Sample records for fluorescence lifetime imaging

  1. Fluorescence lifetime imaging ophthalmoscopy.

    PubMed

    Dysli, Chantal; Wolf, Sebastian; Berezin, Mikhail Y; Sauer, Lydia; Hammer, Martin; Zinkernagel, Martin S

    2017-09-01

    Imaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases. Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. 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.

  3. Maximizing Aggregation of Organic Fluorophores to Prolong Fluorescence Lifetime for Two-Photon Fluorescence Lifetime Imaging.

    PubMed

    Hu, Wenbo; Guo, Lihong; Bai, Lei; Miao, Xiaofei; Ni, Yun; Wang, Qi; Zhao, Hui; Xie, Meng; Li, Lin; Lu, Xiaomei; Huang, Wei; Fan, Quli

    2018-05-28

    Two-photon fluorescence lifetime imaging (TP-FLIM) not only permits imaging deep inside the tissues with precise spatial manipulation but also circumvents tissue autofluorescence, holding tremendous promise in molecular imaging. However, the serious lack of suitable contrast agents with long fluorescence lifetime and efficient two-photon absorption (TPA) greatly limits the advance of TP-FLIM. This study reports a simple approach to fabricate water-soluble organic semiconducting nanoparticles [thioxanthone (TXO) NPs] with ultralong fluorescence lifetime and efficient TPA for in vivo TP-FLIM. The approach utilizes the aggregation of a specifically selected thermally activated delayed fluorescence (TADF) fluorophore to prolong its fluorescence lifetime. Encapsulating the TADF fluorophore within an amphiphilic copolymer not only maximizes its aggregation but also obtains TXO NPs with efficient TPA. Importantly, as-prepared TXO NPs exhibit a considerably long fluorescence lifetime at a magnitude of 4.2 µs, which is almost 1000 times larger than that of existing organic contrast agents. Moreover, such long fluorescence lifetime is almost oxygen-inert, readily realizing both in vitro and in vivo TP-FLIM. This work may set valuable guidance for designing organic semiconducting materials with ultralong fluorescence lifetimes to fulfill the potential of FLIM. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2012-12-01

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

  5. 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.

  6. Fluorescence Lifetime Imaging Microscopy Using Near-Infrared Contrast Agents

    PubMed Central

    Nothdurft, Ralph; Sarder, Pinaki; Bloch, Sharon; Culver, Joseph; Achilefu, Samuel

    2013-01-01

    Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labeled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes’ relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. PMID:22788550

  7. Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

    PubMed

    Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S

    2012-08-01

    Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

  8. Two-Photon Fluorescent Probe for Monitoring Autophagy via Fluorescence Lifetime Imaging.

    PubMed

    Hou, Liling; Ning, Peng; Feng, Yan; Ding, Yaqi; Bai, Lei; Li, Lin; Yu, Haizhu; Meng, Xiangming

    2018-06-19

    We reported the first lysosome targeted two-photon fluorescent probe (Lyso-NP) as a viscosity probe for monitoring autophagy. The fluorescence lifetime of Lyso-NP exhibited an excellent linear relationship with viscosity value ( R 2 = 0.99, x = 0.39). Lyso-NP also showed the specific capability for imaging lysosomal viscosity under two-photon excitation at 860 nm along with good biocompatibility. More importantly, Lyso-NP could be used to monitor the autophagy process in living cells by quantitatively detecting lysosomal viscosity changes during the membrane fusion process via two-photon fluorescence lifetime imaging.

  9. Ns-scaled time-gated fluorescence lifetime imaging for forensic document examination

    NASA Astrophysics Data System (ADS)

    Zhong, Xin; Wang, Xinwei; Zhou, Yan

    2018-01-01

    A method of ns-scaled time-gated fluorescence lifetime imaging (TFLI) is proposed to distinguish different fluorescent substances in forensic document examination. Compared with Video Spectral Comparator (VSC) which can examine fluorescence intensity images only, TFLI can detect questioned documents like falsification or alteration. TFLI system can enhance weak signal by accumulation method. The two fluorescence intensity images of the interval delay time tg are acquired by ICCD and fitted into fluorescence lifetime image. The lifetimes of fluorescence substances are represented by different colors, which make it easy to detect the fluorescent substances and the sequence of handwritings. It proves that TFLI is a powerful tool for forensic document examination. Furthermore, the advantages of TFLI system are ns-scaled precision preservation and powerful capture capability.

  10. Thermally activated delayed fluorescence organic dots for two-photon fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    He, Tingchao; Ren, Can; Li, Zhuohua; Xiao, Shuyu; Li, Junzi; Lin, Xiaodong; Ye, Chuanxiang; Zhang, Junmin; Guo, Lihong; Hu, Wenbo; Chen, Rui

    2018-05-01

    Autofluorescence is a major challenge in complex tissue imaging when molecules present in the biological tissue compete with the fluorophore. This issue may be resolved by designing organic molecules with long fluorescence lifetimes. The present work reports the two-photon absorption (TPA) properties of a thermally activated delayed fluorescence (TADF) molecule with carbazole as the electron donor and dicyanobenzene as the electron acceptor (i.e., 4CzIPN). The results indicate that 4CzIPN exhibits a moderate TPA cross-section (˜9 × 10-50 cm4 s photon-1), high fluorescence quantum yield, and a long fluorescence lifetime (˜1.47 μs). 4CzIPN was compactly encapsulated into an amphiphilic copolymer via nanoprecipitation to achieve water-soluble organic dots. Interestingly, 4CzIPN organic dots have been utilized in applications involving two-photon fluorescence lifetime imaging (FLIM). Our work aptly demonstrates that TADF molecules are promising candidates of nonlinear optical probes for developing next-generation multiphoton FLIM applications.

  11. Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

    NASA Astrophysics Data System (ADS)

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

    2011-07-01

    Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

  12. 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.

  13. Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Pian, Qi; Yao, Ruoyang; Sinsuebphon, Nattawut; Intes, Xavier

    2017-07-01

    Spectrally resolved fluorescence lifetime imaging and spatial multiplexing have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (∼40 ps temporal resolution) using fewer than N2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Förster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.

  14. Fluorescence Lifetime Imaging Microscopy (FLIM) of quantum dots in living cells

    NASA Astrophysics Data System (ADS)

    Nadeau, Jay; Carlini, Lina

    2013-02-01

    Fluorescence lifetime imaging microscopy (FLIM) is an emerging imaging technique that can indicate environmental factors such as pH and redox potential by the effect of these factors on the fluorescence lifetimes of fluorophores. Semiconductor quantum dots (QDs) are highly sensitive to environment and so are ideal for use in FLIM, although certain experimental parameters must be carefully considered for QD imaging to account for their long lifetimes and two-photon behavior. We image the uptake of three types of QDs in cultured fibroblasts and show some preliminary results on the effects of endosomes and lysosomes on QD lifetimes. These results indicate the feasibility of FLIM for studies using QDs in live cells.

  15. Crystallization Kinetics of an Amorphous Pharmaceutical Compound Using Fluorescence-Lifetime-Imaging Microscopy.

    PubMed

    Rautaniemi, Kaisa; Vuorimaa-Laukkanen, Elina; Strachan, Clare J; Laaksonen, Timo

    2018-05-07

    Pharmaceutical scientists are increasingly interested in amorphous drug formulations especially because of their higher dissolution rates. Consequently, the thorough characterization and analysis of these formulations are becoming more and more important for the pharmaceutical industry. Here, fluorescence-lifetime-imaging microscopy (FLIM) was used to monitor the crystallization of an amorphous pharmaceutical compound, indomethacin. Initially, we identified different solid indomethacin forms, amorphous and γ- and α-crystalline, on the basis of their time-resolved fluorescence. All of the studied indomethacin forms showed biexponential decays with characteristic fluorescence lifetimes and amplitudes. Using this information, the crystallization of amorphous indomethacin upon storage in 60 °C was monitored for 10 days with FLIM. The progress of crystallization was detected as lifetime changes both in the FLIM images and in the fluorescence-decay curves extracted from the images. The fluorescence-lifetime amplitudes were used for quantitative analysis of the crystallization process. We also demonstrated that the fluorescence-lifetime distribution of the sample changed during crystallization, and when the sample was not moved between measuring times, the lifetime distribution could also be used for the analysis of the reaction kinetics. Our results clearly show that FLIM is a sensitive and nondestructive method for monitoring solid-state transformations on the surfaces of fluorescent samples.

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

    PubMed Central

    Elson, D S; Jo, J A

    2007-01-01

    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. PMID:19503759

  17. 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.

  18. 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

  19. Multiphoton fluorescence lifetime imaging of chemotherapy distribution in solid tumors

    NASA Astrophysics Data System (ADS)

    Carlson, Marjorie; Watson, Adrienne L.; Anderson, Leah; Largaespada, David A.; Provenzano, Paolo P.

    2017-11-01

    Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.

  20. Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice

    PubMed Central

    Feeks, James A.; Hunter, Jennifer J.

    2017-01-01

    In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina. PMID:28663886

  1. Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.

    PubMed

    Feeks, James A; Hunter, Jennifer J

    2017-05-01

    In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

  2. Fluorescence lifetime imaging with time-gated detection of hyaluronidase using a long lifetime azadioxatriangulenium (ADOTA) fluorophore

    NASA Astrophysics Data System (ADS)

    Chib, Rahul; Requena, Sebastian; Mummert, Mark; Strzhemechny, Yuri M.; Gryczynski, Ignacy; Borejdo, Julian; Gryczynski, Zygmunt; Fudala, Rafal

    2016-12-01

    A fluorescence lifetime imaging probe with a long lifetime was used in combination with time-gating for the detection of hyaluronidase using hyaluronic acid as the probe template. This probe was developed by heavily labeling hyaluronic acid with long lifetime azadioxatriangulenium fluorophores (ADOTA). We used this probe to image hyaluronidase produced by DU-145 prostate cancer cells.

  3. A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation.

    PubMed

    Pelet, S; Previte, M J R; Laiho, L H; So, P T C

    2004-10-01

    Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained analysis. Convergence speed can be greatly accelerated by providing appropriate initial guesses. Realizing that the image morphology often correlates with fluorophore distribution, a global fitting algorithm has been developed to assign initial guesses throughout an image based on a segmentation analysis. This algorithm was tested on both simulated data sets and time-domain lifetime measurements. We have successfully measured fluorophore distribution in fibroblasts stained with Hoechst and calcein. This method further allows second harmonic generation from collagen and elastin autofluorescence to be differentiated in fluorescence lifetime imaging microscopy images of ex vivo human skin. On our experimental measurement, this algorithm increased convergence speed by over two orders of magnitude and achieved significantly better fits. Copyright 2004 Biophysical Society

  4. In vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment.

    PubMed

    Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

    2014-07-01

    Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in vivo fluorescence lifetime imaging with HER2-targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pretreatment size. Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (∼0.13 ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment), the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03 ns. The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. ©2014 American Association for Cancer Research.

