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Sample records for acquire fluorescence images

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  2. Non-negative matrix factorization for the near real-time interpretation of absorption effects in elemental distribution images acquired by X-ray fluorescence imaging.

    PubMed

    Alfeld, Matthias; Wahabzada, Mirwaes; Bauckhage, Christian; Kersting, Kristian; Wellenreuther, Gerd; Barriobero-Vila, Pere; Requena, Guillermo; Boesenberg, Ulrike; Falkenberg, Gerald

    2016-03-01

    Elemental distribution images acquired by imaging X-ray fluorescence analysis can contain high degrees of redundancy and weakly discernible correlations. In this article near real-time non-negative matrix factorization (NMF) is described for the analysis of a number of data sets acquired from samples of a bi-modal α+β Ti-6Al-6V-2Sn alloy. NMF was used for the first time to reveal absorption artefacts in the elemental distribution images of the samples, where two phases of the alloy, namely α and β, were in superposition. The findings and interpretation of the NMF results were confirmed by Monte Carlo simulation of the layered alloy system. Furthermore, it is shown how the simultaneous factorization of several stacks of elemental distribution images provides uniform basis vectors and consequently simplifies the interpretation of the representation. PMID:26917147

  3. Quality analysis of in vivo near-infrared fluorescence and conventional gamma images acquired using a dual-labeled tumor-targeting probe.

    PubMed

    Houston, Jessica P; Ke, Shi; Wang, Wei; Li, Chun; Sevick-Muraca, Eva M

    2005-01-01

    The cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) (c(KRGDf)), which is known to target alpha(v)beta3 integrin, is dual-labeled with a radiotracer, (111)indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, for continuous-wave (cw) imaging of alpha(v)beta3 positive human M21 melanoma in xenografts. Twenty-four hours after administration of the dual-labeled peptide at a dose equivalent to 90 microCi of (111)In and 5 nmol of near-infrared (NIR) dye, whole-body gamma scintigraphy and cw imaging was conducted. Image acquisition time was 15 min for the gamma scintigraphy images and 800 ms for the optical images acquired using an NIR sensitive intensified charge-coupled device. The results show that while the target-to-background ratio (TBR) of nuclear and optical imaging were similar for surface regions of interest and consistent with the origin of gamma and NIR radiation from a common targeted peptide, the signal-to-noise ratio (SNR) was significantly higher for optical than nuclear imaging. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets. While tomographic reconstructions are necessary to probe TBR, SNR, and contrast for interior tissues, this work demonstrates for the first time the direct comparison of molecular optical and planar nuclear imaging for surface and subsurface cancers. PMID:16292970

  4. 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.

  5. 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

  6. A Method for Analyzing Protein–Protein Interactions in the Plasma Membrane of Live B Cells by Fluorescence Resonance Energy Transfer Imaging as Acquired by Total Internal Reflection Fluorescence Microscopy

    PubMed Central

    Sohn, Hae Won; Tolar, Pavel; Brzostowski, Joseph; Pierce, Susan K.

    2012-01-01

    For more than a decade, fluorescence resonance energy transfer (FRET) imaging methods have been developed to study dynamic interactions between molecules at the nanometer scale in live cells. Here, we describe a protocol to measure FRET by the acceptor-sensitized emission method as detected by total internal reflection fluorescence (TIRF) imaging to study the interaction of appropriately labeled plasma membrane-associated molecules that regulate the earliest stages of antigen-mediated signaling in live B lymphocytes. This protocol can be adapted and applied to many cell types where there is an interest in understanding signal transduction mechanisms in live cells. PMID:19957130

  7. Magnetic resonance imaging of acquired cardiac disease.

    PubMed Central

    Carrol, C L; Higgins, C B; Caputo, G R

    1996-01-01

    Over the last 15 years, advances in magnetic resonance imaging techniques have increased the accuracy and applicability of cardiovascular magnetic resonance imaging. These advances have improved the utility of magnetic resonance imaging in evaluating cardiac morphology, blood flow, and myocardial contractility, all significant diagnostic features in the evaluation of the patient with acquired heart disease. Utilization of cardiovascular magnetic resonance imaging has been limited, primarily due to clinical reliance upon nuclear scintigraphy and echocardiography. Recent developments in fast and ultrafast imaging should continue to enhance the significance of magnetic resonance imaging in this field. Widespread use of magnetic resonance imaging in the evaluation of the cardiovascular system will ultimately depend upon its maturation into a comprehensive, noninvasive imaging technique for the varying manifestations of acquired heart disease, including cardiomyopathy, ischemic heart disease, and acquired valvular disease. Images PMID:8792545

  8. Fluorescence Imaging in Surgery

    PubMed Central

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

    2013-01-01

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

  9. 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.

  10. High frame rate fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

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

    2003-07-01

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

  11. Spectrally resolved multidepth fluorescence imaging

    PubMed Central

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

    2011-01-01

    We present a multicolor fluorescence imaging modality to visualize in real-time tissue structures emitting multispectral fluorescent light from different focal depths. Each designated spectrum of fluorescent emission from a specific depth within a volumetric tissue is probed by a depth-spectrum selective holographic grating. The grating for each fluorescent color are multiplexed within a volume hologram, which enables simultaneously obtaining multicolored fluorescent information at different depths within a biological tissue sample. We demonstrate the imaging modality's ability to obtain laser-induced multicolored fluorescence images of a biological sample from different depths without scanning. We also experimentally demonstrate that the imaging modality can be simultaneously operated at both fluorescent and bright field modes to provide complementary information of volumetric tissue structures at different depths in real-time. PMID:21950929

  12. Spectrally resolved multidepth fluorescence imaging

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

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

  13. Fiber-optic fluorescence imaging

    PubMed Central

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

    2010-01-01

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

  14. Fluorescence lifetime imaging of coral fluorescent proteins.

    PubMed

    Cox, Guy; Matz, Mikhail; Salih, Anya

    2007-03-01

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

  15. 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…

  16. 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 ...

  17. 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

  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. 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.

  20. 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.

  1. Fluorescence lifetime-based optical molecular imaging.

    PubMed

    Kumar, Anand T N

    2011-01-01

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

  2. 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).

  3. Acquired immunodeficiency syndrome: Ga-67 citrate imaging

    SciTech Connect

    Woolfenden, J.M.; Carrasquillo, J.A.; Larson, S.M.; Simmons, J.T.; Masur, H.; Smith, P.D.; Shelhamer, J.H.; Ognibene, F.P.

    1987-02-01

    All gallium-67 citrate scans obtained in patients with acquired immunodeficiency syndrome (AIDS) at the Clinical Center, National Institutes of Health (Bethesda, Md.) were retrospectively analyzed and correlated with the results of bronchoscopy, chest radiography, and endoscopy. There were 164 scans of 95 patients. Twenty scans were from patients with Pneumocystis carinii pneumonia; 19 were abnormal, for a sensitivity of 95%. Ga-67 uptake tended to be less in patients receiving therapy for P. carinii pneumonia. Chest radiographs were normal at least initially in three patients with abnormal scans and P. carinii pneumonia. Unusually prominent colonic activity was associated with infection in some patients. No lesions of Kaposi sarcoma showed tracer uptake. Gallium scanning is useful for detecting P. carinii pneumonia and other opportunistic infections in patients with AIDS, but it is not useful for localizing Kaposi sarcoma.

  4. 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

  5. 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.

  6. Fluorescence optical imaging in anticancer drug delivery.

    PubMed

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

    2016-03-28

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

  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. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  12. 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

  13. Recent Progress in Fluorescent Imaging Probes.

    PubMed

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

    2015-01-01

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

  14. Recent Progress in Fluorescent Imaging Probes

    PubMed Central

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

    2015-01-01

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

  15. 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.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    1997-04-15

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

  18. Comprehensive phantom for interventional fluorescence molecular imaging.

    PubMed

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

    2016-09-01

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

  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. Fluorescence goggle for intraoperative breast cancer imaging

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  1. 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.

  2. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2003-07-01

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

  4. Determination of localization accuracy based on experimentally acquired image sets: applications to single molecule microscopy.

    PubMed

    Tahmasbi, Amir; Ward, E Sally; Ober, Raimund J

    2015-03-23

    Fluorescence microscopy is a photon-limited imaging modality that allows the study of subcellular objects and processes with high specificity. The best possible accuracy (standard deviation) with which an object of interest can be localized when imaged using a fluorescence microscope is typically calculated using the Cramér-Rao lower bound, that is, the inverse of the Fisher information. However, the current approach for the calculation of the best possible localization accuracy relies on an analytical expression for the image of the object. This can pose practical challenges since it is often difficult to find appropriate analytical models for the images of general objects. In this study, we instead develop an approach that directly uses an experimentally collected image set to calculate the best possible localization accuracy for a general subcellular object. In this approach, we fit splines, i.e. smoothly connected piecewise polynomials, to the experimentally collected image set to provide a continuous model of the object, which can then be used for the calculation of the best possible localization accuracy. Due to its practical importance, we investigate in detail the application of the proposed approach in single molecule fluorescence microscopy. In this case, the object of interest is a point source and, therefore, the acquired image set pertains to an experimental point spread function. PMID:25837101

  5. 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.

  6. Ion-induced fluorescence imaging of endosomes

    NASA Astrophysics Data System (ADS)

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

    2013-07-01

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

  7. 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

  8. Photocontrollable Fluorescent Proteins for Superresolution Imaging

    PubMed Central

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

    2014-01-01

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

  9. 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.

  10. 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

  11. Evaluation of outliers in acquired brain MR images

    NASA Astrophysics Data System (ADS)

    Moldovanu, S.; (Vişan Pungǎ, M.; Moraru, L.

    2015-01-01

    Pre-processing is an important stage in the analysis of magnetic resonance images (MRI), because the effect of specific image artefacts, such as intensity inhomogeneity, noise and low contrast can adversely affect the quantitative image analysis. The image histogram is a useful tool in the analysis of MR images given that it allows a close relationship with important image features such as contrast and noise. The noise and variable contrast are elements that locally modify the quality of images. The key issue of this study derives from the fact that the spatial histogram can contain outliers indicating corrupted image information through the disorder of the bins. These aberrant errors should be excluded from the studied data sets. Here, the outliers are evaluated by using rigorous methods based on the probability theory and Chauvenet (CC), Grubbs (GC) and Peirce's (PC) criteria. In order to check the quality of the MR images, the Minkowsky (MD), Euclidean (ED) and cosine (CD) distance functions were used. They act as similarity scores between the histogram of the acquired MRI and the processed image. This analysis is necessary because, sometimes, the distance function exceeds the co-domain because of the outliers. In this paper, 32 MRIs are tested and the outliers are removed so that the distance functions generate uncorrupted and real values.

  12. 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

  13. 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

  14. Fluorescence Microscopy Imaging in Biomedical Sciences

    NASA Astrophysics Data System (ADS)

    Sun, Yuansheng; Periasamy, Ammasi

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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

    NASA Astrophysics Data System (ADS)

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

    2006-09-01

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

  7. 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

  8. 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

  9. 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

  10. Fluorescent nanoparticle probes for imaging of cancer.

    PubMed

    Santra, Swadeshmukul; Malhotra, Astha

    2011-01-01

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

  11. Method for acquiring, storing and analyzing crystal images

    NASA Technical Reports Server (NTRS)

    Gester, Thomas E. (Inventor); Rosenblum, William M. (Inventor); Christopher, Gayle K. (Inventor); Hamrick, David T. (Inventor); Delucas, Lawrence J. (Inventor); Tillotson, Brian (Inventor)

    2003-01-01

    A system utilizing a digital computer for acquiring, storing and evaluating crystal images. The system includes a video camera (12) which produces a digital output signal representative of a crystal specimen positioned within its focal window (16). The digitized output from the camera (12) is then stored on data storage media (32) together with other parameters inputted by a technician and relevant to the crystal specimen. Preferably, the digitized images are stored on removable media (32) while the parameters for different crystal specimens are maintained in a database (40) with indices to the digitized optical images on the other data storage media (32). Computer software is then utilized to identify not only the presence and number of crystals and the edges of the crystal specimens from the optical image, but to also rate the crystal specimens by various parameters, such as edge straightness, polygon formation, aspect ratio, surface clarity, crystal cracks and other defects or lack thereof, and other parameters relevant to the quality of the crystals.

  12. Role of Radiologic Imaging in Genetic and Acquired Neuromuscular Disorders

    PubMed Central

    Zanato, Riccardo; Coran, Alessandro; Beltrame, Valeria; Stramare, Roberto

    2015-01-01

    Great technologic and clinical progress have been made in the last two decades in identifying genetic defects of several neuromuscular diseases, as Spinal Muscular Atrophy, genetic muscular dystrophies and other genetic myopathies. The diagnosis is usually challenging, due to great variability in genetic abnormalities and clinical phenotypes and the poor specificity of complementary analyses, i.e., serum creatine kinase (CK) and electrophysiology. Muscle biopsy represents the gold standard for the diagnosis of genetic neuromuscular diseases, but clinical imaging of muscle tissue is an important diagnostic tool to identify and quantifyies muscle damage. Radiologic imaging is, indeed, increasingly used as a diagnostic tool to describe patterns and the extent of muscle involvement, thanks to modern techniques that enable to definethe definition of degrees of muscle atrophy and changes in connective tissue. They usually grade the severity of the disease process with greater accuracy than clinical scores. Clinical imaging is more than complementary to perform muscle biopsy, especially as ultrasound scans are often mandatory to identify the muscle to be biopsied. We will here detail and provideWe will herein provide detailed examples of the radiologic methods that can be used in genetic and acquired neuromuscular disorders, stressing pros and cons. Key Words: Muscle Imaging, MRI, CT, genetic muscle disorders, myopathies, dystrophies PMID:26913153

  13. 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

  14. 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.

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

    PubMed

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

    2015-09-01

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

  16. 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}.

  17. 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.

  18. 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

  19. Self-acquired patient images: the promises and the pitfalls.

    PubMed

    Damanpour, Shadi; Srivastava, Divya; Nijhawan, Rajiv I

    2016-03-01

    Self-acquired patient images, also known as selfies, are increasingly utilized in the practice of dermatology; however, research on their utility is somewhat limited. While the implementation of selfies has yet to be universally accepted, their role in triage appears to be especially useful. The potential for reducing office wait times, expediting referrals, and providing dermatologic services to patients with limited access to care is promising. In addition, as technology advances, the number of smartphone applications related to dermatology that are available to the general public has risen exponentially. With appropriate standardization, regulation, and confidentiality measures, these tools can be feasible adjuncts in clinical practice, dermatologic surgery, and teledermatology. Selfies likely will have a large role in dermatologic practice and delivery in the future. PMID:26963112

  20. 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

  1. Temporal resolution in fluorescence imaging

    PubMed Central

    Mondal, Partha Pratim

    2014-01-01

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

  2. Acquired cystic disease-associated renal cell carcinoma: an immunohistochemical and fluorescence in situ hybridization study.

    PubMed

    Kuroda, Naoto; Yamashita, Motoki; Kakehi, Yoshiyuki; Hes, Ondrej; Michal, Michal; Lee, Gang-Hong

    2011-12-01

    Acquired cystic disease (ACD)-associated renal cell carcinoma (RCC) has been recently identified. However, there are only a few genetic studies to date. In this article, we performed an immunohistochemical and fluorescence in situ hybridization (FISH) study for six cases including one case with sarcomatoid change. As a result, we observed frequent immunohistochemical expression of AMACR. FISH of chromosome 3 showed trisomy for three cases, monosomy for two cases, and disomy for one case. Additionally, FISH of chromosome 16 showed trisomy for three cases, monosomy for two cases, and both trisomy and monosomy for one case. Furthermore, both the carcinomatous area and the sarcomatoid area of one ACD-associated RCC with sarcomatoid change revealed monosomy of chromosomes 3, 9, and 16 but showed disomy of chromosome 14. In conclusion, the numerical abnormalities of chromosomes 3 and 16, irrespective of gain or loss, may be characteristic of ACD-associated RCC. PMID:22179186

  3. An image-guided tool to prevent hospital acquired infections

    NASA Astrophysics Data System (ADS)

    Nagy, Melinda; Szilágyi, László; Lehotsky, Ákos; Haidegger, Tamás; Benyó, Balázs

    2011-03-01

    Hospital Acquired Infections (HAI) represent the fourth leading cause of death in the United States, and claims hundreds of thousands of lives annually in the rest of the world. This paper presents a novel low-cost mobile device|called Stery-Hand|that helps to avoid HAI by improving hand hygiene control through providing an objective evaluation of the quality of hand washing. The use of the system is intuitive: having performed hand washing with a soap mixed with UV re ective powder, the skin appears brighter in UV illumination on the disinfected surfaces. Washed hands are inserted into the Stery-Hand box, where a digital image is taken under UV lighting. Automated image processing algorithms are employed in three steps to evaluate the quality of hand washing. First, the contour of the hand is extracted in order to distinguish the hand from the background. Next, a semi-supervised clustering algorithm classies the pixels of the hand into three groups, corresponding to clean, partially clean and dirty areas. The clustering algorithm is derived from the histogram-based quick fuzzy c-means approach, using a priori information extracted from reference images, evaluated by experts. Finally, the identied areas are adjusted to suppress shading eects, and quantied in order to give a verdict on hand disinfection quality. The proposed methodology was validated through tests using hundreds of images recorded in our laboratory. The proposed system was found robust and accurate, producing correct estimation for over 98% of the test cases. Stery-Hand may be employed in general practice, and it may also serve educational purposes.

  4. Computed tomography based spectral imaging for fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Ford, Bridget Kathleen

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

  5. Hyperspectral fluorescence lifetime imaging for optical biopsy.

    PubMed

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

    2013-09-01

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

  6. Wide field-of-view fluorescence image deconvolution with aberration-estimation from Fourier ptychography

    PubMed Central

    Chung, Jaebum; Kim, Jinho; Ou, Xiaoze; Horstmeyer, Roarke; Yang, Changhuei

    2016-01-01

    This paper presents a method to simultaneously acquire an aberration-corrected, wide field-of-view fluorescence image and a high-resolution coherent bright-field image using a computational microscopy method. First, the procedure applies Fourier ptychographic microscopy (FPM) to retrieve the amplitude and phase of a sample, at a resolution that significantly exceeds the cutoff spatial frequency of the microscope objective lens. At the same time, redundancy within the set of acquired FPM bright-field images offers a means to estimate microscope aberrations. Second, the procedure acquires an aberrated fluorescence image, and computationally improves its resolution through deconvolution with the estimated aberration map. An experimental demonstration successfully improves the bright-field resolution of fixed, stained and fluorescently tagged HeLa cells by a factor of 4.9, and reduces the error caused by aberrations in a fluorescence image by up to 31%, over a field of view of 6.2 mm by 9.3 mm. For optimal deconvolution, we show the fluorescence image needs to have a signal-to-noise ratio of at least ~18. PMID:26977345

  7. Wide field-of-view fluorescence image deconvolution with aberration-estimation from Fourier ptychography.

    PubMed

    Chung, Jaebum; Kim, Jinho; Ou, Xiaoze; Horstmeyer, Roarke; Yang, Changhuei

    2016-02-01

    This paper presents a method to simultaneously acquire an aberration-corrected, wide field-of-view fluorescence image and a high-resolution coherent bright-field image using a computational microscopy method. First, the procedure applies Fourier ptychographic microscopy (FPM) to retrieve the amplitude and phase of a sample, at a resolution that significantly exceeds the cutoff spatial frequency of the microscope objective lens. At the same time, redundancy within the set of acquired FPM bright-field images offers a means to estimate microscope aberrations. Second, the procedure acquires an aberrated fluorescence image, and computationally improves its resolution through deconvolution with the estimated aberration map. An experimental demonstration successfully improves the bright-field resolution of fixed, stained and fluorescently tagged HeLa cells by a factor of 4.9, and reduces the error caused by aberrations in a fluorescence image by up to 31%, over a field of view of 6.2 mm by 9.3 mm. For optimal deconvolution, we show the fluorescence image needs to have a signal-to-noise ratio of at least ~18. PMID:26977345

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  9. Acquiring reproducible fluorescence spectra of dissolved organic matter at very low concentrations.

