. The aim is then to measure the fluorescence lifetime in the sample at each location in space from fluorescence variations observed in a temporal sequence of images obtained by phase modulation of the detection signal. This leads to a sensitivity of lifetime determination to other sources of fluorescence variations such as intracellular motion. In this paper, we propose a robust statistical method for lifetime estimation for both background and small moving structures with a focus on intracellular vesicle trafficking. PMID:26479936

  5. 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.

  6. 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.

  7. 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

  8. 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.

  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. 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

  11. 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

  12. 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

  13. Improved sensitivity to fluorescence for cancer detection in wide-field image-guided neurosurgery

    PubMed Central

    Jermyn, Michael; Gosselin, Yoann; Valdes, Pablo A.; Sibai, Mira; Kolste, Kolbein; Mercier, Jeanne; Angulo, Leticia; Roberts, David W.; Paulsen, Keith D.; Petrecca, Kevin; Daigle, Olivier; Wilson, Brian C.; Leblond, Frederic

    2015-01-01

    In glioma surgery, Protoporphyrin IX (PpIX) fluorescence may identify residual tumor that could be resected while minimizing damage to normal brain. We demonstrate that improved sensitivity for wide-field spectroscopic fluorescence imaging is achieved with minimal disruption to the neurosurgical workflow using an electron-multiplying charge-coupled device (EMCCD) relative to a state-of-the-art CMOS system. In phantom experiments the EMCCD system can detect at least two orders-of-magnitude lower PpIX. Ex vivo tissue imaging on a rat glioma model demonstrates improved fluorescence contrast compared with neurosurgical fluorescence microscope technology, and the fluorescence detection is confirmed with measurements from a clinically-validated spectroscopic probe. Greater PpIX sensitivity in wide-field fluorescence imaging may improve the residual tumor detection during surgery with consequent impact on survival. PMID:26713218

  14. Fluorescence thermometry for advanced high-temperature materials

    SciTech Connect

    Cates, M.R.; Beshears, D.L.; Allison, S.W.

    1996-05-01

    Advanced high-temperature materials, such as ceramics, metals, and composites, are of critical importance to the development of new and improved technologies worldwide. For aircraft, automobiles, or other combustion-engine powered systems, major efficiency improvements depend on the ability to operate at temperatures closer to the adiabatic limit of the chemical processes involved. Materials able to function at higher temperatures must therefore be introduced into improved designs. Jet turbine engines, for example, already require air cooled rotors and stators in order that the nickel alloys used will not deteriorate and fail from overheating. In the case of ceramics, optimum temperature usage will often cause the refractory surfaces to glow red hot and the material itself to become partially translucent. For composites, especially where structural integrity, vibration resistance, and strength are concerned, the temperature behavior of dissimilar components must be well known and well understood before appropriate designs can be effected. As the need for higher temperature materials becomes increasingly more important, so does the requirement to properly measure the temperatures involved. Phosphor thermometry offers measurement solutions at very high temperatures that often cannot be achieved by more conventional methods. In this paper we discuss the phosphor technique and several examples of its application to high-temperature measurement.

  15. 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

  16. 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

  17. 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

  18. Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging.

    PubMed

    Sreejith, Sivaramapanicker; Joseph, James; Lin, Manjing; Menon, Nishanth Venugopal; Borah, Parijat; Ng, Hao Jun; Loong, Yun Xian; Kang, Yuejun; Yu, Sidney Wing-Kwong; Zhao, Yanli

    2015-06-23

    Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1(micelle)) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1(micelle) exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1(micelle) in the NIR window of 800-900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1(micelle), as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1(micelle) proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging.

  19. Non-invasive fluorescence imaging under ambient light conditions using a modulated ICCD and laser diode

    PubMed Central

    Zhu, Banghe; Rasmussen, John C.; Sevick-Muraca, Eva M.