  5. In-vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment

    PubMed Central

    Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

    2015-01-01

    Purpose Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in-vivo fluorescence lifetime imaging with HER2 targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. Experimental Design HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice, bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pre-treatment size. Results Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (~0.13ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment) the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03ns. Conclusions The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in-vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. PMID:24671949

  6. 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.

  7. Fluorescence lifetime imaging of calcium flux in neurons in response to pulsed infrared light

    NASA Astrophysics Data System (ADS)

    Walsh, Alex J.; Sedelnikova, Anna; Tolstykh, Gleb P.; Ibey, Bennett L.; Beier, Hope T.

    2017-02-01

    Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate α1 approximations were achieved in images with low photon counts by computing an α1 index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.

  8. Multispectral analog-mean-delay fluorescence lifetime imaging combined with optical coherence tomography

    PubMed Central

    Nam, Hyeong Soo; Kang, Woo Jae; Lee, Min Woo; Song, Joon Woo; Kim, Jin Won; Oh, Wang-Yuhl; Yoo, Hongki

    2018-01-01

    The pathophysiological progression of chronic diseases, including atherosclerosis and cancer, is closely related to compositional changes in biological tissues containing endogenous fluorophores such as collagen, elastin, and NADH, which exhibit strong autofluorescence under ultraviolet excitation. Fluorescence lifetime imaging (FLIm) provides robust detection of the compositional changes by measuring fluorescence lifetime, which is an inherent property of a fluorophore. In this paper, we present a dual-modality system combining a multispectral analog-mean-delay (AMD) FLIm and a high-speed swept-source optical coherence tomography (OCT) to simultaneously visualize the cross-sectional morphology and biochemical compositional information of a biological tissue. Experiments using standard fluorescent solutions showed that the fluorescence lifetime could be measured with a precision of less than 40 psec using the multispectral AMD-FLIm without averaging. In addition, we performed ex vivo imaging on rabbit iliac normal-looking and atherosclerotic specimens to demonstrate the feasibility of the combined FLIm-OCT system for atherosclerosis imaging. We expect that the combined FLIm-OCT will be a promising next-generation imaging technique for diagnosing atherosclerosis and cancer due to the advantages of the proposed label-free high-precision multispectral lifetime measurement. PMID:29675330

  9. Prostate cancer diagnosis with fluorescence lifetime imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Sridharan, Shamira; Gandour-Edwards, Regina F.; Dall'Era, Marc; Marcu, Laura

    2017-02-01

    More than 1 million men in the United States undergo a prostate biopsy procedure annually and approximately 200,000 men receive a diagnosis of prostate cancer. 5-10% of these men have to undergo a repeat biopsy due to insufficient tissue sampling. We are studying the utility of a multi-spectral time resolved fluorescence spectroscopy (MS-TRFS) technique for real-time prostate cancer diagnosis. The MS-TRFS imaging setup, which includes a fiberoptic set-up with a 355nm excitation light source coupled with a blue (450nm) aiming beam, was used to image ex-vivo prostatectomy specimen. The prostate tissue from 11 patients was sectioned at 2mm thickness and the fluorescence lifetime information was overlaid spatially for histology and thus, diagnostic co-registration. Initial results show that fluorescence lifetime in the 390±40nm channel, which measures collagen and elastin signatures, is longer for glandular regions than in the stromal regions. Additionally, lifetime in the 452±45nm channel, corresponding to NAD redox state, is longer in the cancerous glandular region in comparison with the normal glandular regions. Current work is focused on developing real-time quantitative algorithms to combine the fluorescence signatures from the two channels for performing prostate cancer diagnosis on biopsies.

  10. 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

  11. Community detection for fluorescent lifetime microscopy image segmentation

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  12. In vivo tomographic imaging of deep seated cancer using fluorescence lifetime contrast

    PubMed Central

    Rice, William L.; Shcherbakova, Daria M; Verkusha, Vladislav V.; Kumar, Anand T.N.

    2015-01-01

    Preclinical cancer research would benefit from non-invasive imaging methods that allow tracking and visualization of early stage metastasis in vivo. While fluorescent proteins revolutionized intravital microscopy, two major challenges which still remain are tissue autofluorescence and hemoglobin absorption, which act to limit intravital optical techniques to large or subcutaneous tumors. Here we employ time-domain technology for the effective separation of tissue autofluorescence from extrinsic fluorophores, based on their distinct fluorescence lifetimes. Additionally, we employ cancer cells labelled with near infra-red fluorescent proteins (iRFP) to allow deep-tissue imaging. Our results demonstrate that time-domain imaging allows the detection of metastasis in deep-seated organs of living mice with a more than 20-fold increase in sensitivity compared to conventional continuous wave techniques. Furthermore, the distinct fluorescence lifetimes of each iRFP enables lifetime multiplexing of three different tumors, each expressing unique iRFP labels in the same animal. Fluorescence tomographic reconstructions reveal 3D distributions of iRFP720-expressing cancer cells in lungs and brain of live mice, allowing ready longitudinal monitoring of cancer cell fate with greater sensitivity than otherwise currently possible. PMID:25670171

  13. High-Speed Fluorescence Microscopy: Lifetime Imaging in the Biomedical Sciences

    NASA Astrophysics Data System (ADS)

    Periasamy, Ammasi; Wang, Xue F.; Wodnick, Pawel; Gordon, Gerald W.; Kwon, Seongwook; Diliberto, Pamela A.; Herman, Brian

    1995-02-01

    The ability to observe the behavior of living cells and tissues provides unparalleled access to information regarding the organization and dynamics of complex cellular structures. While great strides have been made over the past 30 to 40 years in the design and application of a variety of novel optical microscopic techniques, until recently, it has not been possible to image biological phenomena that occur over very short time periods (nanosecond to millisecond) or over short distances (10 to 1000 [Angstrom capital A, ring]). However, the recent combination of (1) very rapidly gated and sensitive image intensifiers and (2) the ability to deliver fluorescence excitation energy to intact living biological specimens in a pulsed or sinusoidally modulated fashion has allowed such measurements to become a reality through the imaging of the lifetimes of fluorescent molecules. This capability has resulted in the ability to observe the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques. This paper discusses the implementation of a fluorescence lifetime imaging microscope (FLIM) and provides a review of some of the applications of such an instrument. These include measurements of receptor topography and subunit interactions using fluorescence resonance energy transfer (FRET), fluorescence anisotropy of phospholipids in cell membranes, cytosolic free calcium (Ca2+)i and the detection of human papillomavirus (HPV) infection in clinical cervicovaginal smears.

  14. The Phasor Approach to Fluorescence Lifetime Imaging Analysis

    PubMed Central

    Digman, Michelle A.; Caiolfa, Valeria R.; Zamai, Moreno; Gratton, Enrico

    2008-01-01

    Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of fluorescence resonance energy transfer. The analysis of the fluorescence lifetime imaging microscopy (FLIM) data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or nonlinear fitting. The phasor approach has the potential to simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the nonexpert in spectroscopy and data analysis. PMID:17981902

  15. Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo

    NASA Astrophysics Data System (ADS)

    Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V.; Roberts, Michael S.

    2013-02-01

    There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

  16. Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo.

    PubMed

    Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V; Roberts, Michael S

    2013-02-01

    There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

  17. Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

    PubMed

    Bremer, Daniel; Leben, Ruth; Mothes, Ronja; Radbruch, Helena; Niesner, Raluca

    2017-04-03

    Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  18. 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

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

    PubMed

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-05-05

    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.

  20. The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging.

    PubMed

    Poëa-Guyon, Sandrine; Pasquier, Hélène; Mérola, Fabienne; Morel, Nicolas; Erard, Marie

    2013-05-01

    pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  2. Towards two-photon excited endogenous fluorescence lifetime imaging microendoscopy

    PubMed Central

    Hage, C. H.; Leclerc, P.; Brevier, J.; Fabert, M.; Le Nézet, C.; Kudlinski, A.; Héliot, L.; Louradour, F.

    2017-01-01

    In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements. PMID:29359093

  3. Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

    2016-04-01

    Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

  4. Diagnosis of basal cell carcinoma by two photon excited fluorescence combined with lifetime imaging

    NASA Astrophysics Data System (ADS)

    Fan, Shunping; Peng, Xiao; Liu, Lixin; Liu, Shaoxiong; Lu, Yuan; Qu, Junle

    2014-02-01

    Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

  5. Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

    NASA Astrophysics Data System (ADS)

    Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

    2014-03-01

    Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

  6. 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.

  7. Fluorescence lifetime in cardiovascular diagnostics

    NASA Astrophysics Data System (ADS)

    Marcu, Laura

    2010-01-01

    We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications.

  8. Fluorescence lifetime in cardiovascular diagnostics.

    PubMed

    Marcu, Laura

    2010-01-01

    We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications.

  9. Fluorescence lifetime in cardiovascular diagnostics

    PubMed Central

    Marcu, Laura

    2010-01-01

    We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications. PMID:20210432

  10. 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

  11. Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation

    NASA Astrophysics Data System (ADS)

    Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.

    2016-03-01

    Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

  12. A Single-Photon Avalanche Diode Array for Fluorescence Lifetime Imaging Microscopy.

    PubMed

    Schwartz, David Eric; Charbon, Edoardo; Shepard, Kenneth L

    2008-11-21

    We describe the design, characterization, and demonstration of a fully integrated single-photon avalanche diode (SPAD) imager for use in time-resolved fluorescence imaging. The imager consists of a 64-by-64 array of active SPAD pixels and an on-chip time-to-digital converter (TDC) based on a delay-locked loop (DLL) and calibrated interpolators. The imager can perform both standard time-correlated single-photon counting (TCSPC) and an alternative gated-window detection useful for avoiding pulse pile-up when measuring bright signal levels. To illustrate the use of the imager, we present measurements of the decay lifetimes of fluorescent dyes of several types with a timing resolution of 350 ps.

  13. A Single-Photon Avalanche Diode Array for Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Schwartz, David Eric; Charbon, Edoardo; Shepard, Kenneth L.