    PubMed

    Peiris, B R H; Budman, H; Moresoli, C; Legge, R L

    2009-01-01

    A method that would allow for fast and reliable measurements of dissolved organic matter (DOM), both at low and high concentration levels would be a valuable tool for online monitoring of DOM. This could have applications in a variety of areas including membrane treatment systems for drinking water applications which is of interest to our group. In this study, the feasibility of using fluorescence spectroscopy for monitoring DOM at very low concentration levels was demonstrated with an emphasis on optimizing the instrument parameters necessary to obtain reproducible fluorescence signals. Signals were acquired using a cuvette or a fibre optic probe assembly, the latter which may have applications for on-line or in-line monitoring. The instrument parameters such as photomultiplier tube (PMT) voltage, scanning rate and slit width were studied in detail to find the optimum parameter settings required. The results showed that larger excitation and emission slit widths were preferred, over larger PMT voltage or lower scanning rates, to obtain reproducible and rapid measurements when measuring very low concentration levels of DOM. However, this approach should be implemented with caution to avoid any reduction of the signal resolution. PMID:19759440

  10. Fluorescence lifetime imaging in turbid media

    NASA Astrophysics Data System (ADS)

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

    1996-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  12. Multispectral fluorescence imaging device for malignancy detection

    NASA Astrophysics Data System (ADS)

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

    1997-12-01

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

  13. Mosaic-Detector-Based Fluorescence Spectral Imager

    NASA Technical Reports Server (NTRS)

    Son, Kyung-Ah; Moon, Jeong

    2007-01-01

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

  14. Coherent nonlinear optical imaging: beyond fluorescence microscopy.

    PubMed

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

    2011-01-01

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

  15. 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.

  16. 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

  17. 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

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

    PubMed

    Cao, Jie; Zhou, Mingzhou

    2016-01-01

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

  19. Fluorescence imaging to quantify crop residue cover

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

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

    PubMed

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

    2011-12-27

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

  1. Fluorescence Ratio Imaging Of Dynamic Intracellular Signals

    NASA Astrophysics Data System (ADS)

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

    1989-12-01

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

  2. 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.

  3. 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.

  4. Carbon Quantum Dots for Zebrafish Fluorescence Imaging.

    PubMed

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

    2015-01-01

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

  5. 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

  6. Carbon Quantum Dots for Zebrafish Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  7. 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.

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

    PubMed

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

    2004-01-01

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

  9. Stepwise multi-photon activation fluorescence reveals a new method of melanoma imaging for dermatologists

    NASA Astrophysics Data System (ADS)

    Lai, Zhenhua; Lian, Christine; Ma, Jie; Yu, Jingyi; Gu, Zetong; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2014-02-01

    Previous research has shown that the stepwise multi-photon activated fluorescence (SMPAF) of melanin, activated by a continuous-wave (CW) mode near infrared (NIR) laser, is a low cost and reliable method of detecting melanin. SMPAF images of melanin in a mouse hair and a formalin fixed mouse melanoma were compared with conventional multiphoton fluorescence microscopy (MPFM) images and confocal reflectance microscopy (CRM) images, all of which were acquired at an excitation wavelength of 920 nm, to further prove the effectiveness of SMPAF in detecting melanin. SMPAF images add specificity for melanin detection to MPFM images and CRM images. Melanin SMPAF can be a promising technology to enable melanoma imaging for dermatologists.

  10. Motion corrected photoacoustic difference imaging of fluorescent contrast agents

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  11. Imaging of acquired coronary diseases: From children to adults.

    PubMed

    Dehaene, A; Jacquier, A; Falque, C; Gorincour, G; Gaubert, J Y

    2016-05-01

    Acquired coronary diseases include aneurysms, fistulae, dissections, and stenosis. Aneurysms may occur secondarily to Kawasaki disease, a childhood vasculitis, the prognosis of which depends on the coronary involvement, or they may be degenerative, infectious, inflammatory, or traumatic in origin. Fistulae develop between the coronary arterial system and a pulmonary or bronchial artery, or cardiac cavity. Dissections may occur spontaneously or may be post-traumatic. These coronary abnormalities may be found incidentally or may present as complications, infarction or rupture. The goals of this article are to understand acquired childhood and adult coronary diseases and their usual means of presentation, the ways of investigating them, and the principles of their treatment. PMID:27130480

  12. In-vivo validation of fluorescence lifetime imaging (FLIm) of coronary arteries in swine

    NASA Astrophysics Data System (ADS)

    Bec, Julien; Ma, Dinglong; Yankelevich, Diego R.; Gorpas, Dimitris S.; Ferrier, William T.; Southard, Jeffrey; Marcu, Laura

    2015-02-01

    We report a scanning imaging system that enables high speed multispectral fluorescence lifetime imaging (FLIm) of coronary arteries. This system combines a custom low profile (3 Fr) imaging catheter using a 200 μm core side viewing UV-grade silica fiber optic, an acquisition system able to measure fluorescence decays over four spectral bands at 20 kHz and a fast data analysis and display module. In vivo use of the system has been optimized, with particular emphasis on clearing blood from the optical pathway. A short acquisition time (5 seconds for a 20 mm long coronary segment) enabled data acquisition during a bolus saline solution injection through the 7 Fr catheter guide. The injection parameters were precisely controlled using a power injector and optimized to provide good image quality while limiting the bolus injection duration and volume (12 cc/s, 80 cc total volume). The ability of the system to acquire data in vivo was validated in healthy swine by imaging different sections of the left anterior descending (LAD) coronary. A stent coated with fluorescent markers was placed in the LAD and imaged, demonstrating the ability of the system to discriminate in vivo different fluorescent features and structures from the vessel background fluorescence using spectral and lifetime information. Intensity en face images over the four bands of the instrument were available within seconds whereas lifetime images were computed in 2 minutes, providing efficient feedback during the procedure. This successful demonstration of FLIm in coronaries enables future study of atherosclerotic cardiovascular diseases.

  13. Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging.

    PubMed

    Nguyen, Huy Bang; Thai, Truc Quynh; Saitoh, Sei; Wu, Bao; Saitoh, Yurika; Shimo, Satoshi; Fujitani, Hiroshi; Otobe, Hirohide; Ohno, Nobuhiko

    2016-01-01

    Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM. PMID:27020327

  14. Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging

    PubMed Central

    Nguyen, Huy Bang; Thai, Truc Quynh; Saitoh, Sei; Wu, Bao; Saitoh, Yurika; Shimo, Satoshi; Fujitani, Hiroshi; Otobe, Hirohide; Ohno, Nobuhiko

    2016-01-01

    Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM. PMID:27020327

  15. 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

  16. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

    2016-02-01

    Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

  17. Hyperspectral imaging fluorescence excitation scanning for detecting colorectal cancer: pilot study

    NASA Astrophysics Data System (ADS)

    Leavesley, Silas J.; Wheeler, Mikayla; Lopez, Carmen; Baker, Thomas; Favreau, Peter F.; Rich, Thomas C.; Rider, Paul F.; Boudreaux, Carole W.

    2016-03-01

    Optical spectroscopy and hyperspectral imaging have shown the theoretical potential to discriminate between cancerous and non-cancerous tissue with high sensitivity and specificity. To date, these techniques have not been able to be effectively translated to endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents a new technology that may be well-suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The objective of this pilot study was to evaluate the changes in the fluorescence excitation spectrum of resected specimen pairs of colorectal adenocarcinoma and normal colorectal mucosa. Patients being treated for colorectal adenocarcinoma were enrolled. Representative adenocarcinoma and normal colonic mucosa specimens were collected from each case. Specimens were flash frozen in liquid nitrogen. Adenocarcinoma was confirmed by histologic evaluation of H&E permanent sections. Hyperspectral image data of the fluorescence excitation of adenocarcinoma and surrounding normal tissue were acquired using a custom microscope configuration previously developed in our lab. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation spectral range of 390-450 nm. We conclude that fluorescence excitation-scanning hyperspectral imaging may offer an alternative approach for differentiating adenocarcinoma and surrounding normal mucosa of the colon. Future work will focus on expanding the number of specimen pairs analyzed and will utilize fresh tissues where possible, as flash freezing and reconstituting tissues may have altered the autofluorescence properties.

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

    PubMed

    Lu, Yujie; Zhu, Banghe; Shen, Haiou; Rasmussen, John C; Wang, Ge; Sevick-Muraca, Eva M

    2010-08-21

    Fluorescence molecular imaging/tomography may play an important future role in preclinical research and clinical diagnostics. Time- and frequency-domain fluorescence imaging can acquire more measurement information than the continuous wave (CW) counterpart, improving the image quality of fluorescence molecular tomography. Although diffusion approximation (DA) theory has been extensively applied in optical molecular imaging, high-order photon migration models need to be further investigated to match quantitation provided by nuclear imaging. In this paper, a frequency-domain parallel adaptive finite element solver is developed with simplified spherical harmonics (SP(N)) approximations. To fully evaluate the performance of the SP(N) approximations, a fast time-resolved tetrahedron-based Monte Carlo fluorescence simulator suitable for complex heterogeneous geometries is developed using a convolution strategy to realize the simulation of the fluorescence excitation and emission. The validation results show that high-order SP(N) can effectively correct the modeling errors of the diffusion equation, especially when the tissues have high absorption characteristics or when high modulation frequency measurements are used. Furthermore, the parallel adaptive mesh evolution strategy improves the modeling precision and the simulation speed significantly on a realistic digital mouse phantom. This solver is a promising platform for fluorescence molecular tomography using high-order approximations to the radiative transfer equation. PMID:20671350

  19. Laser induced fluorescence as a diagnostic tool integrated into a scanning fiber endoscope for mouse imaging

    NASA Astrophysics Data System (ADS)

    Brown, Christopher M.; Maggio-Price, Lillian; Seibel, Eric J.

    2007-02-01

    Scanning fiber endoscope (SFE) technology has shown promise as a minimally invasive optical imaging tool. To date, it is capable of capturing full-color 500-line images, at 15 Hz frame rate in vivo, as a 1.6 mm diameter endoscope. The SFE uses a singlemode optical fiber actuated at mechanical resonance to scan a light spot over tissue while backscattered or fluorescent light at each pixel is detected in time series using several multimode optical fibers. We are extending the capability of the SFE from a RGB reflectance imaging device to a diagnostic tool by imaging laser induced fluorescence (LIF) in tissue, allowing for correlation of endogenous fluorescence to tissue state. Design of the SFE for diagnostic imaging is guided by a comparison of single point spectra acquired from an inflammatory bowel disease (IBD) model to tissue histology evaluated by a pathologist. LIF spectra were acquired by illuminating tissue with a 405 nm light source and detecting intrinsic fluorescence with a multimode optical fiber. The IBD model used in this study was mdr1a-/- mice, where IBD was modulated by infection with Helicobacter bilis. IBD lesions in the mouse model ranged from mild to marked hyperplasia and dysplasia, from the distal colon to the cecum. A principle components analysis (PCA) was conducted on single point spectra of control and IBD tissue. PCA allowed for differentiation between healthy and dysplastic tissue, indicating that emission wavelengths from 620 - 650 nm were best able to differentiate diseased tissue and inflammation from normal healthy tissue.

  20. Fluorescence Imaging Study of Impinging Underexpanded Jets

    NASA Technical Reports Server (NTRS)

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

    2008-01-01

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

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

    PubMed

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

    2015-08-24

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Ishizawa, Takeaki; Saiura, Akio; Kokudo, Norihiro

    2016-08-01

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

  4. Classification of Images Acquired with Colposcopy Using Artificial Neural Networks

    PubMed Central

    Simões, Priscyla W; Izumi, Narjara B; Casagrande, Ramon S; Venson, Ramon; Veronezi, Carlos D; Moretti, Gustavo P; da Rocha, Edroaldo L; Cechinel, Cristian; Ceretta, Luciane B; Comunello, Eros; Martins, Paulo J; Casagrande, Rogério A; Snoeyer, Maria L; Manenti, Sandra A

    2014-01-01

    OBJECTIVE To explore the advantages of using artificial neural networks (ANNs) to recognize patterns in colposcopy to classify images in colposcopy. PURPOSE Transversal, descriptive, and analytical study of a quantitative approach with an emphasis on diagnosis. The training test e validation set was composed of images collected from patients who underwent colposcopy. These images were provided by a gynecology clinic located in the city of Criciúma (Brazil). The image database (n = 170) was divided; 48 images were used for the training process, 58 images were used for the tests, and 64 images were used for the validation. A hybrid neural network based on Kohonen self-organizing maps and multilayer perceptron (MLP) networks was used. RESULTS After 126 cycles, the validation was performed. The best results reached an accuracy of 72.15%, a sensibility of 69.78%, and a specificity of 68%. CONCLUSION Although the preliminary results still exhibit an average efficiency, the present approach is an innovative and promising technique that should be deeply explored in the context of the present study. PMID:25374454

  5. 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

  6. Methods for identification of images acquired with digital cameras

    NASA Astrophysics Data System (ADS)

    Geradts, Zeno J.; Bijhold, Jurrien; Kieft, Martijn; Kurosawa, Kenji; Kuroki, Kenro; Saitoh, Naoki

    2001-02-01

    From the court we were asked whether it is possible to determine if an image has been made with a specific digital camera. This question has to be answered in child pornography cases, where evidence is needed that a certain picture has been made with a specific camera. We have looked into different methods of examining the cameras to determine if a specific image has been made with a camera: defects in CCDs, file formats that are used, noise introduced by the pixel arrays and watermarking in images used by the camera manufacturer.

  7. 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

  8. 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.

  9. MACROscopic imaging of tumor xenografts using fluorescence, phase contrast, and transmitted light

    NASA Astrophysics Data System (ADS)

    Constantinou, Paul; Nicklee, Trudey; Hedley, David W.; Wilson, Brian C.; Damaskinos, Savvas

    2004-10-01

    Recent advances in imaging technology have contributed greatly to biological science. Confocal fluorescence microscopes (CFM) can acquire 2D and 3D images of biological samples such as live or fixed cells and tissues. Specimens that are large (e.g., a 10 mm x 10 mm tissue section) and overfill the field of view (FOV) of typical microscope objectives require use of image tiling to cover the entire specimen. This can be time consuming and cause artefacts in the composite image. The MACROscope system (Biomedical Photometrics Inc, Waterloo, Canada), is a confocal device with a 22 mm x 70 mm FOV; ideal for imaging large tissue sections in a single frame. The system used here is a prototype capable of simultaneous acquisition from up to three detection channels. Fluorescence images of SiHa mouse tumour xenografts stained with CD31-Cy3, showing blood vessel location, and EF5-Cy5, showing areas of tissue hypoxia, were collected. Differential phase contrast (DPC) images of the same section were also recorded to show tissue morphology. Finally, RGB transmitted light images of human tongue and pancreas tissues were obtained. This new device avoids the need for image tiling and provides simultaneous imaging of multiple fluorescently-labeled tissue specific markers in large biological samples. This enables time- and cost-efficient high-throughput screening of (immuno)histopathological samples. This device may also serve in the imaging of high-throughput DNA and tissue arrays.

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

    PubMed

    Kawata, Yoshimasa; Nawa, Yasunori; Inami, Wataru

    2014-11-01

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

  11. 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

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

    PubMed

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

    2013-05-01

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

  13. 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.

  14. Imaging Septum Formation by Fluorescence Microscopy.

    PubMed

    Ribas, Juan Carlos; Cortés, Juan Carlos G

    2016-01-01

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

  15. Digital fluorescent imaging system for quantitative analysis of facial sebum production

    NASA Astrophysics Data System (ADS)

    Han, Byungkwan; Jung, Byungjo; Choi, Eung-Ho; Nelson, J. Stuart

    2007-02-01

    Current methods for analysis of sebum excretion have limitations, such as irreproducible results in repeatable measurements due to the point measurement method, user-dependent artifact due to contact measurement or qualitative evaluation of the image, and long measurement time. A UV-induced fluorescent digital imaging system was developed to acquire facial images so that the distribution of sebum excretion on the face could be analyzed. The imaging system consisted of a constant UV-A light source, digital color camera, and head-positioning device. We describe the system characterization for acquisition of a fluorescent facial image and the image analysis method. The imaging modality provided uniform light distribution on the facial mannequin model and presented a discernible color fluorescent image. Valuable parameters of sebum excretion were obtained after image analysis. The imaging system, which provides a non-contact method, was proven to be a useful tool to evaluate sebum excretion and to characterize the pattern of sebum excretion. When compared to conventional "Wood's lamp" and "Sebutape" methods that provide similar parameters for sebum excretion, the method described herein is simpler and more reliable to evaluate the dynamics of sebum excretion in nearly real-time.

  16. Analysis of facial sebum distribution using a digital fluorescent imaging system.

    PubMed

    Han, Byungkwan; Jung, Byungjo; Nelson, J Stuart; Choi, Eung-Ho

    2007-01-01

    Current methods for analysis of sebum excretion have limitations, such as irreproducible results in repeatable measurements due to the point measurement method, user-dependent artifacts due to contact measurement or qualitative evaluation of the image, and long measurement time. A UV-induced fluorescent digital imaging system is developed to acquire facial images so that the distribution of sebum excretion on the face could be analyzed. The imaging system consists of a constant UV-A light source, digital color camera, and head-positioning device. The system for acquisition of a fluorescent facial image and the image analysis method is described. The imaging modality provides uniform light distribution and presents a discernible color fluorescent image. Valuable parameters of sebum excretion are obtained after image analysis. The imaging system, which provides a noncontact method, is proved to be a useful tool to evaluate the amount and pattern of sebum excretion. When compared to conventional "Wood's lamp" and "Sebutape" methods that provide similar parameters for sebum excretion, the described method is simpler and more reliable to evaluate the dynamics of sebum excretion in nearly real-time. PMID:17343481

  17. 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.

  18. Endoscopy imaging of 5-ALA-induced PPIX fluorescence for detecting early neoplasms in the oral cavity

    NASA Astrophysics Data System (ADS)

    Zheng, Wei; Olivo, Malini; Sivanandan, Ranjiv; Karuman, Philip; Lim, Tuan-Kay; Soo, K. C.

    2001-10-01

    A digitized fluorescence endoscopy imaging system combined with 5-Aminolevulinic Acid (5-ALA) induced Protoporphyrin IX (PPIX) has been developed for the detection of neoplasms in oral cavity. It mainly consists of the illumination console, fluorescence detection unit, computer system for image acquisition, processing and analysis, and online image display system as well. The developed system can produce both the digital and video fluorescence images in real time, and can be used to quantify fluorescence images acquired. Preliminary results from the Head and Neck clinic show that high sensitivity and high specificity can be achieved. Furthermore, applying the intensity ratios at two different wavelength regions, the developed system shows the capability of differentiating between different histopathological stages of oral lesions, suggesting a significant potential for realizing the non-invasive optical biopsy for early cancer diagnosis.

  19. Fluorescence imaging of historical buildings by lidar remote sensing

    NASA Astrophysics Data System (ADS)

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

    1998-10-01

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

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

    PubMed

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

    2015-01-01

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

  1. 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

  2. Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging.

    PubMed

    Kumar, S; Dunsby, C; De Beule, P A A; Owen, D M; Anand, U; Lanigan, P M P; Benninger, R K P; Davis, D M; Neil, M A A; Anand, P; Benham, C; Naylor, A; French, P M W

    2007-10-01

    We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor. PMID:19550524

  3. 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

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

    PubMed

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

    2016-01-01

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

  5. RF Device for Acquiring Images of the Human Body

    NASA Technical Reports Server (NTRS)

    Gaier, Todd C.; McGrath, William R.