    2014-01-01

    One limitation of fluorescence molecular imaging that can limit clinical implementation and hamper small animal imaging is the inability to eliminate ambient light. Herein, we demonstrate the ability to conduct rapid non-invasive, far-red and near-infrared fluorescence imaging in living animals and a phantom under ambient light conditions using a modulated image intensified CCD (ICCD) and a laser diode operated in homodyne detection. By mapping AC amplitude from three planar images at varying phase delays, we show improvement in target-to-background ratios (TBR) and reasonable signal-to-noise ratios (SNR) over continuous wave measurements. The rapid approach can be used to accurately collect fluorescence in situations where ambient light cannot be spectrally conditioned or controlled, such as in the case of fluorescent molecular image-guided surgery. PMID:24575349

  20. 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.

  1. 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)

  2. Spirally-patterned pinhole arrays for long-term fluorescence cell imaging.

    PubMed

    Koo, Bon Ung; Kang, YooNa; Moon, SangJun; Lee, Won Gu

    2015-11-01

    Fluorescence cell imaging using a fluorescence microscope is an extensively used technique to examine the cell nucleus, internal structures, and other cellular molecules with fluorescence response time and intensity. However, it is difficult to perform high resolution cell imaging for a long period of time with this technique due to necrosis and apoptosis depending on the type and subcellular location of the damage caused by phototoxicity. A large number of studies have been performed to resolve this problem, but researchers have struggled to meet the challenge between cellular viability and image resolution. In this study, we employ a specially designed disc to reduce cell damage by controlling total fluorescence exposure time without deterioration of the image resolution. This approach has many advantages such as, the apparatus is simple, cost-effective, and easily integrated into the optical pathway through a conventional fluorescence microscope. PMID:26381726

  3. Spirally-patterned pinhole arrays for long-term fluorescence cell imaging.

    PubMed

    Koo, Bon Ung; Kang, YooNa; Moon, SangJun; Lee, Won Gu

    2015-11-01

    Fluorescence cell imaging using a fluorescence microscope is an extensively used technique to examine the cell nucleus, internal structures, and other cellular molecules with fluorescence response time and intensity. However, it is difficult to perform high resolution cell imaging for a long period of time with this technique due to necrosis and apoptosis depending on the type and subcellular location of the damage caused by phototoxicity. A large number of studies have been performed to resolve this problem, but researchers have struggled to meet the challenge between cellular viability and image resolution. In this study, we employ a specially designed disc to reduce cell damage by controlling total fluorescence exposure time without deterioration of the image resolution. This approach has many advantages such as, the apparatus is simple, cost-effective, and easily integrated into the optical pathway through a conventional fluorescence microscope.

  4. Electron beam dispersion measurements in nitrogen using two-dimensional imaging of N2(+) fluorescence

    NASA Astrophysics Data System (ADS)

    Clapp, L. H.; Twiss, R. G.; Cattolica, R. J.

    Experimental results are presented related to the radial spread of fluorescence excited by 10 and 20 KeV electron beams passing through nonflowing rarefied nitrogen at 293 K. An imaging technique for obtaining species distributions from measured beam-excited fluorescence is described, based on a signal inversion scheme mathematically equivalent to the inversion of the Abel integral equation. From fluorescence image data, measurements of beam radius, integrated signal intensity, and spatially resolved distributions of N2(+) first-negative-band fluorescence-emitting species have been made. Data are compared with earlier measurements and with an heuristic beam spread model.

  5. Nitroxide amide-BODIPY probe behavior in fibroblasts analyzed by advanced fluorescence microscopy.

    PubMed

    Liras, M; Simoncelli, S; Rivas-Aravena, A; García, O; Scaiano, J C; Alarcon, E I; Aspée, A

    2016-04-26

    A novel synthesized nitroxide amide-BODIPY prefluorescent probe was used to study cellular redox balance that modulates nitroxide/hydroxylamine ratio in cultured human fibroblasts. FLIM quantitatively differentiated between nitroxide states of the cytoplasm-localized probe imaged by TIRF, monitoring nitroxide depletion by hydrogen peroxide; eluding incorrect interpretation if only fluorescence intensity is considered.