    2013-01-01

    We describe the design, characterization, and demonstration of a fully integrated single-photon avalanche diode (SPAD) imager for use in time-resolved fluorescence imaging. The imager consists of a 64-by-64 array of active SPAD pixels and an on-chip time-to-digital converter (TDC) based on a delay-locked loop (DLL) and calibrated interpolators. The imager can perform both standard time-correlated single-photon counting (TCSPC) and an alternative gated-window detection useful for avoiding pulse pile-up when measuring bright signal levels. To illustrate the use of the imager, we present measurements of the decay lifetimes of fluorescent dyes of several types with a timing resolution of 350 ps. PMID:23976789

  14. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

  15. Fluorescence Lifetime Techniques in Medical Applications

    PubMed Central

    Marcu, Laura

    2012-01-01

    This article presents an overview of time-resolved (lifetime) fluorescence techniques used in biomedical diagnostics. In particular, we review the development of time-resolved fluorescence spectroscopy (TRFS) and fluorescence lifetime imaging (FLIM) instrumentation and associated methodologies which allows for in vivo characterization and diagnosis of biological tissues. Emphasis is placed on the translational research potential of these techniques and on evaluating whether intrinsic fluorescence signals provide useful contrast for the diagnosis of human diseases including cancer (gastrointestinal tract, lung, head and neck, and brain), skin and eye diseases, and atherosclerotic cardiovascular disease. PMID:22273730

  16. Fluorescence lifetime imaging to differentiate bound from unbound ICG-cRGD both in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Stegehuis, Paulien L.; Boonstra, Martin C.; de Rooij, Karien E.; Powolny, François E.; Sinisi, Riccardo; Homulle, Harald; Bruschini, Claudio; Charbon, Edoardo; van de Velde, Cornelis J. H.; Lelieveldt, Boudewijn P. F.; Vahrmeijer, Alexander L.; Dijkstra, Jouke; van de Giessen, Martijn

    2015-03-01

    Excision of the whole tumor is crucial, but remains difficult for many tumor types. Fluorescence lifetime imaging could be helpful intraoperative to differentiate normal from tumor tissue. In this study we investigated the difference in fluorescence lifetime imaging of indocyanine green coupled to cyclic RGD free in solution/serum or bound to integrins e.g. in tumors. The U87-MG glioblastoma cell line, expressing high integrin levels, was cultured to use in vitro and to induce 4 subcutaneous tumors in a-thymic mice (n=4). Lifetimes of bound and unbound probe were measured with an experimental time-domain single-photon avalanche diode array (time resolution <100ps). In vivo measurements were taken 30-60 minutes after intravenous injection, and after 24 hours. The in vitro lifetime of the fluorophores was similar at different concentrations (20, 50 and 100μM) and showed a statistically significant higher lifetime (p<0.001) of bound probe compared to unbound probe. In vivo, lifetimes of the fluorophores in tumors were significantly higher (p<0.001) than at the control site (tail) at 30-60 minutes after probe injection. Lifetimes after 24 hours confirmed tumor-specific binding (also validated by fluorescence intensity images). Based on the difference in lifetime imaging, it can be concluded that it is feasible to separate between bound and unbound probes in vivo.

  17. Fluorescence lifetime imaging of endogenous molecules in live mouse cancer models (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Svindrych, Zdenek; Wang, Tianxiong; Hu, Song; Periasamy, Ammasi

    2017-02-01

    NADH and FAD are important endogenous fluorescent coenzymes participating in key enzymatic reactions of cellular metabolism. While fluorescence intensities of NADH and FAD have been used to determine the redox state of cells and tissues, this simple approach breaks down in the case of deep-tissue intravital imaging due to depth- and wavelength-dependent light absorption and scattering. To circumvent this limitation, our research focuses on fluorescence lifetimes of two-photon excited NADH and FAD emission to study the metabolic state of live tissues. In our custom-built scanning microscope we combine tunable femtosecond Ti:sapphire laser (operating at 740 nm for NADH excitation and 890 nm for FAD excitation), two GaAsP hybrid detectors for registering individual fluorescence photons and two Becker and Hickl time correlator boards for high precision lifetime measurements. Together with our rigorous FLIM analysis approach (including image segmentation, multi-exponential decay fitting and detailed statistical analysis) we are able to detect metabolic changes in cancer xenografts (human pancreatic cancer MPanc96 cells injected subcutaneously into the ear of an immunodeficient nude mouse), relative to surrounding healthy tissue. Advantageously, with the same instrumentation we can also take high-resolution and high-contrast images of second harmonic signal (SHG) originating from collagen fibers of both the healthy skin and the growing tumor. The combination of metabolic measurements (NADH and FAD lifetime) and morphological information (collagen SHG) allows us to follow the tumor growth in live mouse model and the changes in tumor microenvironment.

  18. Chlorophyll fluorescence lifetime imaging provides new insight into the chlorosis induced by plant virus infection.

    PubMed

    Lei, Rong; Jiang, Hongshan; Hu, Fan; Yan, Jin; Zhu, Shuifang

    2017-02-01

    Leaf chlorosis induced by plant virus infection has a short fluorescence lifetime, which reflects damaged photosynthetic complexes and degraded chloroplasts. Plant viruses often induce chlorosis and necrosis, which are intimately related to photosynthetic functions. Chlorophyll fluorescence lifetime measurement is a valuable noninvasive tool for analyzing photosynthetic processes and is a sensitive indicator of the environment surrounding the fluorescent molecules. In this study, our central goal was to explore the effect of viral infection on photosynthesis by employing chlorophyll fluorescence lifetime imaging (FLIM), steady-state fluorescence, non-photochemical quenching (NPQ), transmission electron microscopy (TEM), and pigment analysis. The data indicated that the chlorophyll fluorescence lifetime of chlorotic leaves was significantly shorter than that of healthy control leaves, and the fitted short lifetime component of chlorophyll fluorescence of chlorotic leaves was dominant. This dominant short lifetime component may result from damage to the structure of thylakoid, which was confirmed by TEM. The NPQ value of chlorotic leaves was slightly higher than that of healthy green leaves, which can be explained by increased neoxanthin, lutein and violaxanthin content relative to chlorophyll a. The difference in NPQ is slight, but FLIM can provide simple and direct characterization of PSII structure and photosynthetic function. Therefore, this technique shows great potential as a simple and rapid method for studying mechanisms of plant virus infection.

  19. Fluorescence lifetime imaging and Fourier transform infrared spectroscopy of Michelangelo's David.

    PubMed

    Comelli, Daniela; Valentini, Gianluca; Cubeddu, Rinaldo; Toniolo, Lucia

    2005-09-01

    We developed a combined procedure for the analysis of works of art based on a portable system for fluorescence imaging integrated with analytical measurements on microsamples. The method allows us to localize and identify organic and inorganic compounds present on the surface of artworks. The fluorescence apparatus measures the temporal and spectral features of the fluorescence emission, excited by ultraviolet (UV) laser pulses. The kinetic of the emission is studied through a fluorescence lifetime imaging system, while an optical multichannel analyzer measures the fluorescence spectra of selected points. The chemical characterization of the compounds present on the artistic surfaces is then performed by means of analytical measurements on microsamples collected with the assistance of the fluorescence maps. The previous concepts have been successfully applied to study the contaminants on the surface of Michelangelo's David. The fluorescence analysis combined with Fourier transform infrared (FT-IR) measurements revealed the presence of beeswax, which permeates most of the statue surface, and calcium oxalate deposits mainly arranged in vertical patterns and related to rain washing.

  20. Global analysis of microscopic fluorescence lifetime images using spectral segmentation and a digital micromirror spatial illuminator.

    PubMed

    Bednarkiewicz, Artur; Whelan, Maurice P

    2008-01-01

    Fluorescence lifetime imaging (FLIM) is very demanding from a technical and computational perspective, and the output is usually a compromise between acquisition/processing time and data accuracy and precision. We present a new approach to acquisition, analysis, and reconstruction of microscopic FLIM images by employing a digital micromirror device (DMD) as a spatial illuminator. In the first step, the whole field fluorescence image is collected by a color charge-coupled device (CCD) camera. Further qualitative spectral analysis and sample segmentation are performed to spatially distinguish between spectrally different regions on the sample. Next, the fluorescence of the sample is excited segment by segment, and fluorescence lifetimes are acquired with a photon counting technique. FLIM image reconstruction is performed by either raster scanning the sample or by directly accessing specific regions of interest. The unique features of the DMD illuminator allow the rapid on-line measurement of global good initial parameters (GIP), which are supplied to the first iteration of the fitting algorithm. As a consequence, a decrease of the computation time required to obtain a satisfactory quality-of-fit is achieved without compromising the accuracy and precision of the lifetime measurements.

  1. Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

    PubMed Central

    Chan, Jacky C. K.; Diebold, Eric D.; Buckley, Brandon W.; Mao, Sien; Akbari, Najva; Jalali, Bahram

    2014-01-01

    Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat’s-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures. PMID:25574449

  2. Breast cancer margin delineation with fluorescence lifetime imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Phipps, Jennifer E.; Gorpas, Dimitris; Darrow, Morgan; Unger, Jakob; Bold, Richard; Marcu, Laura

    2017-02-01

    The current standard of care for early stages of breast cancer is breast-conserving surgery (BCS). BCS involves a lumpectomy procedure, during which the tumor is removed with a rim of normal tissue-if cancer cells found in that rim of tissue, it is called a positive margin and means part of the tumor remains in the breast. Currently there is no method to determine if cancer cells exist at the margins of lumpectomy specimens aside from time-intensive histology methods that result in reoperations in up to 38% of cases. We used fluorescence lifetime imaging (FLIm) to measure time-resolved autofluorescence from N=13 ex vivo human breast cancer specimens (N=10 patients undergoing lumpectomy or mastectomy) and compared our results to histology. Tumor (both invasive and ductal carcinoma in situ), fibrous tissue, fat and fat necrosis have unique fluorescence signatures. For instance, between 500-580 nm, fluorescence lifetime of tumor was shortest (4.7 +/- 0.4 ns) compared to fibrous tissue (5.5 +/- 0.7 ns) and fat (7.0 +/- 0.1 ns), P<0.05 (ANOVA). These differences are due to the biochemical properties of lipid, nicotineamide adenine dinucleotide (NADH) and collagen fibers in the fat, tumor and fibrous tissue, respectively. Additionally, the FLIm data is augmented to video of the breast tissue with image processing algorithms that track a blue (450 nm) aiming beam used in parallel with the 355 nm excitation beam. This allows for accurate histologic co-registration and in the future will allow for three-dimensional lumpectomy surfaces to be imaged for cancer margin delineation.