    2010-01-01

    A safe, non-invasive method for forming images through clothing of large groups of people, in order to search for concealed weapons either made of metal or not, has been developed. A millimeter wavelength scanner designed in a unique, ring-shaped configuration can obtain a full 360 image of the body with a resolution of less than a millimeter in only a few seconds. Millimeter waves readily penetrate normal clothing, but are highly reflected by the human body and concealed objects. Millimeter wave signals are nonionizing and are harmless to human tissues when used at low power levels. The imager (see figure) consists of a thin base that supports a small-diameter vertical post about 7 ft (=2.13 m) tall. Attached to the post is a square-shaped ring 2 in. (=5 cm) wide and 3 ft (=91 cm) on a side. The ring is oriented horizontally, and is supported halfway along one side by a connection to a linear bearing on the vertical post. A planar RF circuit board is mounted to the inside of each side of the ring. Each circuit board contains an array of 30 receivers, one transmitter, and digitization electronics. Each array element has a printed-circuit patch antenna coupled to a pair of mixers by a 90 coupler. The mixers receive a reference local oscillator signal to a subharmonic of the transmitter frequency. A single local oscillator line feeds all 30 receivers on the board. The resulting MHz IF signals are amplified and carried to the edge of the board where they are demodulated and digitized. The transmitted signal is derived from the local oscillator at a frequency offset determined by a crystal oscillator. One antenna centrally located on each side of the square ring provides the source illumination power. The total transmitted power is less than 100 mW, resulting in an exposure level that is completely safe to humans. The output signals from all four circuit boards are fed via serial connection to a data processing computer. The computer processes the approximately 1-MB

  6. Detecting thermal phase transitions in corneal stroma by fluorescence micro-imaging analysis

    NASA Astrophysics Data System (ADS)

    Matteini, P.; Rossi, F.; Ratto, F.; Bruno, I.; Nesi, P.; Pini, R.

    2008-02-01

    Thermal modifications induced in corneal stroma were investigated by the use of fluorescence microscopy. Freshly extracted porcine corneas were immersed for 5 minutes in a water bath at temperatures in the 35-90°C range and stored in formalin. The samples were then sliced in 200-μm-thick transversal sections and analyzed under a stereomicroscope to assess corneal shrinkage. Fluorescence images of the thermally treated corneal samples were acquired using a slow-scan cooled CCD camera, after staining the slices with Indocyanine Green (ICG) fluorescent dye which allowed to detect fluorescence signal from the whole tissue. All measurements were performed using an inverted epifluorescence microscope equipped with a mercury lamp. The thermally-induced modifications to the corneal specimens were evaluated by studying the grey level distribution in the fluorescence images. For each acquired image, Discrete Fourier Transform (DFT) and entropy analyses were performed. The spatial distribution of DFT absolute value indicated the spatial orientation of the lamellar planes, while entropy was used to study the image texture, correlated to the stromal structural transitions. As a result, it was possible to indicate a temperature threshold value (62°C) for high thermal damage, resulting in a disorganization of the lamellar planes and in full agreement with the measured temperature for corneal shrinkage onset. Analysis of the image entropy evidenced five strong modifications in stromal architecture at temperatures of ~45°C, 53°C, 57°C, 66°C, 75°C. The proposed procedure proved to be an effective micro-imaging method capable of detecting subtle changes in corneal tissue subjected to thermal treatment.

  7. 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.

  8. 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.

  9. Cell imaging by transient fluorescence detected infrared microscopy

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  10. 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

  11. 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

  12. 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.

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  16. 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

  17. Ion beam induced fluorescence imaging in biological systems

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  18. MULTISPECTRAL LINE-SCAN IMAGING SYSTEM FOR SIMULTANEOUS FLUORESCENCE AND REFLECTANCE MEASUREMENTS OF APPLES: MULTITASK APPLE INSPECTION SYSTEM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this methodology paper, a recently developed line-scan imaging system, capable of simultaneously acquiring a combination of multispectral reflectance and fluorescence from fast moving objects, is presented. The system can potentially provide multitask inspections for quality and safety attribute...

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

    PubMed

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

    2010-10-11

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

  20. 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

  1. 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

  2. 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

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

    PubMed Central

    Zhu, Hongying; Ozcan, Aydogan

    2013-01-01

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

  4. Fluorescence Linear Dichroism Imaging for Quantifying Membrane Order

    PubMed Central

    Benninger, Richard K.P.

    2014-01-01

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

  5. Imaging of community-acquired pneumonia: Roles of imaging examinations, imaging diagnosis of specific pathogens and discrimination from noninfectious diseases

    PubMed Central

    Nambu, Atsushi; Ozawa, Katsura; Kobayashi, Noriko; Tago, Masao

    2014-01-01

    This article reviews roles of imaging examinations in the management of community-acquired pneumonia (CAP), imaging diagnosis of specific CAP and discrimination between CAP and noninfectious diseases. Chest radiography is usually enough to confirm the diagnosis of CAP, whereas computed tomography is required to suggest specific pathogens and to discriminate from noninfectious diseases. Mycoplasma pneumoniae pneumonia, tuberculosis, Pneumocystis jirovecii pneumonia and some cases of viral pneumonia sometimes show specific imaging findings. Peribronchial nodules, especially tree-in-bud appearance, are fairly specific for infection. Evidences of organization, such as concavity of the opacities, traction bronchiectasis, visualization of air bronchograms over the entire length of the bronchi, or mild parenchymal distortion are suggestive of organizing pneumonia. We will introduce tips to effectively make use of imaging examinations in the management of CAP. PMID:25349662

  6. Coherence gated wavefront sensorless adaptive optics for two photon excited fluorescence retinal imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Jian, Yifan; Cua, Michelle; Bonora, Stefano; Pugh, Edward N.; Zawadzki, Robert J.; Sarunic, Marinko V.

    2016-03-01

    We present a novel system for adaptive optics two photon imaging. We utilize the bandwidth of the femtosecond excitation beam to perform coherence gated imaging (OCT) of the sample. The location of the focus is directly observable in the cross sectional OCT images, and adjusted to the desired depth plane. Next, using real time volumetric OCT, we perform Wavefront Sensorless Adaptive Optics (WSAO) aberration correction using a multi-element adaptive lens capable of correcting up to 4th order Zernike polynomials. The aberration correction is performed based on an image quality metric, for example intensity. The optimization time is limited only by the OCT acquisition rate, and takes ~30s. Following aberration correction, two photon fluorescence images are acquired, and compared to results without adaptive optics correction. This technique is promising for multiphoton imaging in multi-layered, scattering samples such as eye and brain, in which traditional wavefront sensing and guide-star sensorless adaptive optics approaches may not be suitable.

  7. Imaging of whole tumor cut sections using a novel scanning beam confocal fluorescence MACROscope

    NASA Astrophysics Data System (ADS)

    Constantinou, Paul; Vukovic, Vojislav; Haugland, Hans K.; Nicklee, Trudey; Hedley, David W.; Wilson, Brian C.

    2001-07-01

    Hypoxia caused by inadequate structure and function of the tumor vasculature has been found to negatively determine the prognosis of cancer patients. Hence, understanding the biological basis of tumor hypoxia is of significant clinical interest. To study solid tumor microenvironments in sufficient detail, large areas (several mm in diameter) need to be imaged at micrometers resolutions. We have used a novel confocal scanning laser MACROscopeTM (CSLM) capable of acquiring images over fields of view up to 2 cm X 2 cm. To demonstrate its performance, frozen sections from a cervical carcinoma xenograft were triple labeled for tissue hypoxia, blood vessels and hypoxia-inducible transcription factor 1 alpha (HIF-1(alpha) ), imaged using the CSLM and compared to images obtained using a standard epifluorescence microscope imaging system. The results indicate that the CSLM is a useful instrument for imaging tissue-based fluorescence at resolutions comparable to standard low-power microscope objectives.

  8. Biological applications of fluorescence lifetime imaging beyond microscopy

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

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

  9. Hounsfield unit recovery in clinical cone beam CT images of the thorax acquired for image guided radiation therapy.

    PubMed

    Thing, Rune Slot; Bernchou, Uffe; Mainegra-Hing, Ernesto; Hansen, Olfred; Brink, Carsten

    2016-08-01

    A comprehensive artefact correction method for clinical cone beam CT (CBCT) images acquired for image guided radiation therapy (IGRT) on a commercial system is presented. The method is demonstrated to reduce artefacts and recover CT-like Hounsfield units (HU) in reconstructed CBCT images of five lung cancer patients. Projection image based artefact corrections of image lag, detector scatter, body scatter and beam hardening are described and applied to CBCT images of five lung cancer patients. Image quality is evaluated through visual appearance of the reconstructed images, HU-correspondence with the planning CT images, and total volume HU error. Artefacts are reduced and CT-like HUs are recovered in the artefact corrected CBCT images. Visual inspection confirms that artefacts are indeed suppressed by the proposed method, and the HU root mean square difference between reconstructed CBCTs and the reference CT images are reduced by 31% when using the artefact corrections compared to the standard clinical CBCT reconstruction. A versatile artefact correction method for clinical CBCT images acquired for IGRT has been developed. HU values are recovered in the corrected CBCT images. The proposed method relies on post processing of clinical projection images, and does not require patient specific optimisation. It is thus a powerful tool for image quality improvement of large numbers of CBCT images. PMID:27405692

  10. Hounsfield unit recovery in clinical cone beam CT images of the thorax acquired for image guided radiation therapy

    NASA Astrophysics Data System (ADS)

    Slot Thing, Rune; Bernchou, Uffe; Mainegra-Hing, Ernesto; Hansen, Olfred; Brink, Carsten

    2016-08-01

    A comprehensive artefact correction method for clinical cone beam CT (CBCT) images acquired for image guided radiation therapy (IGRT) on a commercial system is presented. The method is demonstrated to reduce artefacts and recover CT-like Hounsfield units (HU) in reconstructed CBCT images of five lung cancer patients. Projection image based artefact corrections of image lag, detector scatter, body scatter and beam hardening are described and applied to CBCT images of five lung cancer patients. Image quality is evaluated through visual appearance of the reconstructed images, HU-correspondence with the planning CT images, and total volume HU error. Artefacts are reduced and CT-like HUs are recovered in the artefact corrected CBCT images. Visual inspection confirms that artefacts are indeed suppressed by the proposed method, and the HU root mean square difference between reconstructed CBCTs and the reference CT images are reduced by 31% when using the artefact corrections compared to the standard clinical CBCT reconstruction. A versatile artefact correction method for clinical CBCT images acquired for IGRT has been developed. HU values are recovered in the corrected CBCT images. The proposed method relies on post processing of clinical projection images, and does not require patient specific optimisation. It is thus a powerful tool for image quality improvement of large numbers of CBCT images.

  11. 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.

  12. 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.

  13. Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

    PubMed Central

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

    2016-01-01

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

  14. 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.

  15. Optical imaging-guided cancer therapy with fluorescent nanoparticles

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2000-12-01

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

  17. Cistanches identification based on fluorescent spectral imaging technology combined with principal component analysis and artificial neural network

    NASA Astrophysics Data System (ADS)

    Dong, Jia; Huang, Furong; Li, Yuanpeng; Xiao, Chi; Xian, Ruiyi; Ma, Zhiguo

    2015-03-01

    In this study, fluorescent spectral imaging technology combined with principal component analysis (PCA) and artificial neural networks (ANNs) was used to identify Cistanche deserticola, Cistanche tubulosa and Cistanche sinensis, which are traditional Chinese medicinal herbs. The fluorescence spectroscopy imaging system acquired the spectral images of 40 cistanche samples, and through image denoising, binarization processing to make sure the effective pixels. Furthermore, drew the spectral curves whose data in the wavelength range of 450-680 nm for the study. Then preprocessed the data by first-order derivative, analyzed the data through principal component analysis and artificial neural network. The results shows: Principal component analysis can generally distinguish cistanches, through further identification by neural networks makes the results more accurate, the correct rate of the testing and training sets is as high as 100%. Based on the fluorescence spectral imaging technique and combined with principal component analysis and artificial neural network to identify cistanches is feasible.

  18. Hyperspectral reflectance and fluorescence line-scan imaging system for online detection of fecal contamination on apples

    NASA Astrophysics Data System (ADS)

    Kim, Moon S.; Cho, Byoung-Kwan; Yang, Chun-Chieh; Chao, Kaunglin; Lefcourt, Alan M.; Chen, Yud-Ren

    2006-10-01

    We have developed nondestructive opto-electronic imaging techniques for rapid assessment of safety and wholesomeness of foods. A recently developed fast hyperspectral line-scan imaging system integrated with a commercial apple-sorting machine was evaluated for rapid detection of animal feces matter on apples. Apples obtained from a local orchard were artificially contaminated with cow feces. For the online trial, hyperspectral images with 60 spectral channels, reflectance in the visible to near infrared regions and fluorescence emissions with UV-A excitation, were acquired from apples moving at a processing sorting-line speed of three apples per second. Reflectance and fluorescence imaging required a passive light source, and each method used independent continuous wave (CW) light sources. In this paper, integration of the hyperspectral imaging system with the commercial applesorting machine and preliminary results for detection of fecal contamination on apples, mainly based on the fluorescence method, are presented.

  19. Photoacoustic imaging of a near-infrared fluorescent marker based on dual wavelength pump-probe excitation

    NASA Astrophysics Data System (ADS)

    Märk, Julia; Theiss, Christoph; Schmitt, Franz-Josef; Laufer, Jan

    2014-03-01

    Photoacoustic imaging has been used to determine the spatial distribution of fluorophores, such as exogenous dyes and genetically expressed proteins, from images acquired in phantoms and in vivo. Most methods involve the acquisition of multiwavelength images and rely on differences in the absorption spectra of the tissue chromophores to estimate the spatial distribution and abundance of the latter using spectral decomposition techniques, such as model based inversion schemes. However, the inversion of 3-D images can be computationally expensive. Experimental approaches to localising contrast agents may therefore be useful, especially if quantification is not essential. This work aims to develop a method for determining the spatial distribution of a near-infrared fluorescent cell marker from images acquired using dual wavelength excitation. The excitation wavelengths coincided with the absorption and emission spectrum of the fluorophore. The contrast mechanism relies on reducing the excited state lifetime of the fluorophore by inducing stimulated emission. This changes the amount of energy thermalized by the fluorophore, and hence the photoacoustic signal amplitude. Since this is not observed in endogenous chromophores, the background may be removed by subtracting two images acquired with and without pulse delay between the pump and probe pulses. To characterise the fluorophore, the signal amplitude is measured in a cuvette as a function of pulse delay, concentration, and fluence. The spatial distribution of the fluorophore is determined from images acquired in realistic tissue phantoms. This method may be suitable for in vivo applications, such as imaging of exogenous or genetically expressed fluorescent cell markers.

  20. Portable hyperspectral fluorescence imaging system for detection of biofilms on stainless steel surfaces

    NASA Astrophysics Data System (ADS)

    Jun, Won; Lee, Kangjin; Millner, Patricia; Sharma, Manan; Chao, Kuanglin; Kim, Moon S.

    2008-04-01

    A rapid nondestructive technology is needed to detect bacterial contamination on the surfaces of food processing equipment to reduce public health risks. A portable hyperspectral fluorescence imaging system was used to evaluate potential detection of microbial biofilm on stainless steel typically used in the manufacture of food processing equipment. Stainless steel coupons were immersed in bacterium cultures, such as E. coli, Pseudomonas pertucinogena, Erwinia chrysanthemi, and Listeria innocula. Following a 1-week exposure, biofilm formations were assessed using fluorescence imaging. In addition, the effects on biofilm formation from both tryptic soy broth (TSB) and M9 medium with casamino acids (M9C) were examined. TSB grown cells enhance biofilm production compared with M9C-grown cells. Hyperspectral fluorescence images of the biofilm samples, in response to ultraviolet-A (320 to 400 nm) excitation, were acquired from approximately 416 to 700 nm. Visual evaluation of individual images at emission peak wavelengths in the blue revealed the most contrast between biofilms and stainless steel coupons. Two-band ratios compared with the single-band images increased the contrast between the biofilm forming area and stainless steel coupon surfaces. The 444/588 nm ratio images exhibited the greatest contrast between the biofilm formations and stainless coupon surfaces.

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

    PubMed

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

    2014-06-01

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

  2. Examining Adsorbed Polymer Conformations with Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  3. Classification of corn kernels contaminated with aflatoxins using fluorescence and reflectance hyperspectral images analysis

    NASA Astrophysics Data System (ADS)

    Zhu, Fengle; Yao, Haibo; Hruska, Zuzana; Kincaid, Russell; Brown, Robert; Bhatnagar, Deepak; Cleveland, Thomas

    2015-05-01

    Aflatoxins are secondary metabolites produced by certain fungal species of the Aspergillus genus. Aflatoxin contamination remains a problem in agricultural products due to its toxic and carcinogenic properties. Conventional chemical methods for aflatoxin detection are time-consuming and destructive. This study employed fluorescence and reflectance visible near-infrared (VNIR) hyperspectral images to classify aflatoxin contaminated corn kernels rapidly and non-destructively. Corn ears were artificially inoculated in the field with toxigenic A. flavus spores at the early dough stage of kernel development. After harvest, a total of 300 kernels were collected from the inoculated ears. Fluorescence hyperspectral imagery with UV excitation and reflectance hyperspectral imagery with halogen illumination were acquired on both endosperm and germ sides of kernels. All kernels were then subjected to chemical analysis individually to determine aflatoxin concentrations. A region of interest (ROI) was created for each kernel to extract averaged spectra. Compared with healthy kernels, fluorescence spectral peaks for contaminated kernels shifted to longer wavelengths with lower intensity, and reflectance values for contaminated kernels were lower with a different spectral shape in 700-800 nm region. Principal component analysis was applied for data compression before classifying kernels into contaminated and healthy based on a 20 ppb threshold utilizing the K-nearest neighbors algorithm. The best overall accuracy achieved was 92.67% for germ side in the fluorescence data analysis. The germ side generally performed better than endosperm side. Fluorescence and reflectance image data achieved similar accuracy.

  4. Multi-excitation near infrared (NIR) spectral fluorescence imaging using organic fluorophores

    NASA Astrophysics Data System (ADS)

    Kobayashi, Hisataka; Koyama, Yoshinori; Barrett, Tristan; Hama, Yukihiro; Choyke, Peter L.

    2008-02-01

    The ability to obtain multi-color fluorescent imaging in vivo simultaneously using multi-targeted imaging probes could be of potential benefit from both a research and a clinical perspective. However, the simultaneous acquisition of more than 2 separate organic fluorophores usually requires more than one excitation source, since a single excitation source may not optimally excite all the fluorophores. In this study, we employed a multi-excitation approach in order to acquire optimized images with multiple near infrared (NIR) organic fluorophores at the same time. Using 3 sets of excitation filters (595+/-20nm, 640+/-25nm, 688+/-17nm) to acquire 3 distinct spectra and spectral unmixing software (CRi, Woburn, MA), it was possible to resolve the emission spectra of each of the NIR fluorophores using commercial software (Nuance, CRi, Woburn, MA) To demonstrate the utility of this approach 2 mouse models were investigated; In one model, mice bearing four implanted malignancies were injected with a cocktail of 3 fluorescently labeled monoclonal antibodies, each with its own distinct NIR fluorophore. In the second model five different lymph node drainage basins were imaged with 5-color dendrimer-based lymphatic imaging agents tagged with 5 different NIR fluorophores. We successfully detected each of the targeted tumors in the first model and all of the lymph nodes by their distinct color in the second model; neither of which would have been possible using the single excitation method. In conclusion, multi-excitation NIR spectral fluorescence imaging is feasible in a reasonable time frame and opens the possibility for in vivo immunohistochemical imaging (IHCi).

  5. 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.

  6. Exploiting Molecular Biology by Time-Resolved Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    Müller, Francis; Fattinger, Christof

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

  7. 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

  8. Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

    PubMed Central

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-01-01

    We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. PMID:24921725

  9. 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

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

    PubMed

    Raymo, Françisco M

    2012-09-01

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

  11. 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.