  6. Nitroxide amide-BODIPY probe behavior in fibroblasts analyzed by advanced fluorescence microscopy.

    PubMed

    Liras, M; Simoncelli, S; Rivas-Aravena, A; García, O; Scaiano, J C; Alarcon, E I; Aspée, A

    2016-04-26

    A novel synthesized nitroxide amide-BODIPY prefluorescent probe was used to study cellular redox balance that modulates nitroxide/hydroxylamine ratio in cultured human fibroblasts. FLIM quantitatively differentiated between nitroxide states of the cytoplasm-localized probe imaged by TIRF, monitoring nitroxide depletion by hydrogen peroxide; eluding incorrect interpretation if only fluorescence intensity is considered. PMID:27065020

  7. Recent advancements in structured-illumination microscopy toward live-cell imaging.

    PubMed

    Hirano, Yasuhiro; Matsuda, Atsushi; Hiraoka, Yasushi

    2015-08-01

    Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging. PMID:26133185

  8. Far-red light activatable, multifunctional prodrug for fluorescence optical imaging and combinational treatment.

    PubMed

    Bio, Moses; Rajaputra, Pallavi; Nkepang, Gregory; You, Youngjae

    2014-04-24

    We recently developed "photo-unclick chemistry", a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen, and demonstrated its application to visible light-activatable prodrugs. In this study, we prepared an advanced multifunctional prodrug, Pc-(L-CA4)2, composed of the fluorescent photosensitizer phthalocyanine (Pc), an SO-labile aminoacrylate linker (L), and a cytotoxic drug combretastatin A-4 (CA4). Pc-(L-CA4)2 had reduced dark toxicity compared with CA4. However, once illuminated, it showed improved toxicity similar to CA4 and displayed bystander effects in vitro. We monitored the time-dependent distribution of Pc-(L-CA4)2 using optical imaging with live mice. We also effectively ablated tumors by the illumination with far-red light to the mice, presumably through the combined effects of photodynamic therapy (PDT) and released chemotherapy drug, without any sign of acute systemic toxicity.

  9. 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.

  10. 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

  11. 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.

  12. Projection imaging of photon beams using Čerenkov-excited fluorescence

    PubMed Central

    Glaser, Adam K.; Davis, Scott C.; Voigt, William H.A.; Zhang, Rongxiao; Pogue, Brian W.; Gladstone, David J.

    2013-01-01

    Full 3D beam profiling and quality assurance (QA) of therapeutic megavoltage linear accelerator (LINAC) x-ray photon beams is not routinely performed due to the slow point-by-point measurement nature of conventional scanning ionization chamber systems. In this study we explore a novel optical-based dose imaging approach using a standard commercial camera, water tank, and fluorescent dye, which when excited by the Čerenkov emission induced by the radiation beam, allows 2D projection imaging in a fast timeframe, potentially leading towards 3D tomographic beam profiling. Detailed analysis was done to optimize the imaging parameters in the experimental setup. The results demonstrate that the captured images are linear with delivered dose, independent of dose rate, and comparison of experimentally captured images to a reference dose distribution for a 4×4 cm 6 MV x-ray photon beam yielded results with improved accuracy over a previous study which used direct imaging and Monte Carlo calibration of the Čerenkov emission itself. The agreement with the reference dose distribution was within 1-2% in the lateral direction, and ± 3 % in the depth direction. The study was restricted to single 2D image projection, with the eventual goal of creating full 3D profiles after tomographic reconstruction from multiple projections. Given the increasingly complex advances in radiation therapy, and the increased emphasis on patient-specific treatment plans, further refinement of the technique could prove to be an important tool for fast and robust QA of x-ray photon LINAC beams. PMID:23318469

  13. MRI-coupled spectrally-resolved fluorescence tomography for in vivo imaging

    NASA Astrophysics Data System (ADS)

    Davis, Scott C.; Gibbs-Strauss, Summer L.; Tuttle, Stephen B.; Jiang, Shudong; Springett, Roger; Dehghani, Hamid; Pogue, Brian W.; Paulsen, Keith D.