  3. Rotational multispectral fluorescence lifetime imaging and intravascular ultrasound: bimodal system for intravascular applications

    PubMed Central

    Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Gorpas, Dimitris; Fatakdawala, Hussain; Marcu, Laura

    2014-01-01

    Abstract. We report the development and validation of a hybrid intravascular diagnostic system combining multispectral fluorescence lifetime imaging (FLIm) and intravascular ultrasound (IVUS) for cardiovascular imaging applications. A prototype FLIm system based on fluorescence pulse sampling technique providing information on artery biochemical composition was integrated with a commercial IVUS system providing information on artery morphology. A customized 3-Fr bimodal catheter combining a rotational side-view fiberoptic and a 40-MHz IVUS transducer was constructed for sequential helical scanning (rotation and pullback) of tubular structures. Validation of this bimodal approach was conducted in pig heart coronary arteries. Spatial resolution, fluorescence detection efficiency, pulse broadening effect, and lifetime measurement variability of the FLIm system were systematically evaluated. Current results show that this system is capable of temporarily resolving the fluorescence emission simultaneously in multiple spectral channels in a single pullback sequence. Accurate measurements of fluorescence decay characteristics from arterial segments can be obtained rapidly (e.g., 20 mm in 5 s), and accurate co-registration of fluorescence and ultrasound features can be achieved. The current finding demonstrates the compatibility of FLIm instrumentation with in vivo clinical investigations and its potential to complement conventional IVUS during catheterization procedures. PMID:24898604

  4. Characterization of Retinitis Pigmentosa Using Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO).

    PubMed

    Andersen, Karl M; Sauer, Lydia; Gensure, Rebekah H; Hammer, Martin; Bernstein, Paul S

    2018-06-01

    We investigated fundus autofluorescence (FAF) lifetimes in patients with retinitis pigmentosa (RP) using fluorescence lifetime imaging ophthalmoscopy (FLIO). A total of 33 patients (mean age, 40.0 ± 17.0 years) with RP and an age-matched healthy group were included. The Heidelberg FLIO was used to detect FAF decays in short (SSC; 498-560 nm) and long (LSC; 560-720 nm) spectral channels. We investigated a 30° retinal field and calculated the amplitude-weighted mean fluorescence lifetime (τ m ). Additionally, macular pigment measurements, macular optical coherence tomography (OCT) scans, fundus photographs, visual fields, and fluorescein angiograms were recorded. Genetic studies were performed on nearly all patients. In RP, FLIO shows a typical pattern of prolonged τ m in atrophic regions in the outer macula (SSC, 419 ± 195 ps; LSC, 401 ± 111 ps). Within the relatively preserved retina in the macular region, ring-shaped patterns were found, most distinctive in patients with autosomal dominant RP inheritance. Mean FAF lifetimes were shortened in rings in the LSC. Central areas remained relatively unaffected. FLIO uniquely presents a distinct and specific signature in eyes affected with RP. The ring patterns show variations that indicate genetically determined pathologic processes. Shortening of FAF lifetimes in the LSC may indicate disease progression, as was previously demonstrated for Stargardt disease. Therefore, FLIO might be able to indicate disease progression in RP as well. Hyperfluorescent FLIO rings with short FAF lifetimes may provide insight into the pathophysiologic disease status of RP-affected retinas potentially providing a more detailed assessment of disease progression.

  5. Differentiating quiescent cancer cell populations in heterogeneous samples with fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Heaster, Tiffany M.; Walsh, Alex J.; Skala, Melissa C.

    2016-03-01

    Measurement of relative fluorescence intensities of NAD(P)H and FAD with fluorescence lifetime imaging (FLIM) allows metabolic characterization of cancerous populations and correlation to treatment response. However, quiescent populations of cancer cells introduce heterogeneity to the tumor and exhibit resistance to standard therapies, requiring a better understanding of this influence on treatment outcome. Significant differences were observed between proliferating and quiescent cell populations upon comparison of respective redox ratios (p<0.05) and FAD lifetimes (p<0.05) across monolayers and in mixed samples. These results demonstrate that metabolic activity may function as a marker for separation and characterization of proliferating and quiescent cancer cells within mixed samples, contributing to comprehensive investigation of heterogeneity-dependent drug resistance.

  6. Low-frequency wide-field fluorescence lifetime imaging using a high-power near-infrared light-emitting diode light source

    PubMed Central

    Gioux, Sylvain; Lomnes, Stephen J.; Choi, Hak Soo; Frangioni, John V.

    2010-01-01

    Fluorescence lifetime imaging (FLi) could potentially improve exogenous near-infrared (NIR) fluorescence imaging, because it offers the capability of discriminating a signal of interest from background, provides real-time monitoring of a chemical environment, and permits the use of several different fluorescent dyes having the same emission wavelength. We present a high-power, LED-based, NIR light source for the clinical translation of wide-field (larger than 5 cm in diameter) FLi at frequencies up to 35 MHz. Lifetime imaging of indocyanine green (ICG), IRDye 800-CW, and 3,3′-diethylthiatricarbocyanine iodide (DTTCI) was performed over a large field of view (10 cm by 7.5 cm) using the LED light source. For comparison, a laser diode light source was employed as a gold standard. Experiments were performed both on the bench by diluting the fluorescent dyes in various chemical environments in Eppendorf tubes, and in vivo by injecting the fluorescent dyes mixed in Matrigel subcutaneously into CD-1 mice. Last, measured fluorescence lifetimes obtained using the LED and the laser diode sources were compared with those obtained using a state-of-the-art time-domain imaging system and with those previously described in the literature. On average, lifetime values obtained using the LED and the laser diode light sources were consistent, exhibiting a mean difference of 3% from the expected values and a coefficient of variation of 12%. Taken together, our study offers an alternative to laser diodes for clinical translation of FLi and explores the use of relatively low frequency modulation for in vivo imaging. PMID:20459250

  7. Monitoring macular pigment changes in macular holes using fluorescence lifetime imaging ophthalmoscopy.

    PubMed

    Sauer, Lydia; Peters, Sven; Schmidt, Johanna; Schweitzer, Dietrich; Klemm, Matthias; Ramm, Lisa; Augsten, Regine; Hammer, Martin

    2017-08-01

    To investigate the impact of macular pigment (MP) on fundus autofluorescence (FAF) lifetimes in vivo by characterizing full-thickness idiopathic macular holes (MH) and macular pseudo-holes (MPH). A total of 37 patients with MH and 52 with MPH were included. Using the fluorescence lifetime imaging ophthalmoscope (FLIO), based on a Heidelberg Engineering Spectralis system, a 30° retinal field was investigated. FAF decays were detected in a short (498-560 nm; ch1) and long (560-720 nm; ch2) wavelength channel. τ m , the mean fluorescence lifetime, was calculated from a three-exponential approximation of the FAF decays. Macular coherence tomography scans were recorded, and macular pigment's optical density (MPOD) was measured (one-wavelength reflectometry). Two MH subgroups were analysed according to the presence or absence of an operculum above the MH. A total of 17 healthy fellow eyes were included. A longitudinal FAF decay examination was conducted in nine patients, which were followed up after surgery and showed a closed MH. In MH without opercula, significant τ m differences (p < 0.001) were found between the hole area (MHa) and surrounding areas (MHb) (ch1: MHa 238 ± 64 ps, MHb 181 ± 78 ps; ch2: MHa 275 ± 49 ps, MHb 223 ± 48 ps), as well as between MHa and healthy eyes or closed MH. Shorter τ m , adjacent to the hole, can be assigned to areas with equivalently higher MPOD. Opercula containing MP also show short τ m . In MPH, the intactness of the Hele fibre layer is associated with shortest τ m . Shortest τ m originates from MP-containing retinal layers, especially from the Henle fibre layer. Fluorescence lifetime imaging ophthalmoscope (FLIO) provides information on the MP distribution, the pathogenesis and topology of MH. Macular pigment (MP) fluorescence may provide a biomarker for monitoring pathological changes in retinal diseases. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  8. Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Rudkouskaya, Alena; Sinsuebphon, Nattawut; Intes, Xavier; Mazurkiewicz, Joseph E.; Barroso, Margarida

    2017-02-01

    To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

  9. Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate.

    PubMed

    Eibl, Matthias; Karpf, Sebastian; Weng, Daniel; Hakert, Hubertus; Pfeiffer, Tom; Kolb, Jan Philip; Huber, Robert

    2017-07-01

    Two-photon-excited fluorescence lifetime imaging microscopy (FLIM) is a chemically specific 3-D sensing modality providing valuable information about the microstructure, composition and function of a sample. However, a more widespread application of this technique is hindered by the need for a sophisticated ultra-short pulse laser source and by speed limitations of current FLIM detection systems. To overcome these limitations, we combined a robust sub-nanosecond fiber laser as the excitation source with high analog bandwidth detection. Due to the long pulse length in our configuration, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. In this paper, we show high quality FLIM images acquired at a pixel rate of 1 MHz. This approach is a promising candidate for an easy-to-use and benchtop FLIM system to make this technique available to a wider research community.

  10. Monte Carlo based method for fluorescence tomographic imaging with lifetime multiplexing using time gates

    PubMed Central

    Chen, Jin; Venugopal, Vivek; Intes, Xavier

    2011-01-01

    Time-resolved fluorescence optical tomography allows 3-dimensional localization of multiple fluorophores based on lifetime contrast while providing a unique data set for improved resolution. However, to employ the full fluorescence time measurements, a light propagation model that accurately simulates weakly diffused and multiple scattered photons is required. In this article, we derive a computationally efficient Monte Carlo based method to compute time-gated fluorescence Jacobians for the simultaneous imaging of two fluorophores with lifetime contrast. The Monte Carlo based formulation is validated on a synthetic murine model simulating the uptake in the kidneys of two distinct fluorophores with lifetime contrast. Experimentally, the method is validated using capillaries filled with 2.5nmol of ICG and IRDye™800CW respectively embedded in a diffuse media mimicking the average optical properties of mice. Combining multiple time gates in one inverse problem allows the simultaneous reconstruction of multiple fluorophores with increased resolution and minimal crosstalk using the proposed formulation. PMID:21483610

  11. In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

    PubMed Central

    Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V.; Knutson, Jay R.; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

    2012-01-01

    One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

  12. CMOS image sensor with lateral electric field modulation pixels for fluorescence lifetime imaging with sub-nanosecond time response

    NASA Astrophysics Data System (ADS)

    Li, Zhuo; Seo, Min-Woong; Kagawa, Keiichiro; Yasutomi, Keita; Kawahito, Shoji

    2016-04-01

    This paper presents the design and implementation of a time-resolved CMOS image sensor with a high-speed lateral electric field modulation (LEFM) gating structure for time domain fluorescence lifetime measurement. Time-windowed signal charge can be transferred from a pinned photodiode (PPD) to a pinned storage diode (PSD) by turning on a pair of transfer gates, which are situated beside the channel. Unwanted signal charge can be drained from the PPD to the drain by turning on another pair of gates. The pixel array contains 512 (V) × 310 (H) pixels with 5.6 × 5.6 µm2 pixel size. The imager chip was fabricated using 0.11 µm CMOS image sensor process technology. The prototype sensor has a time response of 150 ps at 374 nm. The fill factor of the pixels is 5.6%. The usefulness of the prototype sensor is demonstrated for fluorescence lifetime imaging through simulation and measurement results.