  12. 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

  13. 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.

  14. Maia X-ray fluorescence imaging: Capturing detail in complex natural samples

    NASA Astrophysics Data System (ADS)

    Ryan, C. G.; Siddons, D. P.; Kirkham, R.; Li, Z. Y.; de Jonge, M. D.; Paterson, D. J.; Kuczewski, A.; Howard, D. L.; Dunn, P. A.; Falkenberg, G.; Boesenberg, U.; De Geronimo, G.; Fisher, L. A.; Halfpenny, A.; Lintern, M. J.; Lombi, E.; Dyl, K. A.; Jensen, M.; Moorhead, G. F.; Cleverley, J. S.; Hough, R. M.; Godel, B.; Barnes, S. J.; James, S. A.; Spiers, K. M.; Alfeld, M.; Wellenreuther, G.; Vukmanovic, Z.; Borg, S.

    2014-04-01

    Motivated by the challenge of capturing complex hierarchical chemical detail in natural material from a wide range of applications, the Maia detector array and integrated realtime processor have been developed to acquire X-ray fluorescence images using X-ray Fluorescence Microscopy (XFM). Maia has been deployed initially at the XFM beamline at the Australian Synchrotron and more recently, demonstrating improvements in energy resolution, at the P06 beamline at Petra III in Germany. Maia captures fine detail in element images beyond 100 M pixels. It combines a large solid-angle annular energy-dispersive 384 detector array, stage encoder and flux counter inputs and dedicated FPGA-based real-time event processor with embedded spectral deconvolution. This enables high definition imaging and enhanced trace element sensitivity to capture complex trace element textures and place them in a detailed spatial context. Maia hardware and software methods provide per pixel correction for dwell, beam flux variation, dead-time and pileup, as well as off-line parallel processing for enhanced throughput. Methods have been developed for real-time display of deconvoluted SXRF element images, depth mapping of rare particles and the acquisition of 3D datasets for fluorescence tomography and XANES imaging using a spectral deconvolution method that tracks beam energy variation.

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

    PubMed

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

    2012-07-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  17. 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.

  18. Precision Agriculture: Using Low-Cost Systems to Acquire Low-Altitude Images.

    PubMed

    Ponti, Moacir; Chaves, Arthur A; Jorge, Fabio R; Costa, Gabriel B P; Colturato, Adimara; Branco, Kalinka R L J C

    2016-01-01

    Low cost remote sensing imagery has the potential to make precision farming feasible in developing countries. In this article, the authors describe image acquisition from eucalyptus, bean, and sugarcane crops acquired by low-cost and low-altitude systems. They use different approaches to handle low-altitude images in both the RGB and NIR (near-infrared) bands to estimate and quantify plantation areas. PMID:27514030

  19. Fluorescence molecular imaging system with a novel mouse surface extraction method and a rotary scanning scheme

    NASA Astrophysics Data System (ADS)

    Zhao, Yue; Zhu, Dianwen; Baikejiang, Reheman; Li, Changqing

    2015-03-01

    We have developed a new fluorescence molecular tomography (FMT) imaging system, in which we utilized a phase shifting method to extract the mouse surface geometry optically and a rotary laser scanning approach to excite fluorescence molecules and acquire fluorescent measurements on the whole mouse body. Nine fringe patterns with a phase shifting of 2π/9 are projected onto the mouse surface by a projector. The fringe patterns are captured using a webcam to calculate a phase map that is converted to the geometry of the mouse surface with our algorithms. We used a DigiWarp approach to warp a finite element mesh of a standard digital mouse to the measured mouse surface thus the tedious and time-consuming procedure from a point cloud to mesh is avoided. Experimental results indicated that the proposed method is accurate with errors less than 0.5 mm. In the FMT imaging system, the mouse is placed inside a conical mirror and scanned with a line pattern laser that is mounted on a rotation stage. After being reflected by the conical mirror, the emitted fluorescence photons travel through central hole of the rotation stage and the band pass filters in a motorized filter wheel, and are collected by a CCD camera. Phantom experimental results of the proposed new FMT imaging system can reconstruct the target accurately.

  20. Multitemporal burnt area detection methods based on a couple of images acquired after the fire event

    NASA Astrophysics Data System (ADS)

    Carlà, R.; Santurri, L.; Bonora, L.; Conese, C.

    2009-09-01

    Fire detection methods based on remote sensing data are gaining more and more attention among the scientific community, and many algorithms have been developed for this purpose. In order to assess the location and the characteristics of burned areas, some of them apply a suitable threshold to a multispectral index such as the NBR (Noise Burn Ratio) index or the NDII (Normalized Difference Infrared Index) evaluated on a single image acquired after the fire season. Other methods use a multitemporal approach based on the processing of a couple of images, the former acquired before and the latter after the fire season, and applying a chosen threshold to the differential value of the same, or other multispectral indexes. This paper focuses the problem of assessing the performance of some burnt areas detection methods based on a couple of satellite images acquired both after the fire season. In particular the threshold method applied to the differential form of the NDII and NDVI (Normalized Differential Vegetation Index) are considered as concern their capacity of locating or detecting (not characterizing) burnt areas and the resulting performances are evaluated and compared with the corresponding ones of the same methods applied to a single image only, acquired after the fire season.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Photon budget analysis for fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

  6. Multilayer fluorescence imaging on a single-pixel detector

    PubMed Central

    Guo, Kaikai; Jiang, Shaowei; Zheng, Guoan

    2016-01-01

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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. Optofluidic Fluorescent Imaging Cytometry on a Cell Phone

    PubMed Central

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

    2012-01-01

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

  12. Towards Whole-Body Fluorescence Imaging in Humans

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-08-10

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

  14. Diffuse reflectance and fluorescence multispectral imaging system for assessment of skin

    NASA Astrophysics Data System (ADS)

    Saknite, Inga; Jakovels, Dainis; Spigulis, Janis

    2014-05-01

    The diffuse reflectance multispectral imaging technique has been used for distant mapping of in vivo skin chromophores (hemoglobin and melanin). The fluorescence multispectral imaging is not so common for skin applications due to complicity of data acquisition and processing, but could provide additional information about skin fluorophores. Both techniques are compatible, and could be combined into a multimodal solution. The multispectral imaging system Nuance based on liquid crystal tunable filters was adapted for diffuse reflectance and fluorescence spectral imaging of in vivo skin. Uniform illumination was achieved by LED ring light. Combination of four LEDs (warm white, 770 nm, 830 nm and 890 nm) was used to support diffuse reflectance mode in spectral range 450-950 nm. 405 nm LEDs were used for excitation of skin autofluorescence. Multispectral imaging system was adapted for spectral working range of 450-950 nm with scanning step of 10 nm and spectral resolution of 15 nm. An average field of view was 50x35 mm in size with spatial resolution 0,05 mm (the pixel size). Due to spectrally different illumination intensity and system sensitivity, various exposure times (from 7…500 ms) were used for each image acquisition. The proposed approach was tested for different skin lesions: benign nevus, hemangioma, basalioma and halo nevus. Spectral image cubes of different skin lesions were acquired and analyzed to test its diagnostic potential.

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

    PubMed

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

    2016-05-01

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

  16. 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.

  17. 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.

  18. 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.

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

    PubMed Central

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

    2015-01-01

    Context The use of fluorescent proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various fluorescent proteins. Objective To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. Design Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). Fluorescence imaging was performed under the cyan fluorescent protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA). Intervention All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and H&E staining. Fluorescence microscopy was performed with an Olympus IMT-2 inverted fluorescence microscope. Main outcome measure Determination of fluorescence intensity of different organs. Results Surprisingly, we found that there is differential enhancement of fluorescence among organs; most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the cyan fluorescent protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. Conclusions The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer. PMID:19287108

  20. Dual modality of non-contact photoacoustic tomography and fluorescence imaging using double cladding fiber

    NASA Astrophysics Data System (ADS)

    Eom, Jonghyun; Park, Seong Jun; Kim, Ju Wan; Park, Soongho; Lee, Byeong Ha

    2015-03-01

    We present a fiber-based dual-modal imaging system that combines non-contact photoacoustic tomography (NCPAT) and fluorescence imaging by using double cladding fiber (DCF). The NCPAT system utilizing an all-fiber heterodyne interferometer as an ultrasound detector measures the photoacoustic signal at the sample surface without physical contact. Fluorescence imaging system is composed of fiber-optics to deliver the excitation light and the emission light. For combined system the probe consists of a specially fabricated DCF coupler and a lensed fiber so that we can simultaneously acquire the signals of two systems with the same probe. The DCF has a core and two claddings, inner and outer, which allows two concentric light-guiding channels via the core and the inner cladding. The lensed fiber of the DCF probe is compactly fabricated to focus the interferometer light and the excitation light, and to efficiently collect the fluorescence signal. To demonstrate the feasibility of the proposed dual-modal system, we have conducted phantom experiments using tissue mimicking phantoms which contained a couple of tubes filled with fluorescein solution and black ink, respectively. The proposed imaging system is implanted with fiber-optic configurations so that it has the potential for minimally invasive and improved diagnosis and guided treatment of diseases.

  1. Fluorescent Molecular Imaging and Dosimetry Tools in Photodynamic Therapy

    PubMed Central

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

    2013-01-01

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

  2. Fluorescence lifetime imaging of human skin and hair

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

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

    PubMed

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

    2013-09-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2009-06-10

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-06-01

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

  7. Fluorescence imaging of metal ions implicated in diseases.

    PubMed

    Qian, Xuhong; Xu, Zhaochao

    2015-07-21

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

  8. Optimized acquisition time for x-ray fluorescence imaging of gold nanoparticles: a preliminary study using photon counting detector

    NASA Astrophysics Data System (ADS)

    Ren, Liqiang; Wu, Di; Li, Yuhua; Chen, Wei R.; Zheng, Bin; Liu, Hong

    2016-03-01

    X-ray fluorescence (XRF) is a promising spectroscopic technique to characterize imaging contrast agents with high atomic numbers (Z) such as gold nanoparticles (GNPs) inside small objects. Its utilization for biomedical applications, however, is greatly limited to experimental research due to longer data acquisition time. The objectives of this study are to apply a photon counting detector array for XRF imaging and to determine an optimized XRF data acquisition time, at which the acquired XRF image is of acceptable quality to allow the maximum level of radiation dose reduction. A prototype laboratory XRF imaging configuration consisting of a pencil-beam X-ray and a photon counting detector array (1 × 64 pixels) is employed to acquire the XRF image through exciting the prepared GNP/water solutions. In order to analyze the signal to noise ratio (SNR) improvement versus the increased exposure time, all the XRF photons within the energy range of 63 - 76KeV that include two Kα gold fluorescence peaks are collected for 1s, 2s, 3s, and so on all the way up to 200s. The optimized XRF data acquisition time for imaging different GNP solutions is determined as the moment when the acquired XRF image just reaches a quality with a SNR of 20dB which corresponds to an acceptable image quality.

  9. Deep UV Native Fluorescence Imaging of Antarctic Cryptoendolithic Communities

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2012-01-01

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

  13. Path method for reconstructing images in fluorescence optical tomography

    SciTech Connect

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

    2006-11-30

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

  14. A Review of Indocyanine Green Fluorescent Imaging in Surgery

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  16. Ultra-high resolution color images of the surface of comet 67P acquired by ROLIS

    NASA Astrophysics Data System (ADS)

    Schröder, Stefan; Mottola, Stefano; Arnold, Gabriele; Grothues, Hans-Georg; Hamm, Maximilian; Jaumann, Ralf; Michaelis, Harald; Pelivan, Ivanka; Proffe, Gerrit; Bibring, Jean-Pierre

    2015-04-01

    On Nov 12, 2014, the Rosetta Philae lander descended towards comet 67P/Churyumov-Gerasimenko. The onboard ROLIS camera successfully acquired high resolution images of the surface looking down from its vantage point on the instrument platform. ROLIS is a compact CCD imager with a 1024×1024 pixel sensor and a 57° field of view (Mottola et al., 2007, SSR 128, 241). It is equipped with an infinity lens (IFL), without which the camera focus is 30 cm. At Philae's final landing site, ROLIS removed the IFL and initiated an imaging sequence that shows the surface at the highest resolution ever obtained for a cometary surface (~0.5 mm per pixel). Illumination of the scene was provided by an onboard array of LEDs in four different colors: red, green, blue, and near-IR. ROLIS acquired one image for each color and a single dark exposure. The images show a unique, almost fractal morphology for the surface below the landing site that defies easy interpretation. However, there are similarities with some structures seen by the CIVA camera. Color and albedo variations over the surface are minor, and individual grains cannot be distinguished. The images are out-of-focus, indicating the surface was further away than the nominal 30 cm. The location of the illumination spot and the change of focus over the image are consistent with an inclined surface, indicating that Philae's final resting position is strongly tilted. In fact, it was inclined so much that we see the local horizon, even though ROLIS is downward-looking. Remarkably, the scene beyond the horizon is illuminated by the Sun, and out-of-focus particles can be seen to travel in the sky. The images suggest the environment of the lander is laden with fine dust, but a final assessment requires careful consideration of possible sources of stray light. Just before Philae went to sleep, ROLIS acquired an additional exposure with the IFL and the red LED. The resulting image is fully in focus. Because Philae had rotated and lifted

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

    NASA Astrophysics Data System (ADS)

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

    2007-05-01

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

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

    PubMed

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

    2014-12-15

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

  19. 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

  20. Imaging protoporphyrin IX fluorescence with a time-domain FMT/microCT system

    NASA Astrophysics Data System (ADS)

    Leblond, Frederic; Kepshire, Dax; O'Hara, Julia A.; Dehghani, Hamid; Srinivasan, Subha; Mincu, N.; Hutchins, M.; Khayat, M.; Pogue, B. W.

    2009-02-01

    Fluorescence molecular tomography (FMT) has the potential to become a powerful quantitative research tool for pre-clinical applications such as evaluating the efficacy of experimental drugs. In this paper, we show how a time-domain FMT/microCT instrument can in principle be used to monitor volumetric fluorescence intensity over time for low fluorophore concentration levels. The experimental results we present relate to Protoporphyrin IX which has a quantum efficiency as much as two orders of magnitude lower compared to more conventional extrinsic dyes used for molecular imaging (e.g., Alexa Fluor dyes, Cyanine dyes). Our results highlight the high sensitivity of the single photon counting technology on which the optical system we have built is based. In conjunction with this system we have developed a diffuse optical fluorescence reconstruction technique that is robust and shown here to perform adequately even in cases when the contribution of noise to the data is important. Related to this, we show that the regularization scheme we have developed is reliable even for low fluorophore concentration values and that no adjustment of the regularization parameter needs to be made for different levels of noise. This generic reconstruction approach insures that images reconstructed from data sets acquired at different times and for different fluorescence levels can be compared on an equal footing.

  1. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images

    PubMed Central

    Afshar, Yaser; Sbalzarini, Ivo F.

    2016-01-01

    Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 1010 pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

  2. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

    PubMed

    Afshar, Yaser; Sbalzarini, Ivo F

    2016-01-01

    Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10) pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

  3. Improving the quality of radiographic images acquired with conical radiation beams through divergence correction and filtering

    NASA Astrophysics Data System (ADS)

    Silvani, M. I.; Almeida, G. L.; Latini, R. M.; Bellido, A. V. B.; Souza, E. S.; Lopes, R. T.

    2015-07-01

    Earlier works have shown the feasibility to correct the deformation of the attenuation map in radiographs acquired with conical radiation beams provided that the inspected object could be expressed into analytical geometry terms. This correction reduces the contribution of the main object in the radiograph, allowing thus the visualization of its otherwise concealed heterogeneities. However, the non-punctual character of the source demanded a cumbersome trial-and-error approach in order to determine the proper correction parameters for the algorithm. Within this frame, this work addresses the improvement of radiographs of specially tailored test-objects acquired with a conical beam through correction of its divergence by using the information contained in the image itself. The corrected images have afterwards undergone a filtration in the frequency domain aiming at the reduction of statistical fluctuation and noise by using a 2D Fourier transform. All radiographs have been acquired using 165Dy and 198Au gamma-ray sources produced at the Argonauta research reactor in Institutode Engenharia Nuclear - CNEN, and an X-ray sensitive imaging plate as detector. The processed images exhibit features otherwise invisible in the original ones. Their processing by conventional histogram equalization carried out for comparison purposes did not succeed to detect those features.

  4. Imaging the urokinase plasminongen activator receptor in preclinical breast cancer models of acquired drug resistance.

    PubMed

    LeBeau, Aaron M; Sevillano, Natalia; King, Mandy L; Duriseti, Sai; Murphy, Stephanie T; Craik, Charles S; Murphy, Laura L; VanBrocklin, Henry F

    2014-01-01

    Subtype-targeted therapies can have a dramatic impact on improving the quality and quantity of life for women suffering from breast cancer. Despite an initial therapeutic response, cancer recurrence and acquired drug-resistance are commonplace. Non-invasive imaging probes that identify drug-resistant lesions are urgently needed to aid in the development of novel drugs and the effective utilization of established therapies for breast cancer. The protease receptor urokinase plasminogen activator receptor (uPAR) is a target that can be exploited for non-invasive imaging. The expression of uPAR has been associated with phenotypically aggressive breast cancer and acquired drug-resistance. Acquired drug-resistance was modeled in cell lines from two different breast cancer subtypes, the uPAR negative luminal A subtype and the uPAR positive triple negative subtype cell line MDA-MB-231. MCF-7 cells, cultured to be resistant to tamoxifen (MCF-7 TamR), were found to significantly over-express uPAR compared to the parental cell line. uPAR expression was maintained when resistance was modeled in triple-negative breast cancer by generating doxorubicin and paclitaxel resistant MDA-MB-231 cells (MDA-MB-231 DoxR and MDA-MB-231 TaxR). Using the antagonistic uPAR antibody 2G10, uPAR was imaged in vivo by near-infrared (NIR) optical imaging and (111)In-single photon emission computed tomography (SPECT). Tumor uptake of the (111)In-SPECT probe was high in the three drug-resistant xenografts (> 46 %ID/g) and minimal in uPAR negative xenografts at 72 hours post-injection. This preclinical study demonstrates that uPAR can be targeted for imaging breast cancer models of acquired resistance leading to potential clinical applications. PMID:24505235

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

    PubMed Central

    Ninomiya, Youichirou; Zhao, Wei; Saga, Yumiko

    2016-01-01

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

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

    PubMed

    Ninomiya, Youichirou; Zhao, Wei; Saga, Yumiko

    2016-01-01

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

  7. An approach of DSM generation from multi-view images acquired by UAVs

    NASA Astrophysics Data System (ADS)

    Hu, DaTian; Ai, Mingyao; Hu, Qingwu; Li, Jiayuan

    2016-03-01

    In the past few years, for its lower-cost, safer and high-resolution images, unmanned aerial vehicles (UAVs) demonstrated great potential for photogrammetric measurements in numerous application fields. Nevertheless, these images are often affected by large rotation, big viewpoint change as well as small overlaps, in which case traditional procedure are not able to orientate images or generate reliable Digital Generation Models (DSM). This paper introduces the whole procedure of the DSM generation, which comprehensively utilizes advantage of both computer vision and multi-image matching algorithms in extracting points and generating a dense DSM. Experiment shows that, based on this procedure, it can quickly extract points from the high-resolution images acquired by UAVs with high location accuracy.

  8. 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.

  9. 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.