    2008-02-01

    A unique fluorescence imaging system incorporates multi-channel spectrometer-based optical detection directly into clinical MRI for simultaneous MR and spectrally-resolved fluorescence tomography acquisition in small animal and human breast-sized volumes. A custom designed MRI rodent coil adapted to accommodate optical fibers in a circular geometry for contact mode acquisition provides small animal imaging capabilities, and human breast-sized volumes are imaged using a clinical breast coil modified with an optical fiber patient array. Spectroscopy fibers couple light emitted from the tissue surface to sixteen highly sensitive CCD-based spectrometers operating in parallel. Tissue structural information obtained from standard and contrast enhanced T1-weighted images is used to spatially constrain the diffuse fluorescence tomography reconstruction algorithm, improving fluorescence imaging capabilities qualitatively and quantitatively. Simultaneous acquisition precludes the use of complex co-registration processes. Calibration procedures for the optical acquisition system are reviewed and the imaging limits of the system are investigated in homogeneous and heterogeneous gelatin phantoms containing Indocyanine Green (ICG). Prior knowledge of fluorescence emission spectra is used to de-couple fluorescence emission from residual excitation laser cross-talk. Preliminary in vivo data suggests improved fluorescence imaging in mouse brain tumors using MR-derived spatial priors. U-251 human gliomas were implanted intracranially into nude mice and combined contrast enhanced MRI/fluorescence tomography acquisition was completed at 24 hour intervals over the course of 72 hours after administration of an EGFR targeted NIR fluorophore. Reconstructed images demonstrate an inability to recover reasonable images of fluorescence activity without the use of MRI spatial priors.

  14. First In-human Intraoperative Imaging of HCC using Fluorescence Goggle System and Transarterial Delivery of Near-infrared Fluorescent Imaging Agent: a Pilot Study

    PubMed Central

    Akers, Walter; Tang, Zhao-You; Fan, Jia; Sun, Hui-Chuan; Ye, Qing-Hai; Wang, Lu; Achilefu, Samuel

    2013-01-01

    Surgical resections remain the primary curative interventions for hepatocellular carcinoma (HCC). However, lack of real-time intraoperative image guidance confines surgeons to subjective visual assessment of the surgical bed, leading to poor visualization of small positive nodules and the extension of diffuse HCC. To address this problem, we developed a wearable fluorescence imaging and display system (fluorescence goggle) for intraoperative imaging of HCCs in human patients. In this pilot study, both intravenous (i.v.) and transarterial hepatic (TAH) delivery of indocyanine green (ICG) were explored to facilitate fluorescence goggle-mediated HCC imaging. The results show that all primary tumors in patients (n=4) who received TAH delivery of ICG were successfully identified by the fluorescence goggle. In addition, 6 satellite tumors were also detected by the goggle, 5 of which were neither identifiable in pre-operative MRI and CT images nor by visual inspection and palpation. In the group (n=5) that received ICG by i.v., only 2 out of 6 tumors visible in the pre-operative MRI or CT images were identified with the fluorescence goggle, demonstrating the limitation of this delivery route for a non-tumor selective imaging agent. Comparative analysis shows that the HCC-to-liver florescence contrast detected by the goggle was significantly higher in patients that received TAH than i.v. delivery of ICG (P=0.013). This pilot study demonstrates the feasibility of using the fluorescence goggle to identify multifocal lesions and small tumor deposits using TAH ICG delivery in HCC patients. PMID:23747795

  15. 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

  16. Algorithms for differentiating between images of heterogeneous tissue across fluorescence microscopes

    PubMed Central

    Chitalia, Rhea; Mueller, Jenna; Fu, Henry L.; Whitley, Melodi Javid; Kirsch, David G.; Brown, J. Quincy; Willett, Rebecca; Ramanujam, Nimmi