  13. In vivo fluorescence lifetime optical projection tomography

    PubMed Central

    McGinty, James; Taylor, Harriet B.; Chen, Lingling; Bugeon, Laurence; Lamb, Jonathan R.; Dallman, Margaret J.; French, Paul M. W.

    2011-01-01

    We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions. PMID:21559145

  14. Fluorescence lifetime based bioassays

    NASA Astrophysics Data System (ADS)

    Meyer-Almes, Franz-Josef

    2017-12-01

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

  15. 3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

    PubMed Central

    Choi, Heejin; Tzeranis, Dimitrios S.; Cha, Jae Won; Clémenceau, Philippe; de Jong, Sander J. G.; van Geest, Lambertus K.; Moon, Joong Ho; Yannas, Ioannis V.; So, Peter T. C.

    2012-01-01

    Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude. PMID:23187477

  16. Pinhole shifting lifetime imaging microscopy

    PubMed Central

    Ramshesh, Venkat K.; Lemasters, John J.

    2009-01-01

    Lifetime imaging microscopy is a powerful tool to probe biological phenomena independent of luminescence intensity and fluorophore concentration. We describe time-resolved imaging of long-lifetime luminescence with an unmodified commercial laser scanning confocal/multiphoton microscope. The principle of the measurement is displacement of the detection pinhole to collect delayed luminescence from a position lagging the rasting laser beam. As proof of principle, luminescence from microspheres containing europium (Eu3+), a red emitting probe, was compared to that of short-lifetime green-fluorescing microspheres and/or fluorescein and rhodamine in solution. Using 720-nm two-photon excitation and a pinhole diameter of 1 Airy unit, the short-lifetime fluorescence of fluorescein, rhodamine and green microspheres disappeared much more rapidly than the long-lifetime phosphorescence of Eu3+ microspheres as the pinhole was repositioned in the lagging direction. In contrast, repositioning of the pinhole in the leading and orthogonal directions caused equal loss of short- and long-lifetime luminescence. From measurements at different lag pinhole positions, a lifetime of 270 μs was estimated for the Eu3+ microspheres, consistent with independent measurements. This simple adaptation is the basis for quantitative 3-D lifetime imaging microscopy. PMID:19123648

  17. Dead-time correction for high-throughput fluorescence lifetime imaging microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Enderlein, Joerg; Ruhlandt, Daja; Chithik, Anna; Ebrecht, René; Wouters, Fred S.; Gregor, Ingo

    2016-02-01

    Fluorescence lifetime microscopy has become an important method of bioimaging, allowing not only to record intensity and spectral, but also lifetime information across an image. One of the most widely used methods of FLIM is based on Time-Correlated Single Photon Counting (TCSPC). In TCSPC, one determines this curve by exciting molecules with a periodic train of short laser pulses, and then measuring the time delay between the first recorded fluorescence photon after each exciting laser pulse. An important technical detail of TCSPC measurements is the fact that the delay times between excitation laser pulses and resulting fluorescence photons are always measured between a laser pulse and the first fluorescence photon which is detected after that pulse. At high count rates, this leads to so-called pile-up: ``early'' photons eclipse long-delay photons, resulting in heavily skewed TCSPC histograms. To avoid pile-up, a rule of thumb is to perform TCSPC measurements at photon count rates which are at least hundred times smaller than the laser-pulse excitation rate. The downside of this approach is that the fluorescence-photon count-rate is restricted to a value below one hundredth of the laser-pulse excitation-rate, reducing the overall speed with which a fluorescence signal can be measured. We present a new data evaluation method which provides pile-up corrected fluorescence decay estimates from TCSPC measurements at high count rates, and we demonstrate our method on FLIM of fluorescently labeled cells.

  18. GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method

    PubMed Central

    Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae

    2016-01-01

    We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection. PMID:28018724

  19. A current-assisted CMOS photonic sampler with two taps for fluorescence lifetime sensing

    NASA Astrophysics Data System (ADS)

    Ingelberts, H.; Kuijk, M.

    2016-04-01

    Imaging based on fluorescence lifetime is becoming increasingly important in medical and biological applications. State-of- the-art fluorescence lifetime microscopes either use bulky and expensive gated image intensifiers coupled to a CCD or single-photon detectors in a slow scanning setup. Numerous attempts are being made to create compact, cost-effective all- CMOS imagers for fluorescence lifetime sensing. Single-photon avalanche diode (SPAD) imagers can have very good timing resolution and noise characteristics but have low detection efficiency. Another approach is to use CMOS imagers based on demodulation detectors. These imagers can be either very fast or very efficient but it remains a challenge to combine both characteristics. Recently we developed the current-assisted photonic sampler (CAPS) to tackle these problems and in this work, we present a new CAPS with two detection taps that can sample a fluorescence decay in two time windows. In the case of mono-exponential decays, two windows provide enough information to resolve the lifetime. We built an electro-optical setup to characterize the detector and use it for fluorescence lifetime measurements. It consists of a supercontinuum pulsed laser source, an optical system to focus light into the detector and picosecond timing electronics. We describe the structure and operation of the two-tap CAPS and provide basic characterization of the speed performance at multiple wavelengths in the visible and near-infrared spectrum. We also record fluorescence decays of different visible and NIR fluorescent dyes and provide different methods to resolve the fluorescence lifetime.

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

    PubMed

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

    2015-11-01

    Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

    Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm2 tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, <1 μm lateral and 3.5 μm axial resolution. FLIM-RCM imaging was performed on a tissue phantom, normal porcine buccal mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

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

    PubMed Central

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

    2015-01-01

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

  3. Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells

    NASA Astrophysics Data System (ADS)

    Rück, Angelika; Hauser, Carmen; Mosch, Simone; Kalinina, Sviatlana

    2014-09-01

    Fluorescence-guided diagnosis of tumor tissue is in many cases insufficient, because false positive results interfere with the outcome. Improvement through observation of cell metabolism might offer the solution, but needs a detailed understanding of the origin of autofluorescence. With respect to this, spectrally resolved multiphoton fluorescence lifetime imaging was investigated to analyze cell metabolism in metabolic phenotypes of malignant and nonmalignant oral mucosa cells. The time-resolved fluorescence characteristics of NADH were measured in cells of different origins. The fluorescence lifetime of bound and free NADH was calculated from biexponential fitting of the fluorescence intensity decay within different spectral regions. The mean lifetime was increased from nonmalignant oral mucosa cells to different squamous carcinoma cells, where the most aggressive cells showed the longest lifetime. In correlation with reports in the literature, the total amount of NADH seemed to be less for the carcinoma cells and the ratio of free/bound NADH was decreased from nonmalignant to squamous carcinoma cells. Moreover for squamous carcinoma cells a high concentration of bound NADH was found in cytoplasmic organelles (mainly mitochondria). This all together indicates that oxidative phosphorylation and a high redox potential play an important role in the energy metabolism of these cells.

  4. Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity

    PubMed Central

    Kwok, Showming; Lee, Claudia; Sánchez, Susana A.; Hazlett, Theodore L.; Gratton, Enrico; Hayashi, Yasunori

    2008-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo. PMID:18302935

  5. Efficacy of photodynamic therapy against larvae of Aedes aegypti: confocal microscopy and fluorescence-lifetime imaging

    NASA Astrophysics Data System (ADS)

    de Souza, L. M.; Pratavieira, S.; Inada, N. M.; Kurachi, C.; Corbi, J.; Guimarães, F. E. G.; Bagnato, V. S.

    2014-03-01

    Recently a few demonstration on the use of Photodynamic Reaction as possibility to eliminate larvae that transmit diseases for men has been successfully demonstrated. This promising tool cannot be vastly used due to many problems, including the lake of investigation concerning the mechanisms of larvae killing as well as security concerning the use of photosensitizers in open environment. In this study, we investigate some of the mechanisms in which porphyrin (Photogem) is incorporated on the Aedes aegypti larvae previously to illumination and killing. Larvae at second instar were exposed to the photosensitizer and after 30 minutes imaged by a confocal fluorescence microscope. It was observed the presence of photosensitizer in the gut and at the digestive tract of the larva. Fluorescence-Lifetime Imaging showed greater photosensitizer concentration in the intestinal wall of the samples, which produces a strong decrease of the Photogem fluorescence lifetime. For Photodynamic Therapy exposition to different light doses and concentrations of porphyrin were employed. Three different light sources (LED, Fluorescent lamp, Sun light) also were tested. Sun light and fluorescent lamp shows close to 100% of mortality after 24 hrs. of illumination. These results indicate the potential use of photodynamic effect against the LARVAE of Aedes aegypti.

  6. Quantification of cellular autofluorescence of human skin using multiphoton tomography and fluorescence lifetime imaging in two spectral detection channels

    PubMed Central

    Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Neil, Mark A. A.; König, Karsten; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

    2011-01-01

    We explore the diagnostic potential of imaging endogenous fluorophores using two photon microscopy and fluorescence lifetime imaging (FLIM) in human skin with two spectral detection channels. Freshly excised benign dysplastic nevi (DN) and malignant nodular Basal Cell Carcinomas (nBCCs) were excited at 760 nm. The resulting fluorescence signal was binned manually on a cell by cell basis. This improved the reliability of fitting using a double exponential decay model and allowed the fluorescence signatures from different cell populations within the tissue to be identified and studied. We also performed a direct comparison between different diagnostic groups. A statistically significant difference between the median mean fluorescence lifetime of 2.79 ns versus 2.52 ns (blue channel, 300-500 nm) and 2.08 ns versus 1.33 ns (green channel, 500-640 nm) was found between nBCCs and DN respectively, using the Mann-Whitney U test (p < 0.01). Further differences in the distribution of fluorescence lifetime parameters and inter-patient variability are also discussed. PMID:22162820

  7. Phasor-based single-molecule fluorescence lifetime imaging using a wide-field photon-counting detector

    PubMed Central

    Colyer, R.; Siegmund, O.; Tremsin, A.; Vallerga, J.; Weiss, S.; Michalet, X.