  10. 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

  11. Evaluation of methods for detection of fluorescence labeled subcellular objects in microscope images

    PubMed Central

    2010-01-01

    Background Several algorithms have been proposed for detecting fluorescently labeled subcellular objects in microscope images. Many of these algorithms have been designed for specific tasks and validated with limited image data. But despite the potential of using extensive comparisons between algorithms to provide useful information to guide method selection and thus more accurate results, relatively few studies have been performed. Results To better understand algorithm performance under different conditions, we have carried out a comparative study including eleven spot detection or segmentation algorithms from various application fields. We used microscope images from well plate experiments with a human osteosarcoma cell line and frames from image stacks of yeast cells in different focal planes. These experimentally derived images permit a comparison of method performance in realistic situations where the number of objects varies within image set. We also used simulated microscope images in order to compare the methods and validate them against a ground truth reference result. Our study finds major differences in the performance of different algorithms, in terms of both object counts and segmentation accuracies. Conclusions These results suggest that the selection of detection algorithms for image based screens should be done carefully and take into account different conditions, such as the possibility of acquiring empty images or images with very few spots. Our inclusion of methods that have not been used before in this context broadens the set of available detection methods and compares them against the current state-of-the-art methods for subcellular particle detection. PMID:20465797

  12. SU-E-J-186: Acquiring and Assessing Upright CBCT Images for Future Treatment Planning

    SciTech Connect

    Fave, X; Yang, J; Balter, P; Court, L

    2014-06-01

    Purpose: To acquire upright CBCT images using the onboard imager of a Varian TrueBeam. An easy to implement upright imaging protocol could allow for widespread upright radiation therapy which would greatly benefit certain patients. These include thoracic cancer patients (because lung volume increases in a seated position) and patients who experience substantial discomfort during supine treatment. Methods: To acquire upright CBCT images, the gantry head remained stationary at 0 degrees with the KV imager arms extended to their lateral positions. Phantoms were placed upright at the end of the treatment couch. During a scan, the couch rotated from 270 to 90 degrees while continuous fluoroscopic projections were taken by the onboard imager. To extend the field-of-view, this sequence was performed twice: once with the KV detector longitudinally offset +14.5cm and once with it longitudinally offset −14.5cm. The resulting two image sets were stitched together before reconstruction. The imaging beam parameters were chosen to deliver a dose similar to that given during a simulation CT. Image quality was evaluated for spatial linearity, high and low contrast resolution, and HU linearity using CatPhan and anthropomorphic phantoms. A deformable registration technique was used to evaluate HU mapping from a simulation CT. Results: Spatial linearity and high contrast resolution were maintained in upright CBCT when compared to simulation CT. However, low contrast resolution and HU linearity degraded. Streak artifacts were caused by the limited 180 degree arc of the couch, and the stitching process created a sharp artifact at the center of the reconstruction. The deformable registration was robust in the HU mapping even with these artifacts and the loss of HU linearity. Conclusions: The image quality obtained from upright CBCT was sufficient for treatment planning. The success of this novel technique is an important step towards a future clinical protocol. This project was funded

  13. 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.

  14. 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

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

    PubMed

    Hong, Guosong; Dai, Hongjie

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

    Spatial and temporal co-registration of nuclear and optical images would enable the fusion of the information from theses complementary molecular imaging modalities. A critical challenge in integration is fitting optical hardware into the nuclear imaging platforms. Flexible fiber-based fluorescence mediated tomography (FMT) systems provide a viable solution because the various imaging bore sizes of small animal nuclear imaging systems can potentially accommodate the FMT fiber imaging arrays. Further, FMT imaging facilitates co-registering the nuclear and optical contrasts in time. Herein, we combine a fiber based FMT system with a preclinical NanoSPECT/CT platform. Feasibility of in vivo imaging is demonstrated by tracking the accumulation of a monomolecular multimodal imaging agent (MOMIA) in a sentinel lymph node (SLN) of a rat. Methods The fiber-based, video-rate FMT imaging system is composed of 12 alternating sources (785nm and 830nm LDs) and 13 detectors. To maintain high temporal sampling, the system simultaneously acquires ratio-metric data at each detector. The data is reconstructed using the normalized Born approach with a three-dimensional finite element model derived from an anatomical CT image of a rat for accurate light propagation modeling. Nuclear and optical contrasts are integrated by using a MOMIA. Data collection begins immediately after injection of the MOMIA intradermally into the forepaw with the FMT data acquired simultaneously with both the SPECT and CT. Results Fluorescence and radioactivity from the MOMIA were co-localized in a spatially coincident region. Intravital imaging with surgical exposure of the lymph node validated the localization of the optical contrast. The optical and nuclear contrasts where integrated by incorporating SPECT as a prior in the DOT reconstruction. Conclusion The feasibility of integrating a fiber-based, video-rate FMT system with a commercial preclinical NanoSPECT/CT platform was established. The co

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

    PubMed Central

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

    2015-01-01

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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  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. In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

    PubMed Central

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

    2013-01-01

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

  6. 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

  7. Laser line illumination scheme allowing the reduction of background signal and the correction of absorption heterogeneities effects for fluorescence reflectance imaging.

    PubMed

    Fantoni, Frédéric; Hervé, Lionel; Poher, Vincent; Gioux, Sylvain; Mars, Jérôme I; Dinten, Jean-Marc

    2015-10-01

    Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality as it allows to noninvasively monitor the fluorescence targeted tumors located below the tissue surface. Some drawbacks of this technique are the background fluorescence decreasing the contrast and absorption heterogeneities leading to misinterpretations concerning fluorescence concentrations. We propose a correction technique based on a laser line scanning illumination scheme. We scan the medium with the laser line and acquire, at each position of the line, both fluorescence and excitation images. We then use the finding that there is a relationship between the excitation intensity profile and the background fluorescence one to predict the amount of signal to subtract from the fluorescence images to get a better contrast. As the light absorption information is contained both in fluorescence and excitation images, this method also permits us to correct the effects of absorption heterogeneities. This technique has been validated on simulations and experimentally. Fluorescent inclusions are observed in several configurations at depths ranging from 1 mm to 1 cm. Results obtained with this technique are compared with those obtained with a classical wide-field detection scheme for contrast enhancement and with the fluorescence by an excitation ratio approach for absorption correction. PMID:26442963

  8. Use of a laser-induced fluorescence thermal imaging system for film cooling heat transfer measurement

    SciTech Connect

    Chyu, M.K.

    1996-04-01

    This paper describes a novel approach based on fluorescence imaging of thermographic phosphor that enables the simultaneous determination of both local film effectiveness and local heat transfer on a film-cooled surface. The film cooling model demonstrated consists of a single row of three discrete holes on a flat plate. The transient temperature measurement relies on the temperature-sensitive fluorescent properties of europium-doped lanthanum oxysulfide (La{sub 2}O{sub 2}S:Eu{sup +3}) thermographic phosphor. A series of full-field surface temperatures, mainstream temperatures, and coolant film temperatures were acquired during the heating of a test surface. These temperatures are used to calculate the heat transfer coefficients and the film effectiveness simultaneously. Because of the superior spatial resolution capability for the heat transfer data reduced from these temperature frames, the laser-induced fluorescence (LIF) imaging system, the present study observes the detailed heat transfer characteristics over a film-protected surface. The trend of the results agrees with those obtained using other conventional thermal methods, as well as the liquid crystal imaging technique. One major advantage of this technique is the capability to record a large number of temperature frames over a given testing period. This offers multiple-sample consistency.

  9. Use of a laser-induced fluorescence thermal imaging system for film cooling heat transfer measurement

    SciTech Connect

    Chyu, M.K.

    1995-10-01

    This paper describes a novel approach based on fluorescence imaging of thermographic phosphor that enables the simultaneous determination of both local film effectiveness and local heat transfer on a film-cooled surface. The film cooling model demonstrated consists of a single row of three discrete holes on a flat plate. The transient temperature measurement relies on the temperature-sensitive fluorescent properties of europium-doped lanthanum oxysulfide (La{sub 2}O{sub 2}S:EU{sup 3+}) thermographic phosphor. A series of full-field surface temperatures, mainstream temperatures, and coolant film temperatures were acquired during the heating of a test surface. These temperatures are used to calculate the heat transfer coefficients and the film effectiveness simultaneously. Because of the superior spatial resolution capability for the heat transfer data reduced from these temperature frames, the laser-induced fluorescence (LIF) imaging system, the present study observes the detailed heat transfer characteristics over a film-protected surface. The trend of the results agrees with those obtained using other conventional thermal methods, as well as the liquid crystal imaging technique. One major advantage of this technique is the capability to record a large number of temperature frames over a given testing period. This offers multiple-sample consistency.

  10. 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.

  11. 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

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

    PubMed

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

    2016-06-01

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

  13. Computer-vision-based weed identification of images acquired by 3CCD camera

    NASA Astrophysics Data System (ADS)

    Zhang, Yun; He, Yong; Fang, Hui

    2006-09-01

    Selective application of herbicide to weeds at an earlier stage in crop growth is an important aspect of site-specific management of field crops. For approaches more adaptive in developing the on-line weed detecting application, more researchers involves in studies on image processing techniques for intensive computation and feature extraction tasks to identify the weeds from the other crops and soil background. This paper investigated the potentiality of applying the digital images acquired by the MegaPlus TM MS3100 3-CCD camera to segment the background soil from the plants in question and further recognize weeds from the crops using the Matlab script language. The image of the near-infrared waveband (center 800 nm; width 65 nm) was selected principally for segmenting soil and identifying the cottons from the thistles was achieved based on their respective relative area (pixel amount) in the whole image. The results show adequate recognition that the pixel proportion of soil, cotton leaves and thistle leaves were 78.24%(-0.20% deviation), 16.66% (+ 2.71% SD) and 4.68% (-4.19% SD). However, problems still exists by separating and allocating single plants for their clustering in the images. The information in the images acquired via the other two channels, i.e., the green and the red bands, need to be extracted to help the crop/weed discrimination. More optical specimens should be acquired for calibration and validation to establish the weed-detection model that could be effectively applied in fields.

  14. New Approaches for Correction of Interlayer Absorption Effects in X-ray Fluorescence Imaging of Paintings.

    PubMed

    Wróbel, Paweł M; Frączek, Piotr; Lankosz, Marek

    2016-02-01

    The X-ray fluorescence imaging technique allows not only the imaging itself but also the identification of the hidden paint layers what makes it much more versatile as compared with X-ray radiography. One of the main disadvantages of the former method is the fact that the characteristic X-rays from the deeper paint layers are absorbed in the covering layers. This effect is manifested by some artifacts that impede a proper interpretation of the acquired images. In this work, it is shown that the methodology for correction of the interlayer absorption effects can be extended to the case of polychromatic excitation. Additionally, a new approach for determination of the optimal correction parameters has also been presented. The methodology was verified using either the test painting or the mock-up painting both measured with a table-top micro-XRF setup. PMID:26708500

  15. Novel method to calculate pulmonary compliance images in rodents from computed tomography acquired at constant pressures

    NASA Astrophysics Data System (ADS)

    Guerrero, Thomas; Castillo, Richard; Sanders, Kevin; Price, Roger; Komaki, Ritsuko; Cody, Dianna

    2006-03-01

    Our goal was to develop a method for generating high-resolution three-dimensional pulmonary compliance images in rodents from computed tomography (CT) images acquired at a series of constant pressures in ventilated animals. One rat and one mouse were used to demonstrate this technique. A pre-clinical GE flat panel CT scanner (maximum 31 line-pairs cm-1 resolution) was utilized for image acquisition. The thorax of each animal was imaged with breath-holds at 2, 6, 10, 14 and 18 cm H2O pressure in triplicate. A deformable image registration algorithm was applied to each pair of CT images to map corresponding tissue elements. Pulmonary compliance was calculated on a voxel by voxel basis using adjacent pairs of CT images. Triplicate imaging was used to estimate the measurement error of this technique. The 3D pulmonary compliance images revealed regional heterogeneity of compliance. The maximum total lung compliance measured 0.080 (±0.007) ml air per cm H2O per ml of lung and 0.039 (±0.004) ml air per cm H2O per ml of lung for the rat and mouse, respectively. In this study, we have demonstrated a unique method of quantifying regional lung compliance from 4 to 16 cm H2O pressure with sub-millimetre spatial resolution in rodents. Presented at the Third IASTED Int. Conf. on Biomechanics (Benidorm, Spain), 7-9 September 2005.

  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. Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging

    PubMed Central

    Wang, Haolu; Liang, Xiaowen; Mohammed, Yousuf H.; Thomas, James A.; Bridle, Kim R.; Thorling, Camilla A.; Grice, Jeffrey E.; Xu, Zhi Ping; Liu, Xin; Crawford, Darrell H. G.; Roberts, Michael S.

    2015-01-01

    Conventional histology with light microscopy is essential in the diagnosis of most liver diseases. Recently, a concept of real-time histology with optical biopsy has been advocated. In this study, live mice livers (normal, with fibrosis, steatosis, hepatocellular carcinoma and ischemia-reperfusion injury) were imaged by MPM-FLIM for stain-free real-time histology. The acquired MPM-FLIM images were compared with conventional histological images. MPM-FLIM imaged subsurface cellular and subcellular histopathological hallmarks of live liver in mice models at high resolution. Additional information such as distribution of stellate cell associated autofluorescence and fluorescence lifetime changes was also gathered by MPM-FLIM simultaneously, which cannot be obtained from conventional histology. MPM-FLIM could simultaneously image and quantify the cellular morphology and microenvironment of live livers without conventional biopsy or fluorescent dyes. We anticipate that in the near future MPM-FLIM will be evaluated from bench to bedside, leading to real-time histology and dynamic monitoring of human liver diseases. PMID:25798303

  18. 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

  19. 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

  20. 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

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

    PubMed

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

    2013-05-15

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

  2. Clinical results of fluorescence lifetime imaging in ophthalmology

    NASA Astrophysics Data System (ADS)

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

    2009-07-01

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

  3. Simultaneous fluorescence and high-resolution bright-field imaging with aberration correction over a wide field-of-view with Fourier ptychographic microscopy (FPM) (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Chung, Jaebum; Kim, Jinho; Ou, Xiaoze; Horstmeyer, Roarke; Yang, Changhuei

    2016-03-01

    We present a method to acquire both fluorescence and high-resolution bright-field images with correction for the spatially varying aberrations over a microscope's wide field-of-view (FOV). First, the procedure applies Fourier ptychographic microscopy (FPM) to retrieve the amplitude and phase of a sample, at a resolution that significantly exceeds the cutoff frequency of the microscope objective lens. At the same time, FPM algorithm is able to leverage on the redundancy within the set of acquired FPM bright-field images to estimate the microscope aberrations, which usually deteriorate in regions further away from the FOV's center. Second, the procedure acquires a raw wide-FOV fluorescence image within the same setup. Lack of moving parts allows us to use the FPM-estimated aberration map to computationally correct for the aberrations in the fluorescence image through deconvolution. Overlaying the aberration-corrected fluorescence image on top of the high-resolution bright-field image can be done with accurate spatial correspondence. This can provide means to identifying fluorescent regions of interest within the context of the sample's bright-field information. An experimental demonstration successfully improves the bright-field resolution of fixed, stained and fluorescently tagged HeLa cells by a factor of 4.9, and reduces the error caused by aberrations in a fluorescence image by 31%, over a field of view of 6.2 mm by 9.3 mm. For optimal deconvolution, we show the fluorescence image needs to have a signal-to-noise ratio of ~18.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

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

    PubMed

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

    2016-04-13

    Fluorescent carbon nanoparticle-based probes with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. However, synthesis of red fluorescent carbon nanoparticles and their transformation into functional nanoparticles are very challenging. Here we report red fluorescent carbon nanoparticle-based nanobioconjugates of <25 nm hydrodynamic size and their application as fluorescent cell labels. Hydrophobic carbon nanoparticles are synthesized via high temperature colloid-chemical approach and transformed into water-soluble functional nanoparticles via coating with amphiphilic polymer followed by covalent linking with desired biomolecules. Following this approach, carbon nanoparticles are functionalized with polyethylene glycol, primary amine, glucose, arginine, histidine, biotin and folic acid. These functional nanoparticles can be excited with blue/green light (i.e., 400-550 nm) to capture their emission spanning from 550 to 750 nm. Arginine and folic acid functionalized nanoparticles have been demonstrated as fluorescent cell labels where blue and green excitation has been used for imaging of labeled cells. The presented method can be extended for the development of carbon nanoparticle-based other bioimaging probes. PMID:27011336

  9. [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

  10. 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

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

    PubMed

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

    2016-03-01

    A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth-inhibitory studies reveal a better therapeutic effect of FSMs after CPT encapsulation when compared with the free CPT drug. The multifunctional FSM nanomedicine platform as a nanovehicle has great potential for fluorescence imaging-guided cancer therapy. PMID:26881415

  12. 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

  13. 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.

  14. Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer

    PubMed Central

    Elliott, Amicia D.; Gao, Liang; Ustione, Alessandro; Bedard, Noah; Kester, Robert; Piston, David W.; Tkaczyk, Tomasz S.

    2012-01-01

    Summary The development of multi-colored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of a snapshot hyperspectral imaging device, the image mapping spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signal capture from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined fluorescent protein (FP)-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in pancreatic β-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope. PMID:22854044

  15. Stereoscopic Planar Laser-Induced Fluorescence Imaging at 500 kHz

    NASA Technical Reports Server (NTRS)

    Medford, Taylor L.; Danehy, Paul M.; Jones, Stephen B.; Jiang, N.; Webster, M.; Lempert, Walter; Miller, J.; Meyer, T.

    2011-01-01

    A new measurement technique for obtaining time- and spatially-resolved image sequences in hypersonic flows is developed. Nitric-oxide planar laser-induced fluorescence (NO PLIF) has previously been used to investigate transition from laminar to turbulent flow in hypersonic boundary layers using both planar and volumetric imaging capabilities. Low flow rates of NO were typically seeded into the flow, minimally perturbing the flow. The volumetric imaging was performed at a measurement rate of 10 Hz using a thick planar laser sheet that excited NO fluorescence. The fluorescence was captured by a pair of cameras having slightly different views of the flow. Subsequent stereoscopic reconstruction of these images allowed the three-dimensional flow structures to be viewed. In the current paper, this approach has been extended to 50,000 times higher repetition rates. A laser operating at 500 kHz excites the seeded NO molecules, and a camera, synchronized with the laser and fitted with a beam-splitting assembly, acquires two separate images of the flow. The resulting stereoscopic images provide three-dimensional flow visualizations at 500 kHz for the first time. The 200 ns exposure time in each frame is fast enough to freeze the flow while the 500 kHz repetition rate is fast enough to time-resolve changes in the flow being studied. This method is applied to visualize the evolving hypersonic flow structures that propagate downstream of a discrete protuberance attached to a flat plate. The technique was demonstrated in the NASA Langley Research Center s 31-Inch Mach 10 Air Tunnel facility. Different tunnel Reynolds number conditions, NO flow rates and two different cylindrical protuberance heights were investigated. The location of the onset of flow unsteadiness, an indicator of transition, was observed to move downstream during the tunnel runs, coinciding with an increase in the model temperature.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. A new method to acquire 3-D images of a dental cast

    NASA Astrophysics Data System (ADS)

    Li, Zhongke; Yi, Yaxing; Zhu, Zhen; Li, Hua; Qin, Yongyuan

    2006-01-01

    This paper introduced our newly developed method to acquire three-dimensional images of a dental cast. A rotatable table, a laser-knife, a mirror, a CCD camera and a personal computer made up of a three-dimensional data acquiring system. A dental cast is placed on the table; the mirror is installed beside the table; a linear laser is projected to the dental cast; the CCD camera is put up above the dental cast, it can take picture of the dental cast and the shadow in the mirror; while the table rotating, the camera records the shape of the laser streak projected on the dental cast, and transmit the data to the computer. After the table rotated one circuit, the computer processes the data, calculates the three-dimensional coordinates of the dental cast's surface. In data processing procedure, artificial neural networks are enrolled to calibrate the lens distortion, map coordinates form screen coordinate system to world coordinate system. According to the three-dimensional coordinates, the computer reconstructs the stereo image of the dental cast. It is essential for computer-aided diagnosis and treatment planning in orthodontics. In comparison with other systems in service, for example, laser beam three-dimensional scanning system, the characteristic of this three-dimensional data acquiring system: a. celerity, it casts only 1 minute to scan a dental cast; b. compact, the machinery is simple and compact; c. no blind zone, a mirror is introduced ably to reduce blind zone.