    2016-01-01

    Fluorescence microscopy can be used to acquire real-time images of tissue morphology and with appropriate algorithms can rapidly quantify features associated with disease. The objective of this study was to assess the ability of various segmentation algorithms to isolate fluorescent positive features (FPFs) in heterogeneous images and identify an approach that can be used across multiple fluorescence microscopes with minimal tuning between systems. Specifically, we show a variety of image segmentation algorithms applied to images of stained tumor and muscle tissue acquired with 3 different fluorescence microscopes. Results indicate that a technique called maximally stable extremal regions followed by thresholding (MSER + Binary) yielded the greatest contrast in FPF density between tumor and muscle images across multiple microscopy systems. PMID:27699108

  17. Algorithms for differentiating between images of heterogeneous tissue across fluorescence microscopes

    PubMed Central

    Chitalia, Rhea; Mueller, Jenna; Fu, Henry L.; Whitley, Melodi Javid; Kirsch, David G.; Brown, J. Quincy; Willett, Rebecca; Ramanujam, Nimmi

    2016-01-01

    Fluorescence microscopy can be used to acquire real-time images of tissue morphology and with appropriate algorithms can rapidly quantify features associated with disease. The objective of this study was to assess the ability of various segmentation algorithms to isolate fluorescent positive features (FPFs) in heterogeneous images and identify an approach that can be used across multiple fluorescence microscopes with minimal tuning between systems. Specifically, we show a variety of image segmentation algorithms applied to images of stained tumor and muscle tissue acquired with 3 different fluorescence microscopes. Results indicate that a technique called maximally stable extremal regions followed by thresholding (MSER + Binary) yielded the greatest contrast in FPF density between tumor and muscle images across multiple microscopy systems.

  18. 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

  19. Design and synthesis of caged fluorescent nucleotides and application to live-cell RNA imaging.

    PubMed

    Ikeda, Shuji; Kubota, Takeshi; Wang, Dan Ohtan; Yanagisawa, Hiroyuki; Umemoto, Tadashi; Okamoto, Akimitsu

    2011-12-16

    A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization-sensitive thiazole orange units has been designed for area-specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization-sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex-formation ability. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area-specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.

  20. Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore

    NASA Astrophysics Data System (ADS)

    Siegel, J.; Suhling, K.; Lévêque-Fort, S.; Webb, S. E. D.; Davis, D. M.; Phillips, D.; Sabharwal, Y.; French, P. M. W.

    2003-01-01

    We report a picosecond time-gated fluorescence lifetime imaging (FLIM) system extended to perform time-resolved fluorescence anisotropy imaging (TR-FAIM). Upon excitation with linearly polarized laser pulses, the parallel and perpendicular components of the fluorescence emission from a sample are imaged simultaneously using a polarization-resolved imager. The imaging technique presented here quantitatively reports the rotational mobility of a fluorophore as it varies according to the local environment. In a single acquisition run it yields maps of both rotational correlation time and fluorescence lifetime as they vary across a sample. TR-FAIM has been applied to imaging standard multiwell plate samples of rhodamine 6G dissolved in methanol, ethylene glycol, trimethylene glycol, and glycerol. The observed rotational correlation times and fluorescence lifetimes, which report the local viscosity and refractive index of the local rhodamine 6G environment, respectively, are in good agreement with previously published single point measurements. By considering the linear dependence of the rotational correlation time on viscosity up to 20 cP, we are able to obtain a two-dimensional viscosity map. Wide-field maps of rotational correlation time, and therefore viscosity, have been obtained. This illustrates the potential to image the local viscosity and fluorescence lifetime distributions of fluorophore tagged proteins in cells.

  1. 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

  2. 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

  3. Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells.

    PubMed

    Micic, Miodrag; Hu, Dehong; Suh, Yung Doug; Newton, Greg; Romine, Margaret; Lu, H Peter

    2004-04-15

    We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.