    2011-01-01

    Fluorescence lifetime imaging (FLIM) is a powerful approach to studying the immediate environment of molecules. For example, it is used in biology to study changes in the chemical environment, or to study binding processes, aggregation, and conformational changes by measuring Förster resonance energy transfer (FRET) between donor and acceptor fluorophores. FLIM can be acquired by time-domain measurements (time-correlated single-photon counting) or frequency-domain measurements (with PMT modulation or digital frequency domain acquisition) in a confocal setup, or with wide-field systems (using time-gated cameras). In the best cases, the resulting data is analyzed in terms of multicomponent fluorescence lifetime decays with demanding requirements in terms of signal level (and therefore limited frame rate). Recently, the phasor approach has been proposed as a powerful alternative for fluorescence lifetime analysis of FLIM, ensemble, and single-molecule experiments. Here we discuss the advantages of combining phasor analysis with a new type of FLIM acquisition hardware presented previously, consisting of a high temporal and spatial resolution wide-field single-photon counting device (the H33D detector). Experimental data with live cells and quantum dots will be presented as an illustration of this new approach. PMID:21625298

  8. Time-resolved nanosecond fluorescence lifetime imaging and picosecond infrared spectroscopy of combretastatin A-4 in solution and in cellular systems

    NASA Astrophysics Data System (ADS)

    Bisby, Roger H.; Botchway, Stanley W.; Greetham, Greg M.; Hadfield, John A.; McGown, Alan T.; Parker, Anthony W.; Scherer, Kathrin M.; Towrie, Mike

    2012-08-01

    Fluorescence lifetime images of intrinsic fluorescence obtained with two-photon excitation at 630 nm are shown following uptake of a series of E-combretastatins into live cells, including human umbilical vein endothelial cells (HUVECs) that are the target for the anticancer activity of combretastatins. Images show distribution of the compounds within the cell cytoplasm and in structures identified as lipid droplets by comparison with images obtained following Nile red staining of the same cells. The intracellular fluorescent lifetimes are generally longer than in fluid solution as a consequence of the high viscosity of the cellular environment. Following incubation, the intracellular concentrations of a fluorinated derivative of E-combretastatin A-4 in HUVECs are between two and three orders of magnitude higher than the concentration in the surrounding medium. Evidence is presented to indicate that at moderate laser powers (up to 6 mW), it is possible to isomerize up to 25% of the combretastatin within the femtolitre focal volume of the femtosecond laser beam. This suggests that it may be possible to activate the E-combretastatin (with low cellular toxicity) to the Z-isomer with high anticancer drug activity using two-photon irradiation. The isomerization of Z- and E-combretastatins by 266 nm irradiation has been probed by ultrafast time-resolved infrared spectroscopy. Results for the E-isomer show a rapid loss of excess vibrational energy in the excited state with a lifetime of 7 ps, followed by a slower process with a lifetime of 500 ps corresponding to the return to the ground state as also determined from the fluorescence lifetime. In contrast, the Z-isomer, whilst also appearing to undergo a rapid cooling of the initial excited state, has a much shorter overall excited state lifetime of 14 ps. DedicationThis paper is dedicated to the memory of Professor Christopher G Morgan (1949-2011). He was a valued colleague and friend at the University of Salford and made

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

    NASA Astrophysics Data System (ADS)

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

    2004-04-01

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

  10. Long-term fluorescence lifetime imaging of a genetically encoded sensor for caspase-3 activity in mouse tumor xenografts

    NASA Astrophysics Data System (ADS)

    Zherdeva, Victoria; Kazachkina, Natalia I.; Shcheslavskiy, Vladislav; Savitsky, Alexander P.

    2018-03-01

    Caspase-3 is known for its role in apoptosis and programmed cell death regulation. We detected caspase-3 activation in vivo in tumor xenografts via shift of mean fluorescence lifetimes of a caspase-3 sensor. We used the genetically encoded sensor TR23K based on the red fluorescent protein TagRFP and chromoprotein KFP linked by 23 amino acid residues (TagRFP-23-KFP) containing a specific caspase cleavage DEVD motif to monitor the activity of caspase-3 in tumor xenografts by means of fluorescence lifetime imaging-Forster resonance energy transfer. Apoptosis was induced by injection of paclitaxel for A549 lung adenocarcinoma and etoposide and cisplatin for HEp-2 pharynx adenocarcinoma. We observed a shift in lifetime distribution from 1.6 to 1.9 ns to 2.1 to 2.4 ns, which indicated the activation of caspase-3. Even within the same tumor, the lifetime varied presumably due to the tumor heterogeneity and the different depth of tumor invasion. Thus, processing time-resolved fluorescence images allows detection of both the cleaved and noncleaved states of the TR23K sensor in real-time mode during the course of several weeks noninvasively. This approach can be used in drug screening, facilitating the development of new anticancer agents as well as improvement of chemotherapy efficiency and its adaptation for personal treatment.

  11. Development of a Time Domain Fluorimeter for Fluorescent Lifetime Multiplexing Analysis

    PubMed Central

    Weissleder, Ralph; Mahmood, Umar

    2009-01-01

    We show that a portable, inexpensive USB-powered time domain fluorimeter (TDF) and analysis scheme were developed for use in evaluating a new class of fluorescent lifetime multiplexed dyes. Fluorescent proteins, organic dyes, and quantum dots allow the labeling of more and more individual features within biological systems, but the wide absorption and emission spectra of these fluorophores limit the number of distinct processes which may be simultaneously imaged using spectral separation alone. By additionally separating reporters in a second dimension, fluorescent lifetime multiplexing provides a means to multiply the number of available imaging channels. PMID:19830273

  12. Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

    NASA Astrophysics Data System (ADS)

    Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

    2018-05-01

    We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

  13. 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.

  14. NIR fluorescence lifetime sensing through a multimode fiber for intravascular molecular probing

    NASA Astrophysics Data System (ADS)

    Ingelberts, H.; Hernot, S.; Debie, P.; Lahoutte, T.; Kuijk, M.

    2016-04-01

    Coronary artery disease (CAD) contributes to millions of deaths each year. The identification of vulnerable plaques is essential to the diagnosis of CAD but is challenging. Molecular probes can improve the detection of these plaques using intravascular imaging methods. Fluorescence lifetime sensing is a safe and robust method to image these molecular probes. We present two variations of an optical system for intravascular near-infrared (NIR) fluorescence lifetime sensing through a multimode fiber. Both systems are built around a recently developed fast and efficient CMOS detector, the current-assisted photonic sampler (CAPS) that is optimized for sub-nanosecond NIR fluorescence lifetime sensing. One system mimics the optical setup of an epifluorescence microscope while the other uses a practical fiber optic coupler to separate fluorescence excitation and emission. We test both systems by measuring the lifetime of several NIR dyes in DMSO solutions and we show that these systems are capable of detecting lifetimes of solutions with concentrations down to 370 nM and this with short acquisition times. These results are compared with time-correlated single photon counting (TCSPC) measurements for reference.

  15. Toward two-photon excited fluorescence lifetime endomicroscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hage, Charles-Henri; Leclerc, Pierre; Fabert, Marc; Brevier, Julien; Habert, Rémi; Braud, Flavie; Kudlinski, Alexandre; Louradour, Frédéric

    2017-02-01

    Fluorescence lifetime imaging microscopy (FLIM) represents a powerful tool for biological studies. Endoscopic FLIM applied to the intracellular native biomarker NADH and FAD represents a promising mean for in vivo in situ malignant tissue diagnosis in the medical field. Else, 2-photon-excited fluorescence (2PEF) provides increased 3D resolution and imaging depth. But very few demonstrations about 2PEF lifetime measurement through a fiber have been reported and none about endoscopic 2P-FLIM through a practical fiber length (< 3m). Our group has recently demonstrated the possibility to efficiently deliver through a very long optical fiber the short and intense excitation pulses required for 2P-FLIM. Our goal is now to check that collecting fluorescence through the same endoscopic fiber does not deteriorate the lifetime measurement. Relying on the basis previously published in case of 1PEF by P. French and co-workers (J. Biophotonics, 2015), we have experimentally quantitatively evaluated the influence on the lifetime measurement of the fiber chromatic and intermodal dispersions. The main result is that the fiber contribution to the system impulse response function, even in the case of a 3-meter long double-clad optical fiber, does not hinder the separation between free and bound NADH states using FLIM. Related calibrations and measurements will be detailed. Ongoing experiments about the development of a 2P-FLIM endomicroscope on the basis of an previously reported 2P-endomicroscope (Ducourthial et al., Sc. Reports, 2015), used under various configurations (i.e. point measurement in the center of the 2P-endomicroscope image, averaged lifetime, binned endoscopic 2P-FLIM image), will be also presented.

  16. Bessel beam fluorescence lifetime tomography of live embryos (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Xu, Dongli; Peng, Leilei

    2016-03-01

    Optical tomography allows isotropic 3D imaging of embryos. Scanning-laser optical tomography (SLOT) has superior light collecting efficiency than wide-field optical tomography, making it ideal for fluorescence imaging of live embryos. We previously reported an imaging system that combines SLOT with a novel Fourier-multiplexed fluorescence lifetime imaging (FmFLIM) technique named FmFLIM-SLOT. FmFLIM-SLOT performs multiplexed FLIM-FRET readout of multiple FRET sensors in live embryos. Here we report a recent effort on improving the spatial resolution of the FmFLIM-SLOT system in order to image complex biochemical processes in live embryos at the cellular level. Optical tomography has to compromise between resolution and the depth of view. In SLOT, the commonly-used focused Gaussian beam diverges quickly from the focal plane, making it impossible to achieve high resolution imaging in a large volume specimen. We thus introduce Bessel beam laser-scanning tomography, which illuminates the sample with a spatial-light-modulator-generated Bessel beam that has an extended focal depth. The Bessel beam is scanned across the whole specimen. Fluorescence projection images are acquired at equal angular intervals as the sample rotates. Reconstruction artifacts due to annular-rings of the Bessel beam are removed by a modified 3D filtered back projection algorithm. Furthermore, in combination of Fourier-multiplexing fluorescence lifetime imaging (FmFLIM) method, the Bessel FmFLIM-SLOT system is capable of perform 3D lifetime imaging of live embryos at cellular resolution. The system is applied to in-vivo imaging of transgenic Zebrafish embryos. Results prove that Bessel FmFLIM-SLOT is a promising imaging method in development biology research.

  17. Modulated CMOS camera for fluorescence lifetime microscopy.

    PubMed

    Chen, Hongtao; Holst, Gerhard; Gratton, Enrico

    2015-12-01

    Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition. © 2015 Wiley Periodicals, Inc.

  18. High speed fluorescence imaging with compressed ultrafast photography

    NASA Astrophysics Data System (ADS)

    Thompson, J. V.; Mason, J. D.; Beier, H. T.; Bixler, J. N.