  1. Dynamic Linear Model Analysis of Optical Imaging Data Acquired from the Human Neocortex

    PubMed Central

    Lavine, Michael; Haglund, Michael M.; Hochman, Daryl W.

    2011-01-01

    The amount of light absorbed and scattered by neocortical tissue is altered by neuronal activity. Imaging of intrinsic optical signals (ImIOS), a technique for mapping these activity-evoked optical changes with an imaging detector, has the potential to be useful for both clinical and experimental investigations of the human neocortex. However, its usefulness for human studies is currently limited because intraoperatively acquired ImIOS data is noisy. To improve the reliability and usefulness of ImIOS for human studies, it is desirable to find appropriate methods for the removal of noise artifacts and its statistical analysis. Here we develop a Bayesian, dynamic linear modeling approach that appears to address these problems. A dynamic linear model (DLM) was constructed that included cyclic components to model the heartbeat and respiration artifacts, and a local linear component to model the activity-evoked response. The robustness of the model was tested on a set of ImIOS data acquired from the exposed cortices of six human subjects illuminated with either 535 nm or 660 nm light. The DLM adequately reduced noise artifacts in these data while reliably preserving their activity-evoked optical responses. To demonstrate how these methods might be used for intraoperative neurosurgical mapping, optical data acquired from a single human subject during direct electrical stimulation of the cortex were quantitatively analyzed. This example showed that the DLM can be used to provide quantitative information about human ImIOS data that is not available through qualitative analysis alone. PMID:21640137

  2. Comparison of two automatic cell-counting solutions for fluorescent microscopic images.

    PubMed

    Lojk, J; Čibej, U; Karlaš, D; Šajn, L; Pavlin, M

    2015-10-01

    Cell counting in microscopic images is one of the fundamental analysis tools in life sciences, but is usually tedious, time consuming and prone to human error. Several programs for automatic cell counting have been developed so far, but most of them demand additional training or data input from the user. Most of them do not allow the users to online monitor the counting results, either. Therefore, we designed two straightforward, simple-to-use cell-counting programs that also allow users to correct the detection results. In this paper, we present the Cellcounter and Learn123 programs for automatic and semiautomatic counting of objects in fluorescent microscopic images (cells or cell nuclei) with a user-friendly interface. Although Cellcounter is based on predefined and fine-tuned set of filters optimized on sets of chosen experiments, Learn123 uses an evolutionary algorithm to determine the adapt filter parameters based on a learning set of images. Cellcounter also includes an extension for analysis of overlaying images. The efficiency of both programs was assessed on images of cells stained with different fluorescent dyes by comparing automatically obtained results with results that were manually annotated by an expert. With both programs, the correlation between automatic and manual counting was very high (R(2) < 0.9), although Cellcounter had some difficulties processing images with no cells or weakly stained cells, where sometimes the background noise was recognized as an object of interest. Nevertheless, the differences between manual and automatic counting were small compared to variations between experimental repeats. Both programs significantly reduced the time required to process the acquired images from hours to minutes. The programs enable consistent, robust, fast and accurate detection of fluorescent objects and can therefore be applied to a range of different applications in different fields of life sciences where fluorescent labelling is used for

  3. Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Krasieva, Tatiana B.; Stringari, Chiara; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Gratton, Enrico; Tromberg, Bruce J.

    2013-03-01

    Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

  4. Intracellular Monitoring of AS1411 Aptamer by Time-Resolved Microspectrofluorimetry and Fluorescence Imaging.

    PubMed

    Kočišová, Eva; Praus, Petr; Bok, Jiří; Bonneau, Stéphanie; Sureau, Franck

    2015-09-01

    Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one. PMID:26179074

  5. Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo

    PubMed Central

    Krasieva, Tatiana B.; Stringari, Chiara; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Gratton, Enrico

    2012-01-01

    Abstract. Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000  nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo. PMID:23235925

  6. A novel method to acquire 3D data from serial 2D images of a dental cast

    NASA Astrophysics Data System (ADS)

    Yi, Yaxing; Li, Zhongke; Chen, Qi; Shao, Jun; Li, Xinshe; Liu, Zhiqin

    2007-05-01

    This paper introduced a newly developed method to acquire three-dimensional data from serial two-dimensional images of a dental cast. The system consists of a computer and a set of data acquiring device. The data acquiring device is used to take serial pictures of the a dental cast; an artificial neural network works to translate two-dimensional pictures to three-dimensional data; then three-dimensional image can reconstruct by the computer. The three-dimensional data acquiring of dental casts is the foundation of computer-aided diagnosis and treatment planning in orthodontics.

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

    PubMed Central

    Saldua, Meagan A.; Olsovsky, Cory A.; Callaway, Evelyn S.; Chapkin, Robert S.

    2012-01-01

    Abstract. Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333  lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7  mm/sec. A single 1×60 mm2 field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion. PMID:22352656

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

    PubMed

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

    2012-01-01

    Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333 lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7 mm/sec. A single 1 × 60 mm(2) field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion. PMID:22352656

  9. Acquiring 3-D information about thick objects from differential interference contrast images using texture extraction

    NASA Astrophysics Data System (ADS)

    Sierra, Heidy; Brooks, Dana; Dimarzio, Charles

    2010-07-01

    The extraction of 3-D morphological information about thick objects is explored in this work. We extract this information from 3-D differential interference contrast (DIC) images by applying a texture detection method. Texture extraction methods have been successfully used in different applications to study biological samples. A 3-D texture image is obtained by applying a local entropy-based texture extraction method. The use of this method to detect regions of blastocyst mouse embryos that are used in assisted reproduction techniques such as in vitro fertilization is presented as an example. Results demonstrate the potential of using texture detection methods to improve morphological analysis of thick samples, which is relevant to many biomedical and biological studies. Fluorescence and optical quadrature microscope phase images are used for validation.

  10. 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.

  11. 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

  12. CT reconstruction from portal images acquired during volumetric-modulated arc therapy

    NASA Astrophysics Data System (ADS)

    Poludniowski, G.; Thomas, M. D. R.; Evans, P. M.; Webb, S.

    2010-10-01

    Volumetric-modulated arc therapy (VMAT), a form of intensity-modulated arc therapy (IMAT), has become a topic of research and clinical activity in recent years. As a form of arc therapy, portal images acquired during the treatment fraction form a (partial) Radon transform of the patient. We show that these portal images, when used in a modified global cone-beam filtered backprojection (FBP) algorithm, allow a surprisingly recognizable CT-volume to be reconstructed. The possibility of distinguishing anatomy in such VMAT-CT reconstructions suggests that this could prove to be a valuable treatment position-verification tool. Further, some potential for local-tomography techniques to improve image quality is shown.

  13. Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons.

    PubMed

    Fan, G Y; Fujisaki, H; Miyawaki, A; Tsay, R K; Tsien, R Y; Ellisman, M H

    1999-05-01

    A video-rate (30 frames/s) scanning two-photon excitation microscope has been successfully tested. The microscope, based on a Nikon RCM 8000, incorporates a femtosecond pulsed laser with wavelength tunable from 690 to 1050 nm, prechirper optics for laser pulse-width compression, resonant galvanometer for video-rate point scanning, and a pair of nonconfocal detectors for fast emission ratioing. An increase in fluorescent emission of 1.75-fold is consistently obtained with the use of the prechirper optics. The nonconfocal detectors provide another 2.25-fold increase in detection efficiency. Ratio imaging and optical sectioning can therefore be performed more efficiently without confocal optics. Faster frame rates, at 60, 120, and 240 frames/s, can be achieved with proportionally reduced scan lines per frame. Useful two-photon images can be acquired at video rate with a laser power as low as 2.7 mW at specimen with the genetically modified green fluorescent proteins. Preliminary results obtained using this system confirm that the yellow "cameleons" exhibit similar optical properties as under one-photon excitation conditions. Dynamic two-photon images of cardiac myocytes and ratio images of yellow cameleon-2.1, -3.1, and -3.1nu are also presented. PMID:10233058

  14. Multiple Velocity Profile Measurements in Hypersonic Flows using Sequentially-Imaged Fluorescence Tagging

    NASA Technical Reports Server (NTRS)

    Bathel, Brett F.; Danehy, Paul M.; Inmian, Jennifer A.; Jones, Stephen B.; Ivey, Christopher B.; Goyne, Christopher P.

    2010-01-01

    Nitric-oxide planar laser-induced fluorescence (NO PLIF) was used to perform velocity measurements in hypersonic flows by generating multiple tagged lines which fluoresce as they convect downstream. For each laser pulse, a single interline, progressive scan intensified CCD camera was used to obtain separate images of the initial undelayed and delayed NO molecules that had been tagged by the laser. The CCD configuration allowed for sub-microsecond acquisition of both images, resulting in sub-microsecond temporal resolution as well as sub-mm spatial resolution (0.5-mm x 0.7-mm). Determination of axial velocity was made by application of a cross-correlation analysis of the horizontal shift of individual tagged lines. Quantification of systematic errors, the contribution of gating/exposure duration errors, and influence of collision rate on fluorescence to temporal uncertainty were made. Quantification of the spatial uncertainty depended upon the analysis technique and signal-to-noise of the acquired profiles. This investigation focused on two hypersonic flow experiments: (1) a reaction control system (RCS) jet on an Orion Crew Exploration Vehicle (CEV) wind tunnel model and (2) a 10-degree half-angle wedge containing a 2-mm tall, 4-mm wide cylindrical boundary layer trip. The experiments were performed at the NASA Langley Research Center's 31-inch Mach 10 wind tunnel.

  15. 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.

  16. Elastic registration for auto-fluorescence image averaging.

    PubMed

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

    2006-01-01

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

  17. 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.

  18. 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.

  19. 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.

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

    PubMed

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

    2016-04-12

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2005-08-01

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

  3. Highest Resolution Image of Dust and Sand Yet Acquired on Mars

    NASA Technical Reports Server (NTRS)

    2008-01-01

    [figure removed for brevity, see original site] [figure removed for brevity, see original site] [figure removed for brevity, see original site] Click on image for Figure 1Click on image for Figure 2Click on image for Figure 3

    This mosaic of four side-by-side microscope images (one a color composite) was acquired by the Optical Microscope, a part of the Microscopy, Electrochemistry, and Conductivity Analyzer (MECA) instrument suite on NASA's Phoenix Mars Lander. Taken on the ninth Martian day of the mission, or Sol 9 (June 3, 2008), the image shows a 3 millimeter (0.12 inch) diameter silicone target after it has been exposed to dust kicked up by the landing. It is the highest resolution image of dust and sand ever acquired on Mars. The silicone substrate provides a sticky surface for holding the particles to be examined by the microscope.

    Martian Particles on Microscope's Silicone Substrate In figure 1, the particles are on a silcone substrate target 3 millimeters (0.12 inch) in diameter, which provides a sticky surface for holding the particles while the microscope images them. Blow-ups of four of the larger particles are shown in the center. These particles range in size from about 30 microns to 150 microns (from about one one-thousandth of an inch to six one-thousandths of an inch).

    Possible Nature of Particles Viewed by Mars Lander's Optical Microscope In figure 2, the color composite on the right was acquired to examine dust that had fallen onto an exposed surface. The translucent particle highlighted at bottom center is of comparable size to white particles in a Martian soil sample (upper pictures) seen two sols earlier inside the scoop of Phoenix's Robotic Arm as imaged by the lander's Robotic Arm Camera. The white particles may be examples of the abundant salts that have been found in the Martian soil by previous missions. Further investigations will be needed to determine the white material's composition and whether translucent particles

  4. 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.

  5. In Vivo X-Ray Fluorescence Microtomographic Imaging of Elements in Single-Celled Fern Spores

    SciTech Connect

    Hirai, Yasuharu; Yoneyama, Akio; Hisada, Akiko; Uchida, Kenko

    2007-01-19

    We have observed in vivo three-dimensional distributions of constituent elements of single-celled spores of the fern Adiantum capillus-veneris using an X-ray fluorescence computed microtomography method. The images of these distributions are generated from a series of slice data, each of which is acquired by a sample translation-rotation method. An incident X-ray microbeam irradiates the sample with a spot size of 1 {mu}m. The high Ca concentration in the testa and the localized and overlapping Fe and Zn concentrations inside the spore are shown in three-dimensional images. The K concentration is high throughout the cell, and there are localized regions of higher density. The atomic number densities of these elements in the testa and inside the cell in a tomographic slice are estimated with a resolution of about 1 {mu}m.

  6. 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

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

    PubMed

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

    2009-10-22

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

  8. Accumulative Difference Image Protocol for Particle Tracking in Fluorescence Microscopy Tested in Mouse Lymphonodes

    PubMed Central

    Villa, Carlo E.; Caccia, Michele; Sironi, Laura; D'Alfonso, Laura; Collini, Maddalena; Rivolta, Ilaria; Miserocchi, Giuseppe; Gorletta, Tatiana; Zanoni, Ivan; Granucci, Francesca; Chirico, Giuseppe

    2010-01-01

    The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done. PMID:20808918

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

    PubMed Central

    2015-01-01

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

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

    PubMed

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. Fluorescence resonance energy transfer imaging by maximum likelihood estimation

    NASA Astrophysics Data System (ADS)

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

    2004-06-01

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

  16. 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

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

    PubMed

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

    2015-01-01

    Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. Further, we performed spontaneous Raman spectral analysis at single points of the sample which provided molecular vibration information characteristics of lipid droplets. PMID:25993434

  18. 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.

  19. 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

  20. 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

  1. Image processing and classification procedures for analysis of sub-decimeter imagery acquired with an unmanned aircraft over arid rangelands

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using five centimeter resolution images acquired with an unmanned aircraft system (UAS), we developed and evaluated an image processing workflow that included the integration of resolution-appropriate field sampling, feature selection, object-based image analysis, and processing approaches for UAS i...

  2. 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

  3. 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

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

    PubMed

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

    2009-02-20

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

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

    PubMed

    Elmes, Robert B P

    2016-07-12

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

  6. 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.

  7. 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.

  8. 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

  9. Alignment of low-dose X-ray fluorescence tomography images using differential phase contrast

    PubMed Central

    Hong, Young Pyo; Gleber, Sophie-Charlotte; O’Halloran, Thomas V.; Que, Emily L.; Bleher, Reiner; Vogt, Stefan; Woodruff, Teresa K.; Jacobsen, Chris

    2014-01-01

    X-ray fluorescence nanotomography provides unprecedented sensitivity for studies of trace metal distributions in whole biological cells. Dose fractionation, in which one acquires very low dose individual projections and then obtains high statistics reconstructions as signal from a voxel is brought together (Hegerl & Hoppe, 1976 ▶), requires accurate alignment of these individual projections so as to correct for rotation stage runout. It is shown here that differential phase contrast at 10.2 keV beam energy offers the potential for accurate cross-correlation alignment of successive projections, by demonstrating that successive low dose, 3 ms per pixel, images acquired at the same specimen position and rotation angle have a narrower and smoother cross-correlation function (1.5 pixels FWHM at 300 nm pixel size) than that obtained from zinc fluorescence images (25 pixels FWHM). The differential phase contrast alignment resolution is thus well below the 700 nm × 500 nm beam spot size used in this demonstration, so that dose fractionation should be possible for reduced-dose, more rapidly acquired, fluorescence nanotomography experiments. PMID:24365941

  10. Alignment of low-dose X-ray fluorescence tomography images using differential phase contrast.

    PubMed

    Hong, Young Pyo; Gleber, Sophie-Charlotte; O'Halloran, Thomas V; Que, Emily L; Bleher, Reiner; Vogt, Stefan; Woodruff, Teresa K; Jacobsen, Chris

    2014-01-01

    X-ray fluorescence nanotomography provides unprecedented sensitivity for studies of trace metal distributions in whole biological cells. Dose fractionation, in which one acquires very low dose individual projections and then obtains high statistics reconstructions as signal from a voxel is brought together (Hegerl & Hoppe, 1976), requires accurate alignment of these individual projections so as to correct for rotation stage runout. It is shown here that differential phase contrast at 10.2 keV beam energy offers the potential for accurate cross-correlation alignment of successive projections, by demonstrating that successive low dose, 3 ms per pixel, images acquired at the same specimen position and rotation angle have a narrower and smoother cross-correlation function (1.5 pixels FWHM at 300 nm pixel size) than that obtained from zinc fluorescence images (25 pixels FWHM). The differential phase contrast alignment resolution is thus well below the 700 nm × 500 nm beam spot size used in this demonstration, so that dose fractionation should be possible for reduced-dose, more rapidly acquired, fluorescence nanotomography experiments. PMID:24365941

  11. 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...

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  13. Dissecting key components of the Ca2+ homeostasis game by multifunctional fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Bastianello, Stefano; Ciubotaru, Catalin D.; Beltramello, Martina; Mammano, Fabio

    2004-07-01

    Different sub-cellular compartments and organelles, such as cytosol, endoplasmic reticulum and mitochondria, are known to be differentially involved in Ca2+ homeostasis. It is thus of primary concern to develop imaging paradigms that permit to make out these diverse components. To this end, we have constructed a complete system that performs multi-functional imaging under software control. The main hardware components of this system are a piezoelectric actuator, used to set objective lens position, a fast-switching monochromator, used to select excitation wavelength, a beam splitter, used to separate emission wavelengths, and a I/O interface to control the hardware. For these demonstrative experiments, cultured HeLa cells were transfected with a Ca2+ sensitive fluorescent biosensor (cameleon) targeted to the mitochondria (mtCam), and also loaded with cytosolic Fura2. The main system clock was provided by the frame-valid signal (FVAL) of a cooled CCD camera that captured wide-field fluorescence images of the two probes. Excitation wavelength and objective lens position were rapidly set during silent periods between successive exposures, with a minimum inter-frame interval of 2 ms. Triplets of images were acquired at 340, 380 and 430 nm excitation wavelengths at each one of three adjacent focal planes, separated by 250 nm. Optical sectioning was enhanced off-line by applying a nearest-neighbor deconvolution algorithm based on a directly estimated point-spread function (PSF). To measure the PSF, image stacks of sub-resolution fluorescent beads, incorporated in the cell cytoplasm by electroporation, were acquired under identical imaging conditions. The different dynamics of cytosolic and mitochondrial Ca2+ signals evoked by histamine could be distinguished clearly, with sub-micron resolution. Other FRET-based probes capable of sensing different chemical modifications of the cellular environment can be integrated in this approach, which is intrinsically suitable for the

  14. Ablation plume structure and dynamics in ambient gas observed by laser-induced fluorescence imaging spectroscopy

    NASA Astrophysics Data System (ADS)

    Miyabe, M.; Oba, M.; Iimura, H.; Akaoka, K.; Khumaeni, A.; Kato, M.; Wakaida, I.

    2015-08-01

    The dynamic behavior of an ablation plume in ambient gas has been investigated by laser-induced fluorescence imaging spectroscopy. The second harmonic beam from an Nd:YAG laser (0.5-6 J/cm2) was focused on a sintered oxide pellet or a metal chip of gadolinium. The produced plume was subsequently intersected with a sheet-shaped UV beam from a dye laser so that time-resolved fluorescence images were acquired with an intensified CCD camera at various delay times. The obtained cross-sectional images of the plume indicate that the ablated ground state atoms and ions of gadolinium accumulate in a hemispherical contact layer between the plume and the ambient gas, and a cavity containing a smaller density of ablated species is formed near the center of the plume. At earlier expansion stage, another luminous component also expands in the cavity so that it coalesces into the hemispherical layer. The splitting and coalescence for atomic plume occur later than those for ionic plume. Furthermore, the hemispherical layer of neutral atoms appears later than that of ions; however, the locations of the layers are nearly identical. This coincidence of the appearance locations of the layers strongly suggests that the neutral atoms in the hemispherical layer are produced as a consequence of three-body recombination of ions through collisions with gas atoms. The obtained knowledge regarding plume expansion dynamics and detailed plume structure is useful for optimizing the experimental conditions for ablation-based spectroscopic analysis.