  4. Advance in diagnosis of female genital tract tumor with laser fluorescence

    NASA Astrophysics Data System (ADS)

    Ding, Ai-Hua; Tseng, Quen; Lian, Shao-Hui

    1998-11-01

    In order to improve the diagnostic accuracy of malignant tumors with laser fluorescence, in 1996, our group successfully created the computerized laser fluorescence spectrograph type II with more reliable images shown overshadowing the naked eye method before 74 cases of female genital tract diseases had been examined by the LFS II resulting in 10 positive cases which were also proven pathologically as malignant tumors, without nay false negative, 3 cases presented suspicious positive but all were proven pathologically as non-tumors lesions, the false positive rate was 4 percent. Our work showed that the method of LFS II can provide a more rapid and accurate diagnosis for the clinical malignant tumors.

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

    PubMed

    Cho, B; Kim, M S; Chao, K; Lawrence, K; Park, B; Kim, K

    2009-04-01

    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 poultry carcasses was investigated. One of the challenges for using fluorescence imaging for inspection of agricultural material is the low fluorescence yield in that fluorescence can be masked by ambient light. A laser-induced fluorescence imaging system (LIFIS) developed by our group allowed acquisition of fluorescence from feces-contaminated poultry carcasses in ambient light. Fluorescence emission images at 630 nm were captured with 415-nm laser excitation. Image processing algorithms including threshold and image erosion were used to identify fecal spots diluted up to 1: 10 by weight with double distilled water. Feces spots on the carcasses, without dilution and up to 1: 5 dilutions, could be detected with 100% accuracy regardless of feces type. Detection accuracy for fecal matters diluted up to 1: 10 was 96.6%. The results demonstrated good potential of the LIFIS for detection of diluted poultry fecal matter, which can harbor pathogens, on poultry carcasses.

  6. 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.

  7. 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

  8. 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.

  9. Gold nanoparticle cluster-plasmon-enhanced fluorescent silica core-shell nanoparticles for X-ray computed tomography-fluorescence dual-mode imaging of tumors.

    PubMed

    Hayashi, Koichiro; Nakamura, Michihiro; Miki, Hirokazu; Ozaki, Shuji; Abe, Masahiro; Matsumoto, Toshio; Ishimura, Kazunori

    2013-06-11

    Owing to the surface plasmon resonance-enhanced electromagnetic field, clustered gold nanoparticles-fluorescent silica core-shell nanoparticles became excited within the therapeutic window and fluoresced strongly in this window. The nanoparticles enabled tumor detection using fluorescence imaging and X-ray computed tomography.

  10. Changes in the redox state and endogenous fluorescence of in vivo human skin due to intrinsic and photo-aging, measured by multiphoton tomography with fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Sanchez, Washington Y.; Obispo, Clara; Ryan, Elizabeth; Grice, Jeffrey E.; Roberts, Michael S.

    2013-06-01

    Ultraviolet radiation from solar exposure is a key extrinsic factor responsible for premature skin aging (i.e., photo-aging). Recent advances using in vivo multiphoton tomography (MPT) demonstrate the efficacy of this approach to assess intrinsic and extrinsic skin aging as an alternative to existing invasive techniques. In this study, we measured changes in epidermal autofluorescence, dermal collagen second harmonic generation (SHG), and the redox state of solar-exposed and solar-protected human skin by MPT with fluorescence lifetime imaging (MPT-FLIM). Twenty-four volunteers across four age categories (20 to 29, 30 to 39, 40 to 49, and 50 to 59 years old; six volunteers each) were recruited for MPT-FLIM imaging of the dorsal (solar-exposed; photo-damaged) and volar (solar-protected) forearm. We demonstrate a higher intensity of dermal collagen SHG within the volar forearm compared to dorsal solar-exposed skin. Redox imaging of each epidermal skin stratum by FLIM demonstrates an increase in fluorescence lifetime in the solar-exposed dorsal forearm that is more apparent in aged skin. The results of this study suggest the redox state of the viable epidermis is a key marker in assessing intrinsic and pho