    2017-02-01

    Fluorescent lifetime imaging is an optical technique that facilitates imaging molecular interactions and cellular functions. Because the excited lifetime of a fluorophore is sensitive to its local microenvironment,1, 2 measurement of fluorescent lifetimes can be used to accurately detect regional changes in temperature, pH, and ion concentration. However, typical state of the art fluorescent lifetime methods are severely limited when it comes to acquisition time (on the order of seconds to minutes) and video rate imaging. Here we show that compressed ultrafast photography (CUP) can be used in conjunction with fluorescent lifetime imaging to overcome these acquisition rate limitations. Frame rates up to one hundred billion frames per second have been demonstrated with compressed ultrafast photography using a streak camera.3 These rates are achieved by encoding time in the spatial direction with a pseudo-random binary pattern. The time domain information is then reconstructed using a compressed sensing algorithm, resulting in a cube of data (x,y,t) for each readout image. Thus, application of compressed ultrafast photography will allow us to acquire an entire fluorescent lifetime image with a single laser pulse. Using a streak camera with a high-speed CMOS camera, acquisition rates of 100 frames per second can be achieved, which will significantly enhance our ability to quantitatively measure complex biological events with high spatial and temporal resolution. In particular, we will demonstrate the ability of this technique to do single-shot fluorescent lifetime imaging of cells and microspheres.

  19. Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

    NASA Astrophysics Data System (ADS)

    Wahl, Michael; Rahn, Hans-Juergen; Ortmann, Uwe; Erdmann, Rainer; Boehmer, Martin; Enderlein, Joerg

    2002-03-01

    Fluorescence lifetime measurement of organic fluorophores is a powerful tool for distinguishing molecules of interest from background or other species. This is of interest in sensitive analysis and Single Molecule Detection (SMD). A demand in many applications is to provide 2-D imaging together with lifetime information. The method of choice is then Time-Correlated Single Photon Counting (TCSPC). We have devloped a compact system on a single PC board that can perform TCSPC at high throughput, while synchronously driving a piezo scanner holding the immobilized sample. The system allows count rates up to 3 MHz and a resolution down to 30 ps. An overall Instrument Response Function down to 300ps is achieved with inexpensive detectors and diode lasers. The board is designed for the PCI bus, permitting high throughput without loss of counts. It is reconfigurable to operate in different modes. The Time-Tagged Time-Resolved (TTTR) mode permits the recording of all photon events with a real-time tag allowing data analysis with unlimited flexibility. We use the Time-Tag clock for an external piezo scanner that moves the sample. As the clock source is common for scanning and tagging, the individual photons can be matched to pixels. Demonstrating the capablities of the system we studied single molecule solutions. Lifetime imaging can be performed at high resolution with as few as 100 photons per pixel.

  20. pH and chloride recordings in living cells using two-photon fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Lahn, Mattes; Hille, Carsten; Koberling, Felix; Kapusta, Peter; Dosche, Carsten

    2010-02-01

    Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences. The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a combination with two-photon (2P) excitation is quite promising. Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique. We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values. However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range. Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated pharmacologically. In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time measurements in combination with reduced cell and dye damages.

  1. Evaluation of the oxidative stress of psoriatic fibroblasts based on spectral two-photon fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Kapsokalyvas, Dimitrios; Barygina, Victoria; Cicchi, Riccardo; Fiorillo, Claudia; Pavone, Francesco S.

    2013-02-01

    Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Metabolic activity is increased in the epidermis and the dermis. Oxidative stress is high mainly due to reactive oxygen species (ROS) originating from the skin environment and cellular metabolism. We employed a custom multiphoton microscope coupled with a FLIM setup to image primary culture fibroblast cells from perilesional and lesional psoriatic skin in-vitro. Twophoton excited fluorescence images revealed the morphological differences between healthy and psoriatic fibroblasts. Based on the spectral analysis of the NADH and FAD components the oxidative stress was assessed and found to be higher in psoriatic cells. Furthermore the fluorescence lifetime properties were investigated with a TCSPC FLIM module. Mean fluorescence lifetime was found to be longer in psoriatic lesional cells. Analysis of the fast (τ1) and slow (τ2) decay lifetimes revealed a decrease of the ratio of the contribution of the fast (α1) parameter to the contribution of the slow (α2) parameter. The fluorescence in the examined part of the spectrum is attributed mainly to NADH. The decrease of the ratio (α1)/ (α2) is believed to correlate strongly with the anti-oxidant properties of NADH which can lead to the variation of its population in high ROS environment. This methodology could serve as an index of the oxidative status in cells and furthermore could be used to probe the oxidative stress of tissues in-vivo.

  2. A comparative study of metabolic state of stem cells during osteogenic and adipogenic differentiations via fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Chakraborty, Sandeep; Ou, Meng-Hsin; Kuo, Jean-Cheng; Chiou, Arthur

    2016-10-01

    Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P < 0.001), while the ratio of free to protein-bound NADH ratio decreased significantly in 7- days (P < 0.001) and 14-days (P < 0.001). Thus, our results indicated a higher metabolic rate in both osteogenic and adipogenic differentiation processes when compared with undifferentiated hMSCs. This approach may be further utilized to study proliferation efficiency and differentiation potential of stem cells into other specialized cell lineages.

  3. Fluorescence lifetime technique for surgical imaging, guidance and augmented reality (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Marcu, Laura

    2017-02-01

    The surgeon's limited ability to accurately delineate the tumor margin during surgical interventions is one key challenge in clinical management of cancer. New methods for guiding tumor resection decisions are needed. Numerous studies have shown that tissue autofluorescence properties have the potential to asses biochemical features associates with distinct pathologies in tissue and to distinguish various cancers from normal tissues. However, despite these promising reports, autofluorescence techniques were sparsely adopted in clinical settings. Moreover, when adopted they were primarily used for pre-operative diagnosis rather than guiding interventions. To address this need, we have researched and engineered instrumentation that utilizes label-free fluorescence lifetime contrast to characterize tissue biochemical features in vivo in patients and methodologies conducive to real-time (few seconds) diagnosis of tissue pathologies during surgical procedures. This presentation overviews clinically-compatible multispectral fluorescence lifetime imaging techniques developed in our laboratory and their ability to operate as stand-alone tools, integrated in a biopsy needle and in conjunction with the da Vinci surgical robot. We present pre-clinical and clinical studies in patients that demonstrate the potential of these techniques for intraoperative assessment of brain tumors and head and neck cancer. Current results demonstrate that intrinsic fluorescence signals can provide useful contrast for delineation distinct types of tissues including tumors intraoperatively. Challenges and solutions in the clinical implementation of these techniques are discussed.

  4. Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine

    NASA Astrophysics Data System (ADS)

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2010-01-01

    Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by ~300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.

  5. Fluorophore:dendrimer ratio impacts cellular uptake and intracellular fluorescence lifetime.

    PubMed

    Dougherty, Casey A; Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

    2015-02-18

    G5-NH2-TAMRAn (n = 1-4, 5+, and 1.5(avg)) were prepared with n = 1-4 as a precise dye:dendrimer ratio, 5+ as a mixture of dendrimers with 5 or more dye per dendrimer, and 1.5(avg) as a Poisson distribution of dye:dendrimer ratios with a mean of 1.5 dye per dendrimer. The absorption intensity increased sublinearly with n whereas the fluorescence emission and lifetime decreased with an increasing number of dyes per dendrimer. Flow cytometry was employed to quantify uptake into HEK293A cells. Dendrimers with 2-4 dyes were found to have greater uptake than dendrimer with a single dye. Fluorescence lifetime imaging microscopy (FLIM) showed that the different dye:dendrimer ratio alone was sufficient to change the fluorescence lifetime of the material observed inside cells. We also observed that the lifetime of G5-NH2-TAMRA5+ increased when present in the cell as compared to solution. However, cells treated with G5-NH2-TAMRA1.5(avg) did not exhibit the high lifetime components present in G5-NH2-TAMRA1 and G5-NH2-TAMRA5+. In general, the effects of the dye:dendrimer ratio on fluorescence lifetime were of similar magnitude to environmentally induced lifetime shifts.

  6. In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse

    PubMed Central

    McGinty, James; Stuckey, Daniel W.; Soloviev, Vadim Y.; Laine, Romain; Wylezinska-Arridge, Marzena; Wells, Dominic J.; Arridge, Simon R.; French, Paul M. W.; Hajnal, Joseph V.; Sardini, Alessandro

    2011-01-01

    Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct. PMID:21750768

  7. A CTRW-based model of time-resolved fluorescence lifetime imaging in a turbid medium

    NASA Astrophysics Data System (ADS)

    Chernomordik, Victor; Gandjbakhche, Amir H.; Hassan, Moinuddin; Pajevic, Sinisa; Weiss, George H.

    2010-12-01

    We develop an analytic model of time-resolved fluorescent imaging of photons migrating through a semi-infinite turbid medium bounded by an infinite plane in the presence of a single stationary point fluorophore embedded in the medium. In contrast to earlier models of fluorescent imaging in which photon motion is assumed to be some form of continuous diffusion process, the present analysis is based on a continuous-time random walk (CTRW) on a simple cubic lattice, the objective being to estimate the position and lifetime of the fluorophore. This can provide information related to local variations in pH and temperature with potential medical significance. Aspects of the theory were tested using time-resolved measurements of the fluorescence from small inclusions inside tissue-like phantoms. The experimental results were found to be in good agreement with theoretical predictions provided that the fluorophore was not located too close to the planar boundary, a common problem in many diffusive systems.

  8. Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

    PubMed Central

    Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

    2015-01-01

    Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving

  9. Imaging bio-distribution of a topically applied dermatological cream on minipig skin using fluorescence lifetime imaging microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Alex, Aneesh; Chaney, Eric J.; Criley, Jennifer M.; Spillman, Darold R.; Hutchison, Phaedra B.; Li, Joanne; Marjanovic, Marina; Frey, Steve; Cook, Steven; Boppart, Stephen A.; Arp, Zane A.

    2017-02-01

    Currently there is a lack of in vivo techniques to evaluate the spatial bio-distribution of dermal drugs over time without the need to take multiple serial biopsies. To address this gap, we investigated the use of multi-photon optical imaging methods to non-invasively track drug distribution on miniature pig (Species: Sus scrofa, Strain: Göttingen) skin in vivo. Minipig skin is the standard comparative research model to human skin, and is anatomically and functionally similar. We employed fluorescence lifetime imaging microscopy (FLIM) to visualize the spatial distribution and residency time of a topically applied experimental dermatological cream. This was made possible by the endogenous fluorescent optical properties of the experimental drug (fluorescence lifetime > 3000 ps). Two different drug formulations were applied on 2 minipigs for 7 consecutive days, with the control creams applied on the contralateral side, followed by 7 days of post-application monitoring using a multi-modal optical imaging system (MPTflex-CARS, JenLab, Germany). FLIM images were obtained from the treated regions 24 hr post-application from day 1 to day 14 that allowed visualization of cellular and sub-cellular features associated with different dermal layers non-invasively to a depth of 200 µm. Five punch biopsies per animal were obtained from the corresponding treated regions between days 8 and 14 for bioanalytical analysis and comparison with results obtained using FLIM. In conclusion, utilization of non-invasive optical biopsy methods for dermal drug evaluation can provide true longitudinal monitoring of drug spatial distribution, remove sampling limitations, and be more time-efficient compared to traditional methods.