  15. Snapshot spectrally encoded fluorescence imaging through a fiber bundle

    PubMed Central

    Bedard, Noah

    2012-01-01

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

  16. Snapshot spectrally encoded fluorescence imaging through a fiber bundle

    NASA Astrophysics Data System (ADS)

    Bedard, Noah; Tkaczyk, Tomasz S.

    2012-08-01

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

  17. 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.

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

    PubMed

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

    2015-09-01

    A wearable all-plastic imaging system for real-time fluorescence image-guided surgery is presented. The compact size of the system is especially suitable for applications in the operating room. The system consists of a dual-mode imaging system, see-through goggle, autofocusing, and auto-contrast tuning modules. The paper will discuss the system design and demonstrate the system performance. PMID:26358823

  19. Another treatment of fluorescence polarization microspectroscopy and imaging.

    PubMed

    Fisz, Jacek J

    2009-04-16

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

  20. 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

  1. 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.

  2. Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating.

    PubMed

    Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

    2014-01-01

    Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV(-)) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV(-) centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo. PMID:24994610

  3. Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

    NASA Astrophysics Data System (ADS)

    Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

    2014-07-01

    Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV-) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV- centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

  4. 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.

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

    PubMed

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

    2011-01-01

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

  6. 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

  7. 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

  8. 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.

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

    PubMed

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

    2006-12-15

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

  10. 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,...

  11. Detection of Artificial Satellites in Images Acquired in Track Rate Mode

    NASA Astrophysics Data System (ADS)

    Levesque, M.

    2011-09-01

    For surveillance of space needs, satellites must be re-observed periodically to measure their position and update their orbital parameters. This represents an incredible volume of data for which an automatic processing capability is desired. Previous developments [1,2,3] produced automatic detection algorithms for images acquired in Step Stare mode (SSM) with sidereal tracking. However, it was proven that the track rate mode (TRM) [6] is more sensitive. Hence, the algorithmic framework was redesigned and applied to this mode. When an imaging sensor tracks a satellite (or a satellite cluster), the stars appear as streaks while the satellites are point-like objects. A series of algorithms was developed for the detection of satellites and star streaks. The centroids of the star streaks are first detected. They are necessary for the astrometric calibration of the image. Thereafter, the satellites are detected using two sets of logical conditions; they are detected with the maximum of sensitivity against the dark sky background, and with the contrast criteria if they are overlapping star streaks. This algorithm framework automatically extracts all required information from the image and adapts the processing parameters and strategy consequently, so no a priori knowledge is require for their execution, which is a requirement for automatic processing capacity.

  12. Decoding of quantum dots encoded microbeads using a hyperspectral fluorescence imaging method.

    PubMed

    Liu, Yixi; Liu, Le; He, Yonghong; Zhu, Liang; Ma, Hui

    2015-05-19

    We presented a decoding method of quantum dots encoded microbeads with its fluorescence spectra using line scan hyperspectral fluorescence imaging (HFI) method. A HFI method was developed to attain both the spectra of fluorescence signal and the spatial information of the encoded microbeads. A decoding scheme was adopted to decode the spectra of multicolor microbeads acquired by the HFI system. Comparison experiments between the HFI system and the flow cytometer were conducted. The results showed that the HFI system has higher spectrum resolution; thus, more channels in spectral dimension can be used. The HFI system detection and decoding experiment with the single-stranded DNA (ssDNA) immobilized multicolor beads was done, and the result showed the efficiency of the HFI system. Surface modification of the microbeads by use of the polydopamine was characterized by the scanning electron microscopy and ssDNA immobilization was characterized by the laser confocal microscope. These results indicate that the designed HFI system can be applied to practical biological and medical applications. PMID:25902043

  13. 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

  14. Live Cell Imaging of a Fluorescent Gentamicin Conjugate

    PubMed Central

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

    2012-01-01

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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

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

    PubMed

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

    2012-04-15

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

  1. 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.

  2. Color calibration of swine gastrointestinal tract images acquired by radial imaging capsule endoscope

    NASA Astrophysics Data System (ADS)

    Ou-Yang, Mang; Jeng, Wei-De; Lai, Chien-Cheng; Wu, Hsien-Ming; Lin, Jyh-Hung

    2016-01-01

    The type of illumination systems and color filters used typically generate varying levels of color difference in capsule endoscopes, which influence medical diagnoses. In order to calibrate the color difference caused by the optical system, this study applied a radial imaging capsule endoscope (RICE) to photograph standard color charts, which were then employed to calculate the color gamut of RICE. Color gamut was also measured using a spectrometer in order to get a high-precision color information, and the results obtained using both methods were compared. Subsequently, color-correction methods, namely polynomial transform and conformal mapping, were used to improve the color difference. Before color calibration, the color difference value caused by the influences of optical systems in RICE was 21.45±1.09. Through the proposed polynomial transformation, the color difference could be reduced effectively to 1.53±0.07. Compared to another proposed conformal mapping, the color difference value was substantially reduced to 1.32±0.11, and the color difference is imperceptible for human eye because it is <1.5. Then, real-time color correction was achieved using this algorithm combined with a field-programmable gate array, and the results of the color correction can be viewed from real-time images.

  3. 20 kHz toluene planar laser-induced fluorescence imaging of a jet in nearly sonic crossflow

    NASA Astrophysics Data System (ADS)

    Miller, V. A.; Troutman, V. A.; Mungal, M. G.; Hanson, R. K.

    2014-10-01

    This manuscript describes continuous, high-repetition-rate (20 kHz) toluene planar laser-induced fluorescence (PLIF) imaging in an expansion tube impulse flow facility. Cinematographic image sequences are acquired that visualize an underexpanded jet of hydrogen in Mach 0.9 crossflow, a practical flow configuration relevant to aerospace propulsion systems. The freestream gas is nitrogen seeded with toluene; toluene broadly absorbs and fluoresces in the ultraviolet, and the relatively high quantum yield of toluene produces large signals and high signal-to-noise ratios. Toluene is excited using a commercially available, frequency-quadrupled (266 nm), high-repetition-rate (20 kHz), pulsed (0.8-0.9 mJ per pulse), diode-pumped solid-state Nd:YAG laser, and fluorescence is imaged with a high-repetition-rate intensifier and CMOS camera. The resulting PLIF movie and image sequences are presented, visualizing the jet start-up process and the dynamics of the jet in crossflow; the freestream duration and a measure of freestream momentum flux steadiness are also inferred. This work demonstrates progress toward continuous PLIF imaging of practical flow systems in impulse facilities at kHz acquisition rates using practical, turn-key, high-speed laser and imaging systems.

  4. 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

  5. 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

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

    NASA Astrophysics Data System (ADS)

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

    1999-09-01

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

  7. Physical Activity Recognition Based on Motion in Images Acquired by a Wearable Camera

    PubMed Central

    Zhang, Hong; Li, Lu; Jia, Wenyan; Fernstrom, John D.; Sclabassi, Robert J.; Mao, Zhi-Hong; Sun, Mingui

    2011-01-01

    A new technique to extract and evaluate physical activity patterns from image sequences captured by a wearable camera is presented in this paper. Unlike standard activity recognition schemes, the video data captured by our device do not include the wearer him/herself. The physical activity of the wearer, such as walking or exercising, is analyzed indirectly through the camera motion extracted from the acquired video frames. Two key tasks, pixel correspondence identification and motion feature extraction, are studied to recognize activity patterns. We utilize a multiscale approach to identify pixel correspondences. When compared with the existing methods such as the Good Features detector and the Speed-up Robust Feature (SURF) detector, our technique is more accurate and computationally efficient. Once the pixel correspondences are determined which define representative motion vectors, we build a set of activity pattern features based on motion statistics in each frame. Finally, the physical activity of the person wearing a camera is determined according to the global motion distribution in the video. Our algorithms are tested using different machine learning techniques such as the K-Nearest Neighbor (KNN), Naive Bayesian and Support Vector Machine (SVM). The results show that many types of physical activities can be recognized from field acquired real-world video. Our results also indicate that, with a design of specific motion features in the input vectors, different classifiers can be used successfully with similar performances. PMID:21779142

  8. PsbS is required for systemic acquired acclimation and post-excess-light-stress optimization of chlorophyll fluorescence decay times in Arabidopsis

    PubMed Central

    Ciszak, Kamil; Kulasek, Milena; Barczak, Anna; Grzelak, Justyna; Maćkowski, Sebastian; Karpiński, Stanisław

    2015-01-01

    Systemic acquired acclimation (SAA) is an important light acclimatory mechanism that depends on the global adjustments of non-photochemical quenching and chloroplast retrograde signaling. As the exact regulation of these processes is not known, we measured time-resolved fluorescence of chlorophyll a in Arabidopsis thaliana leaves exposed to excess light, in leaves undergoing SAA, and in leaves after excess light episode. We compare the behavior induced in wild-type plants with null mutant of non-photochemical quenching (npq4–1). The wild type rosettes exhibit a small reduction of fluorescence decay times in leaves directly exposed to excess light and in leaves undergoing SAA in ambient low light. However in npq4–1 exposition to excess light results in much faster fluorescence decay, which is insensitive to excitation power. At the same time npq4–1 leaves undergoing SAA displayed intermediate fluorescence decay. The npq4–1 plants also lost the ability to optimize florescence decay, and thus chlorophyll a dynamics up to 2 h after excess light episode. The fluorescence decay dynamics in both WT and npq4–1 can be described by a set of 3 maximum decay times. Based on the results, we concluded that functional PsbS is required for optimization of absorbed photon fate and optimal light acclimatory responses such as SAA or after excess light stress. PMID:25654166

  9. Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics

    PubMed Central

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

    2012-01-01

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

  10. MaterialCloning: Acquiring Elasticity Parameters from Images for Medical Applications.

    PubMed

    Yang, Shan; Lin, Ming C

    2016-09-01

    We present a practical approach for automatically estimating the material properties of soft bodies from two sets of images, taken before and after deformation. We reconstruct 3D geometry from the given sets of multiple-view images; we use a coupled simulation-optimization-identification framework to deform one soft body at its original, non-deformed state to match the deformed geometry of the same object in its deformed state. For shape correspondence, we use a distance-based error metric to compare the estimated deformation fields against the actual deformation field from the reconstructed geometry. The optimal set of material parameters is thereby determined by minimizing the error metric function. This method can simultaneously recover the elasticity parameters of multiple types of soft bodies using Finite Element Method-based simulation (of either linear or nonlinear materials undergoing large deformation) and particle-swarm optimization methods. We demonstrate this approach on real-time interaction with virtual organs in patient-specific surgical simulation, using parameters acquired from low-resolution medical images. We also highlight the results on physics-based animation of virtual objects using sketches from an artist's conception. PMID:26661471

  11. Spectrally resolved fluorescence imaging of human colonic adenomas.

    PubMed

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

    1999-06-01

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

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

    NASA Astrophysics Data System (ADS)

    Alexandratou, Eleni; Yova, Dido; Gorpas, Dimitris

    2009-07-01

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

  13. Multiple Velocity Profile Measurements in Hypersonic Flows Using Sequentially-Imaged Fluorescence Tagging

    NASA Technical Reports Server (NTRS)

    Bathel, Brett F.; Danehy, Paul M.; Inman, Jennifer A.; Jones, Stephen B.; Ivey,Christopher b.; Goyne, Christopher P.

    2010-01-01

    Nitric-oxide planar laser-induced fluorescence (NO PLIF) was used to perform velocity measurements in hypersonic flows by generating multiple tagged lines which fluoresce as they convect downstream. For each laser pulse, a single interline, progressive scan intensified CCD (charge-coupled device) camera was used to obtain two sequential images of the NO molecules that had been tagged by the laser. The CCD configuration allowed for sub-microsecond acquisition of both images, resulting in sub-microsecond temporal resolution as well as sub-mm spatial resolution (0.5-mm horizontal, 0.7-mm vertical). Determination of axial velocity was made by application of a cross-correlation analysis of the horizontal shift of individual tagged lines. A numerical study of measured velocity error due to a uniform and linearly-varying collisional rate distribution was performed. Quantification of systematic errors, the contribution of gating/exposure duration errors, and the influence of collision rate on temporal uncertainty were made. Quantification of the spatial uncertainty depended upon the signal-to-noise ratio of the acquired profiles. This velocity measurement technique has been demonstrated for two hypersonic flow experiments: (1) a reaction control system (RCS) jet on an Orion Crew Exploration Vehicle (CEV) wind tunnel model and (2) a 10-degree half-angle wedge containing a 2-mm tall, 4-mm wide cylindrical boundary layer trip. The experiments were performed at the NASA Langley Research Center's 31-Inch Mach 10 Air Tunnel.

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

    PubMed Central

    Schlimpert, Susan; Flärdh, Klas; Buttner, Mark

    2016-01-01

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

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

    PubMed

    Schlimpert, Susan; Flärdh, Klas; Buttner, Mark

    2016-01-01

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

  16. 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

  17. Optimisation of the diffractive optical element for snapshot spectral imaging used in fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Leitner, R.; Arnold, T.; De Biasio, M.; McGunnigle, G.; Rogojanu, R.; Steiner, G. E.; Hermerschmidt, A.

    2012-03-01

    Snapshot approaches address various possibilities to acquire the spectral and spatial information of a scene within a single camera frame. One advantage over the classical push broom or staring imager approaches is that the temporal inconsistency between consecutive scan lines in first case or between the acquired monochromatic images in the second case is avoided. However, this has to be paid by some effort to rearrange or reconstruct the explicit spectral cube from the entangled raw data in the single camera frame. Besides others, the utilization of a diffractive optical element (DOE) is one such snapshot approach (CTIS - computed tomography imaging spectrometer). The DOE is used to create an optical transfer function that projects both the spectral and spatial information of a scene onto a sensor array and a reconstruction algorithm is used that recovers the spectral cube from the dispersed image pattern. The design of the DOE is crucial for the overall system performance as the absolute transmission efficiency of the zeroth and first order versus the relative efficiency between the two over the required wavelength range are difficult to optimize if the limited dynamic range of a real camera is considered. We describe the optimization of such a DOE for the wavelength range from 400 to 780nm and the required reconstruction algorithm to recover the spectral cube from the entangled snapshot image. The described snapshot approach has been evaluated using experiments to assess the spatial and spectral resolution using diffuse reflectance standards. Additionally the results achieved using the described setup for multi-color in-situ fluorescence hybridized preparations (M-FISH) are discussed.

  18. 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.

  19. 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

  20. 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

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

    PubMed Central

    Leblond, F; Ovanesyan, Z; Davis, S C; Valdés, P A; Kim, A; Hartov, A; Wilson, B C; Pogue, B W; Paulsen, K D; Roberts, D W

    2016-01-01

    Here we derived analytical solutions to diffuse light transport in biological tissue based on spectral deformation of diffused near-infrared measurements. These solutions provide a closed-form mathematical expression which predicts that the depth of a fluorescent molecule distribution is linearly related to the logarithm of the ratio of fluorescence at two different wavelengths. The slope and intercept values of the equation depend on the intrinsic values of absorption and reduced scattering of tissue. This linear behavior occurs if the following two conditions are satisfied: the depth is beyond a few millimeters, and the tissue is relatively homogenous. We present experimental measurements acquired with a broad-beam non-contact multi-spectral fluorescence imaging system using a hemoglobin-containing diffusive phantom. Preliminary results confirm that a significant correlation exists between the predicted depth of a distribution of protoporphyrin IX (PpIX) molecules and the measured ratio of fluorescence at two different wavelengths. These results suggest that depth assessment of fluorescence contrast can be achieved in fluorescence-guided surgery to allow improved intra-operative delineation of tumor margins. PMID:21971201

  2. 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

  3. 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.

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

    PubMed

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

    2010-01-01

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

  5. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  8. Combined system of fluorescence diffuse optical tomography and microcomputed tomography for small animal imaging

    NASA Astrophysics Data System (ADS)

    Yang, Xiaoquan; Gong, Hui; Quan, Guotao; Deng, Yong; Luo, Qingming

    2010-05-01

    We developed a dual-modality system that combines fluorescence diffuse optical tomography (fDOT) and flat panel detector-based microcomputed tomography (micro-CT) to simultaneously reveal molecular and structural information in small animals. In fDOT, a 748 nm diode laser was used as an excitation source, while a cooled charge coupled device camera was adopted to collect transmission fluorescence. In micro-CT, a flat panel detector based on amorphous silicon, with active area of 13×13 cm2, and a microfocus x-ray tube were used. The fDOT system was mounted orthogonally to the micro-CT and the projection images were acquired without rotation of the sample, which is different from the method used for micro-CT alone. Both the finite element method and the algebraic reconstruction technique were used to reconstruct images from the fDOT. Phantom data showed that the resolution of the fDOT system was about 3 mm at an imaging depth of 7 mm. Quantitative error was no more than 5% and imaging sensitivity for 1,1'-dioctadecyl-3,3,3',3'-etramethylindotricarbocyanine iodide bis-oleate (DiR-BOA) was estimated to be higher than 100 nM at a depth of 7 mm. Calculations of the phantom's center of mass showed that the location accuracy of fDOT was about 0.7 mm. We applied a Feldkamp algorithm to reconstruct the micro-CT image. By measuring the presampled modulation transfer function with a 30 μm tungsten thread, we estimated that the micro-CT has a resolution of 5 mm-1 when the field of view was 6.5 cm. Our results indicate the uniformity of the transaxial micro-CT image and the contrast-to-noise ratio was measured as 1.95 for a radiation dose of 1 cGy. A non-image-based method was employed for merging images from the two imaging modalities. A nude mouse with DiR-BOA, imaged ex vivo, was used to validate the feasibility of the dual-modality system.

  9. A near-infrared fluorescence-based surgical navigation system imaging software for sentinel lymph node detection

    NASA Astrophysics Data System (ADS)

    Ye, Jinzuo; Chi, Chongwei; Zhang, Shuang; Ma, Xibo; Tian, Jie

    2014-02-01

    Sentinel lymph node (SLN) in vivo detection is vital in breast cancer surgery. A new near-infrared fluorescence-based surgical navigation system (SNS) imaging software, which has been developed by our research group, is presented for SLN detection surgery in this paper. The software is based on the fluorescence-based surgical navigation hardware system (SNHS) which has been developed in our lab, and is designed specifically for intraoperative imaging and postoperative data analysis. The surgical navigation imaging software consists of the following software modules, which mainly include the control module, the image grabbing module, the real-time display module, the data saving module and the image processing module. And some algorithms have been designed to achieve the performance of the software, for example, the image registration algorithm based on correlation matching. Some of the key features of the software include: setting the control parameters of the SNS; acquiring, display and storing the intraoperative imaging data in real-time automatically; analysis and processing of the saved image data. The developed software has been used to successfully detect the SLNs in 21 cases of breast cancer patients. In the near future, we plan to improve the software performance and it will be extensively used for clinical purpose.

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

    PubMed

    Noller, Crystal M; Boulina, Maria; McNamara, George; Szeto, Angela; McCabe, Philip M; Mendez, Armando J

    2016-09-01

    Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 μm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines. PMID:27182204

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

    PubMed Central

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

    2016-01-01

    Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 μm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines. PMID:27182204

  12. Retrieval of aerosol optical thickness from PROBA-CHRIS images acquired over a coniferous forest

    NASA Astrophysics Data System (ADS)

    Maffei, Carmine; Leone, Antonio P.; Menenti, Massimo; Pippi, Ivan; Maselli, Fabio; Antonelli, Paolo

    2005-10-01

    In the present work we show the potential of multiangular hyperspectral PROBA-CHRIS data to estimate aerosol optical properties over dense dark vegetation. Data acquired over San Rossore test site (Pisa, Italy) have been used together with simultaneous ground measurements. Additionally, spectral measurement over the canopy have been performed to describe the directional behavior of a Pinus pinaster canopy. Determination of aerosol properties from optical remote sensing images over land is an under-determined problem, and some assumptions have to be made on both the aerosol and the surface being imaged. Radiance measured on multiple directions add extra information that help in reducing retrieval ambiguity. Nevertheless, multiangular observations don't allow to ignore directional spectral properties of vegetation canopies. Since surface reflectivity is the parameter we wish to determine with remote sensing after atmospheric correction, at least the shape of the bi-directional reflectance factor has to be assumed. We have adopted a Rahman BRF, and have estimated its geometrical parameters from ground spectral measurements. The inversion of measured radiance to obtain aerosol optical properties has been performed, allowing simultaneous retrieval of aerosol model and optical thickness together with the vegetation reflectivity parameter of the Rahman model.

  13. Multimodal Image Analysis in Acquired Vitelliform Lesions and Adult-Onset Foveomacular Vitelliform Dystrophy

    PubMed Central

    Rocha Bastos, Ricardo; Ferreira, Carla Sofia; Brandão, Elisete; Falcão-Reis, Fernando; Carneiro, Ângela M.

    2016-01-01

    Purpose. To characterize vitelliform lesions (VLs) in adult-onset foveomacular vitelliform dystrophy (AOFVD) and acquired vitelliform (AVL) patients using multimodal image analysis. Methods. Retrospective study of twenty-eight eyes from nineteen patients diagnosed with AVL or AOFVD. They were evaluated by color fundus photographs, fundus autofluorescence (FAF), fluorescein angiography (FA), and spectral-domain optical coherence tomography (SD-OCT). Results. Bilateral VLs were associated with AOFVD (p = 0.013). Regular and centered VLs were associated with AOFVD (p = 0.004 and p = 0.016), whereas irregular and noncentered lesions were more frequent in AVL patients. Visual acuity, greatest linear dimension (GLD), lesion height (LH), and pseudohypopyon were similar between groups. Whereas median LH and GLD in AVL group diminished significantly during follow-up (p = 0.009 and p = 0.001), AOFVD lesions tended to become larger and thicker. Conclusions. When consulting a patient presenting a VL with unknown age of onset, familial history, or previous retinal diseases, some aspects of multimodal imaging assessment may lead the ophthalmologist to a correct diagnosis. PMID:27190637

  14. Effective spatial database support for acquiring spatial information from remote sensing images

    NASA Astrophysics Data System (ADS)

    Jin, Peiquan; Wan, Shouhong; Yue, Lihua

    2009-12-01

    In this paper, a new approach to maintain spatial information acquiring from remote-sensing images is presented, which is based on Object-Relational DBMS. According to this approach, the detected and recognized results of targets are stored and able to be further accessed in an ORDBMS-based spatial database system, and users can access the spatial information using the standard SQL interface. This approach is different from the traditional ArcSDE-based method, because the spatial information management module is totally integrated into the DBMS and becomes one of the core modules in the DBMS. We focus on three issues, namely the general framework for the ORDBMS-based spatial database system, the definitions of the add-in spatial data types and operators, and the process to develop a spatial Datablade on Informix. The results show that the ORDBMS-based spatial database support for image-based target detecting and recognition is easy and practical to be implemented.

  15. Traffic sign detection in MLS acquired point clouds for geometric and image-based semantic inventory

    NASA Astrophysics Data System (ADS)

    Soilán, Mario; Riveiro, Belén; Martínez-Sánchez, Joaquín; Arias, Pedro

    2016-04-01

    Nowadays, mobile laser scanning has become a valid technology for infrastructure inspection. This technology permits collecting accurate 3D point clouds of urban and road environments and the geometric and semantic analysis of data became an active research topic in the last years. This paper focuses on the detection of vertical traffic signs in 3D point clouds acquired by a LYNX Mobile Mapper system, comprised of laser scanning and RGB cameras. Each traffic sign is automatically detected in the LiDAR point cloud, and its main geometric parameters can be automatically extracted, therefore aiding the inventory process. Furthermore, the 3D position of traffic signs are reprojected on the 2D images, which are spatially and temporally synced with the point cloud. Image analysis allows for recognizing the traffic sign semantics using machine learning approaches. The presented method was tested in road and urban scenarios in Galicia (Spain). The recall results for traffic sign detection are close to 98%, and existing false positives can be easily filtered after point cloud projection. Finally, the lack of a large, publicly available Spanish traffic sign database is pointed out.

  16. Multimodal Image Analysis in Acquired Vitelliform Lesions and Adult-Onset Foveomacular Vitelliform Dystrophy.

    PubMed

    Rocha Bastos, Ricardo; Ferreira, Carla Sofia; Brandão, Elisete; Falcão-Reis, Fernando; Carneiro, Ângela M

    2016-01-01

    Purpose. To characterize vitelliform lesions (VLs) in adult-onset foveomacular vitelliform dystrophy (AOFVD) and acquired vitelliform (AVL) patients using multimodal image analysis. Methods. Retrospective study of twenty-eight eyes from nineteen patients diagnosed with AVL or AOFVD. They were evaluated by color fundus photographs, fundus autofluorescence (FAF), fluorescein angiography (FA), and spectral-domain optical coherence tomography (SD-OCT). Results. Bilateral VLs were associated with AOFVD (p = 0.013). Regular and centered VLs were associated with AOFVD (p = 0.004 and p = 0.016), whereas irregular and noncentered lesions were more frequent in AVL patients. Visual acuity, greatest linear dimension (GLD), lesion height (LH), and pseudohypopyon were similar between groups. Whereas median LH and GLD in AVL group diminished significantly during follow-up (p = 0.009 and p = 0.001), AOFVD lesions tended to become larger and thicker. Conclusions. When consulting a patient presenting a VL with unknown age of onset, familial history, or previous retinal diseases, some aspects of multimodal imaging assessment may lead the ophthalmologist to a correct diagnosis. PMID:27190637

  17. Body image among 22 persons with acquired and congenital severe mobility impairment.

    PubMed

    Stensman, R

    1989-02-01

    Interviews concerning body image were conducted with 10 persons with tetraplegia due to traumatic spinal cord injury and 12 persons with cerebral palsy. They all needed a wheelchair and daily assistance from another person. Their satisfaction/dissatisfaction with their body in different situations was reported on a visual analogue scale. The body size as estimated on a Body Puzzle was compared with true measurements. The body mass index of the 22 persons was calculated. These results showed only slight differences between the groups of persons with acquired and congenital motor impairment and did not differ from the results in a reference group. Information was obtained about different experiences of the interviewed persons, e.g. those from the viewpoint of a wheelchair user, lack of body language and moving about in dreams. In general, the adjustments made to the problems concerning body image by the persons with severe mobility impairment were successful. This finding is important in the light of the efforts among persons with severe mobility impairment to reach equality and 'a normal life'. PMID:2922204

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

    PubMed

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

    2002-11-01

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

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

    PubMed

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

    2014-11-21

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

  20. 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.

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

    PubMed Central

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

    2014-01-01

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

  2. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    PubMed Central

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

    2012-01-01

    We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD)-based cameras for fluorescence lifetime imaging microscopy (FLIM) by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 μm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber) are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast. PMID:22778606

  5. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

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

    PubMed Central

    Discher, D E; Mohandas, N

    1996-01-01

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

  8. Live Imaging Fluorescent Proteins in Early Mouse Embryos

    PubMed Central

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

    2016-01-01

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

  9. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

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

  11. 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

  12. 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.

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

    PubMed

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

    2015-01-01

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

  14. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  16. 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

  17. Performance evaluation of principal component analysis for dynamic fluorescence tomographic imaging in measurement space

    NASA Astrophysics Data System (ADS)

    Liu, Xin; He, Xiaowei; Yan, Zhuangzhi

    2015-05-01

    Challenges remain in resolving drug (fluorescent biomarkers) distributions within small animals by fluorescence diffuse optical tomography (FDOT). Principal component analysis (PCA) provides the capability of detecting organs (functional structures) from dynamic FDOT images. However, the resolving performance of PCA may be affected by various experimental factors, e.g., the noise levels in measurement data, the variance in optical properties, the number of acquired frames, and so on. To address the problem, based on a simulation model, we analyze and compare the performance of PCA when applied to three typical sets of experimental conditions (frames number, noise level, and optical properties). The results show that the noise is a critical factor affecting the performance of PCA. When input data containing a low noise (<5%), by a short (e.g., 6 frame) projection sequence, we can resolve the poly(DL-lactic-coglycolic acid)/indocynaine green (PLGA/ICG) distributions in heart and lungs, even though there are great variances in optical properties. In contrast, when 20% Gaussian noise is added to the input data, it hardly resolves the distributions of PLGA/ICG in heart and lungs even though accurate optical properties are used. However, with an increased number of frames, the resolving performance of PCA may gradually recover.

  18. 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

  19. NIR fluorescent image-based evaluation of gastric tube perfusion after esophagectomy in preclinical model (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kim, Minji; Quan, Yuhua; Han, Kook Nam; Choi, Byeong Hyun; Choi, Yeonho; Kim, Hyun Koo; Kim, Beop-Min

    2016-03-01

    This study was to evaluate the feasibility of near infrared (NIR) fluorescent images as a tool for evaluating the perfusion of the gastric tube after esophagectomy. In addition, we investigated the time required to acquire enough signal to confirm the presence of ischemia in gastric tube after injection of indocyanine green (ICG) through peripheral versus and central venous route. 4 porcine underwent esophagogastrostomy and their right gastric arteries were ligated to mimic ischemic condition of gastric tube. ICG (0.6mg/kg) was intravenously injected and the fluorescence signal-to-background ratios (SBR) were measured by using the custom-built intraoperative color and fluorescence imaging system (ICFIS). We evaluated perfusion of gastric tubes by comparing their SBR with esophageal SBR. In ischemic models, SBR of esophagus was higher than that of gastric tube (2.8+/-0.54 vs. 1.7+/-0.37, p<0.05). It showed high esophagus-stomach signal to signal ratio. (SSR, 1.8+/-0.76). We also could observe recovery of blood perfusion in few minutes after releasing the ligation of right gastric artery. In addition, in comparison study according to the injection route of ICG, The time to acquire signal stabilization was faster in central than in peripheral route (119 +/- 65.1 seconds in central route vs. 295+/-130.4 in peripheral route, p<0.05). NIR fluorescent images could provide the real-time information if there was ischemia or not in gastric tube during operation. And, central injection of ICG might give that information faster than peripheral route.

  20. Planar Fluorescence Imaging and Three-Dimensional Reconstructions of Capsule RCS Jets

    NASA Technical Reports Server (NTRS)

    Inman, Jennifer A.; Danehy, Paul M.; Alderfer, David W.; Buck, Gregory M.; Mccrea, Andrew C.

    2009-01-01

    Planar laser-induced fluorescence (PLIF) flowfield visualization has been used to investigate reaction control system (RCS) jet flows in the wake of hypersonic capsule reentry vehicles. Pitch, roll, and yaw RCS jets were all studied. PLIF was used to obtain off-body flow images at planar slices in these flowfields, which are not easily visualized by other techniques owing to characteristically low gas density. When viewed individually, these slices are shown to provide spatially and temporally resolved information, including the locations and characteristics of turbulent flow structures and the location of the jet flow relative to the vehicle. In addition, ensembles of slices, acquired at multiple locations throughout the flowfield, are combined using computer visualization techniques to reconstruct the three-dimensional shape of the flow. Collectively, the off-body flow visualization data set acquired in these tests represents a valuable compliment to surface measurements, especially as a basis for explaining otherwise perplexing discrepancies between such measurements and computational fluid dynamics (CFD) results. The tests described herein were conducted in the 31-Inch Mach 10 Air Tunnel at NASA Langley Research Center.

  1. 3D Imaging of Transition Metals in the Zebrafish Embryo by X-ray Fluorescence Microtomography

    PubMed Central

    Bourassa, Daisy; Gleber, Sophie-Charlotte; Vogt, Stefan; Yi, Hong; Will, Fabian; Richter, Heiko; Shin, Chong Hyun; Fahrni, Christoph J.

    2014-01-01

    Synchrotron X-ray fluorescence (SXRF) microtomography has emerged as a powerful technique for the 3D visualization of the elemental distribution in biological samples. The mechanical stability, both of the instrument and the specimen, is paramount when acquiring tomographic projection series. By combining the progressive lowering of temperature method (PLT) with femtosecond laser sectioning, we were able to embed, excise, and preserve a zebrafish embryo at 24 hours post fertilization in an X-ray compatible, transparent resin for tomographic elemental imaging. Based on a data set comprised of 60 projections, acquired with a step size of 2 μm during 100 hours of beam time, we reconstructed the 3D distribution of zinc, iron, and copper using the iterative maximum likelihood expectation maximization (MLEM) reconstruction algorithm. The volumetric elemental maps, which entail over 124 million individual voxels for each transition metal, revealed distinct elemental distributions that could be correlated with characteristic anatomical features at this stage of embryonic development. PMID:24992831

  2. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2016-05-01

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

  7. Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation

    PubMed Central

    Radbruch, Helena; Andresen, Volker; Mossakowski, Agata; Siffrin, Volker; Seelemann, Thomas; Spiecker, Heinrich; Moll, Ingrid; Herz, Josephine; Hauser, Anja E.; Zipp, Frauke; Behne, Martin J.; Niesner, Raluca

    2013-01-01

    Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm2) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm2) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal

  8. In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish

    NASA Astrophysics Data System (ADS)

    Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

    2014-03-01

    Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

  9. Clinical Application of High-Resolution Computed Tomographic Imaging Features of Community-Acquired Pneumonia

    PubMed Central

    Nie, Yunqiang; Li, Cuiyun; Zhang, Jingling; Wang, Hui; Han, Ping; Lv, Xin; Xu, Xinyi; Guo, Miao

    2016-01-01

    Background This article discusses the value of high-resolution computed tomography (HRCT) in the diagnosis and treatment of pulmonary infections. Lung infection caused by pathogens is an important cause of death. Traditional methods to treat lung infection involved empirical antibiotic therapy. Thin-slice CT scanning is widely used in the clinical setting, and HRCT scan can very clearly show alveolar and bronchiolar involvement of infection. Material/Methods In total, 178 patients with community-acquired pneumonia (CAP) were enrolled. All the patients underwent CT scan, qualified sputum, and blood samples for culture or immunological biochemical tests. CT imaging features, pathogenic bacteria, and treatment results were used for statistical analysis. Results In 77 patients with lobar consolidation, the rate of detection was 43.26% (77/178), and in 101 patients with lobular pneumonia it was 56.74% (101/178). In 51 patients, pathogenic bacteria were detected (28.65%, 51/178). Sixteen of 33 patients detected with bacteria had cavities (48.5%, 16/33) and 35 of 145 patients detected with bacteria had no cavities (24.1%, 35/145). The difference between the 2 groups was statistically significant (χ2=7.795, P=0.005). According to the pathogenic bacteria, 38 patients were cured (74.51%, 38/51), and according to the CT imaging features 81 patients were cured (71.05%, 81/114). No statistically significant difference was found between them (χ2=0.209, P=0.647). Conclusions Treatment effect of CAP based on HRCT findings is not inferior to treatment effect guided by microbial characterization. PMID:27031210

  10. 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.

  11. Cell nuclear features for classification from fluorescence images

    NASA Astrophysics Data System (ADS)

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

    2000-04-01

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

  12. The utilization of a non-invasive fluorescence imaging system to follow clinical dermatological MAL-PDT

    NASA Astrophysics Data System (ADS)

    Tyrrell, Jessica; Campbell, Sandra; Curnow, Alison

    2009-06-01

    This study employed a commercially available, non-invasive, fluorescence imaging system (Dyaderm, Biocam, Germany), to measure protoporphyrin IX (PpIX) concentration at several different stages during clinical dermatological methyl aminolevulinate photodynamic therapy (MAL-PDT). We validated the system prior to use to ensure that the PpIX changes witnessed were accurate and not due to environmental or user induced artifacts. The system was then employed to acquire color (morphological) and fluorescent (physiological) images simultaneously during dermatological PDT. Clinical data was collected from a range of licensed dermatological conditions (actinic keratosis, Bowen's disease and superficial basal cell carcinoma) during initial and subsequent PDT treatment cycles. The initial clinical data indicated that each type of licensed lesion considered responded in a similar manner following the application of Metvix (Galderma, U.K.) and the subsequent light irradiation (Aktilite, Galderma, U.K.). Images acquired three hours after Metvix application showed a significant increase in PpIX concentration within the lesion (P < 0.05), whilst PpIX levels in the surrounding normal tissue remained unaltered. After irradiation, the PpIX concentration was significantly decreased and returned to a level similar to the initial concentration originally observed. Lesions that received subsequent treatment cycles accumulated significantly less PpIX (P < 0.05) prior to irradiation.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

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

    NASA Astrophysics Data System (ADS)

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

    2001-01-01

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

  2. 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

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

    PubMed

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

    2015-04-01

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

  4. Road Signs Detection and Recognition Utilizing Images and 3d Point Cloud Acquired by Mobile Mapping System

    NASA Astrophysics Data System (ADS)

    Li, Y. H.; Shinohara, T.; Satoh, T.; Tachibana, K.

    2016-06-01

    High-definition and highly accurate road maps are necessary for the realization of automated driving, and road signs are among the most important element in the road map. Therefore, a technique is necessary which can acquire information about all kinds of road signs automatically and efficiently. Due to the continuous technical advancement of Mobile Mapping System (MMS), it has become possible to acquire large number of images and 3d point cloud efficiently with highly precise position information. In this paper, we present an automatic road sign detection and recognition approach utilizing both images and 3D point cloud acquired by MMS. The proposed approach consists of three stages: 1) detection of road signs from images based on their color and shape features using object based image analysis method, 2) filtering out of over detected candidates utilizing size and position information estimated from 3D point cloud, region of candidates and camera information, and 3) road sign recognition using template matching method after shape normalization. The effectiveness of proposed approach was evaluated by testing dataset, acquired from more than 180 km of different types of roads in Japan. The results show a very high success in detection and recognition of road signs, even under the challenging conditions such as discoloration, deformation and in spite of partial occlusions.

  5. 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.

  6. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  8. Development of an image processing support system based on fluorescent dye to prevent elderly people with dementia from wandering.

    PubMed

    Nishigaki, Yutaka; Tanaka, Kentaro; Kim, Juhyon; Nakajima, Kazuki

    2013-01-01

    The wandering of elderly people with dementia is a significant behavioral problem and is a heavy burden on caregivers in residential and nursing homes. Thus, warning systems have been developed to prevent elderly people with dementia from leaving the premises. Some of these systems use radio waves. However, systems based on radio waves present several practical problems. For instance, the transmitter must be carried and may become lost; in addition, the battery of the transmitter must be changed. To solve these problems, we developed a support system that prevents elderly people with dementia from wandering. The system employs image processing technology based on fluorescent dye. The composition of the support system can be described as follows: fluorescent dye is painted in a simple shape on the clothes of an elderly person. The fluorescent color becomes visible by irradiation with a long wavelength of ultraviolet light. In the present paper, the relationship between the color of the dye and the cloth was investigated. A 3D video camera was used to acquire a 3D image and detect the simple shape. As a preliminary experiment, 3 colors (red, green and blue) of fluorescent dye were applied to cloths of 9 different colors. All fluorescent colors were detected on 6 of the cloths, but red and blue dye could not be detected on the other 3 cloths. In contrast, green dye was detectable on all 9 of the cloths. Additionally, we determined whether green dye could be detected in an actual environment. A rectangular shaped patch of green fluorescent dye was painted on the shoulder area of a subject, from the scapula to the clavicle. As a result, the green dye was detected on all 9 different colored cloths. PMID:24111431

  9. Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

    PubMed

    Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

    2015-12-01

    Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation. PMID:26510475

  10. 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

  11. 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

  12. 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

  13. 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 siz