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

    PubMed

    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 (λ(ex)=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.

  11. 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.

  12. 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

  13. High resolution diagnosis of common nevi by multiphoton laser tomography and fluorescence lifetime imaging.

    PubMed

    Arginelli, Federica; Manfredini, Marco; Bassoli, Sara; Dunsby, Christopher; French, Paul; König, Karsten; Magnoni, Cristina; Ponti, Giovanni; Talbot, Clifford; Seidenari, Stefania

    2013-05-01

    Multiphoton Laser Tomography (MPT) has developed as a non-invasive tool that allows real-time observation of the skin with subcellular resolution. MPT is readily combined with time resolved detectors to achieve fluorescence lifetime imaging (FLIM). The aim of our study was to identify morphologic MPT/FLIM descriptors of melanocytic nevi, referring to cellular and architectural features. In the preliminary study, MPT/FLIM images referring to 16 ex vivo nevi were simultaneously evaluated by 3 observers for the identification of morphologic descriptors characteristic of melanocytic nevi. Proposed descriptors were discussed and the parameters referring to epidermal keratinocytes, epidermal melanocytes, dermo-epidermal junction, papillary dermis and overall architecture were selected. In the main study, the presence/absence of the specified criteria were blindly evaluated on a test set, comprising 102 ex vivo samples (51 melanocytic nevi, 51 miscellaneous skin lesions) by 2 observers. Twelve descriptors were identified: "short-lifetime cells in the stratum corneum", "melanin-containing keratinocytes", "dendritic cells", "small short-lifetime cells" in the upper and lower layers", "edged papillae", "non-edged papillae", "junctional nests of short-lifetime cells", "dermal cell clusters", "short-lifetime cells in the papilla", "monomorphic and regular histoarchitecture", "architectural disarray". Identified descriptors for benign melanocytic lesions proved sensitive and specific, enabling the differentiation between melanocytic nevi and non-melanocytic lesions. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

  14. FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging

    PubMed Central

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-01-01

    We report a fast non-iterative lifetime data analysis method for the Fourier multiplexed frequency-sweeping confocal FLIM (Fm-FLIM) system [ Opt. Express22, 10221 ( 2014)24921725]. The new method, named R-method, allows fast multi-channel lifetime image analysis in the system’s FPGA data processing board. Experimental tests proved that the performance of the R-method is equivalent to that of single-exponential iterative fitting, and its sensitivity is well suited for time-lapse FLIM-FRET imaging of live cells, for example cyclic adenosine monophosphate (cAMP) level imaging with GFP-Epac-mCherry sensors. With the R-method and its FPGA implementation, multi-channel lifetime images can now be generated in real time on the multi-channel frequency-sweeping FLIM system, and live readout of FRET sensors can be performed during time-lapse imaging. PMID:25321778

  15. Fluorescence lifetime microscopy with a time- and space-resolved single-photon counting detector

    PubMed Central

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

    2017-01-01

    We have recently developed a wide-field photon-counting detector (the H33D detector) having high-temporal and high-spatial resolutions and capable of recording up to 500,000 photons per sec. Its temporal performance has been previously characterized using solutions of fluorescent materials with different lifetimes, and its spatial resolution using sub-diffraction objects (beads and quantum dots). Here we show its application to fluorescence lifetime imaging of live cells and compare its performance to a scanning confocal TCSPC approach. With the expected improvements in photocathode sensitivity and increase in detector throughput, this technology appears as a promising alternative to the current lifetime imaging solutions. PMID:29449756

  16. Fluorescence lifetime measurements in flow cytometry

    NASA Astrophysics Data System (ADS)

    Beisker, Wolfgang; Klocke, Axel

    1997-05-01

    Fluorescence lifetime measurements provide insights int eh dynamic and structural properties of dyes and their micro- environment. The implementation of fluorescence lifetime measurements in flow cytometric systems allows to monitor large cell and particle populations with high statistical significance. In our system, a modulated laser beam is used for excitation and the phase shift of the fluorescence signal recorded with a fast computer controlled digital oscilloscope is processed digitally to determine the phase shift with respect to a reference beam by fast fourier transform. Total fluorescence intensity as well as other parameters can be determined simultaneously from the same fluorescence signal. We use the epi-illumination design to allow the use of high numerical apertures to collect as much light as possible to ensure detection of even weak fluorescence. Data storage and processing is done comparable to slit-scan flow cytometric data using data analysis system. The results are stored, displayed, combined with other parameters and analyzed as normal listmode data. In our report we discuss carefully the signal to noise ratio for analog and digital processed lifetime signals to evaluate the theoretical minimum fluorescence intensity for lifetime measurements. Applications to be presented include DNA staining, parameters of cell functions as well as different applications in non-mammalian cells such as algae.

  17. Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

    PubMed

    Görlitz, Frederik; Kelly, Douglas J; Warren, Sean C; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J; Stuhmeier, Frank; Neil, Mark A A; Tate, Edward W; Dunsby, Christopher; French, Paul M W

    2017-01-18

    We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

  18. Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Warren, Sean C.; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A.; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J.; Stuhmeier, Frank; Neil, Mark A. A.; Tate, Edward W.; Dunsby, Christopher; French, Paul M. W.

    2017-01-01

    We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set. PMID:28190060

  19. In vivo optical imaging of dihydroethidium oxidation in the mouse brain employing fluorescence intensity and lifetime contrast

    NASA Astrophysics Data System (ADS)

    Hall, David J.; Han, Sung-Ho; Dugan, Laura

    2009-02-01

    Reactive oxygen species (ROS) are believed to be involved in many diseases and injuries to the brain, but the molecular processes are not well understood due to a lack of in vivo imaging techniques to evaluate ROS. The fluorescent oxidation products of dihydroethidium (DHE) can monitor ROS production in vivo. Here we demonstrate the novel optical imaging of brain in live mice to measure ROS production via generation of fluorescent DHE oxidation products (ox-DHE) by ROS. We show that in Sod2+/- mice, which have partial loss of a key antioxidant enzyme, superoxide dismutase-2, that ox-DHE fluorescence intensity was significantly higher than in hSOD1 mice, which have four-fold overexpression of superoxide dismutase-1 activity, which had almost no ox-DHE fluorescence, confirming specificity of ox-DHE to ROS production. The DHE oxidation products were also confirmed by detecting a characteristic fluorescence lifetime of the oxidation product, which was validated with ex vivo measurements.

  20. Single pulse two-photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate and an all fiber based setup

    NASA Astrophysics Data System (ADS)

    Eibl, Matthias; Karpf, Sebastian; Hakert, Hubertus; Weng, Daniel; Pfeiffer, Tom; Kolb, Jan Philip; Huber, Robert

    2017-07-01

    Newly developed microscopy methods have the goal to give researches in bio-molecular science a better understanding of processes ongoing on a cellular level. Especially two-photon excited fluorescence (TPEF) microscopy is a readily applied and widespread modality. Compared to one photon fluorescence imaging, it is possible to image not only the surface but also deeper lying structures. Together with fluorescence lifetime imaging (FLIM), which provides information on the chemical composition of a specimen, deeper insights on a molecular level can be gained. However, the need for elaborate light sources for TPEF and speed limitations for FLIM hinder an even wider application. In this contribution, we present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is perfectly suited for fiber delivery as typically limiting non-linear effects like self-phase or cross-phase modulation (SPM, XPM) are negligible. Furthermore, compared to the typically applied femtosecond pulses, our longer pulses produce much more fluorescence photons per single shot. In this paper, we show that this higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate our system, we acquired FLIM images of a dye solution with single exponential behavior to assess the accuracy of our lifetime determination and also FLIM images of a plant stem at a pixel rate of 1 MHz to show the speed performance of our single pulse two-photon FLIM (SP-FLIM) system.

  1. Fluorescence lifetime imaging of microviscosity changes during ER autophagy in live cells

    NASA Astrophysics Data System (ADS)

    He, Ying; Samanta, Soham; Gong, Wanjun; Liu, Wufan; Pan, Wenhui; Yang, Zhigang; Qu, Junle

    2018-02-01

    Unfolded or misfolded protein accumulation inside Endoplasmic Reticulum (ER) will cause ER stress and subsequently will activate cellular autophagy to release ER stress, which would ultimately result in microviscosity changes. However, even though, it is highly significant to gain a quantitative assessment of microviscosity changes during ER autophagy to study ER stress and autophagy behaviors related diseases, it has rarely been reported yet. In this work, we have reported a BODIPY based fluorescent molecular rotor that can covalently bind with vicinal dithiols containing nascent proteins in ER and hence can result in ER stress through the inhibition of the folding of nascent proteins. The change in local viscosity, caused by the release of the stress in cells through autophagy, was quantified by the probe using fluorescence lifetime imaging. This work basically demonstrates the possibility of introducing synthetic chemical probe as a promising tool to diagnose ER-viscosity-related diseases.

  2. Phytoplankton-Fluorescence-Lifetime Vertical Profiler

    NASA Technical Reports Server (NTRS)

    Fernandez, Salvador M.; Guignon, Ernest F.; St. Louis, Ernest

    2004-01-01

    A battery-operated optoelectronic instrument is designed to be lowered into the ocean to measure the intensity and lifetime of fluorescence of chlorophyll A in marine phytoplankton as a function of depth from 0 to 300 m. Fluorescence lifetimes are especially useful as robust measures of photosynthetic productivity of phytoplankton and of physical and chemical mechanisms that affect photosynthesis. The knowledge of photosynthesis in phytoplankton gained by use of this and related instruments is expected to contribute to understanding of global processes that control the time-varying fluxes of carbon and associated biogenic elements in the ocean. The concentration of chlorophyll in the ocean presents a major detection challenge because in order to obtain accurate values of photosynthetic parameters, the intensity of light used to excite fluorescence must be kept very low so as not to disturb the photosynthetic system. Several innovations in fluorometric instrumentation were made in order to make it possible to reach the required low detection limit. These innovations include a highly efficient optical assembly with an integrated flow-through sample interface, and a high-gain, low-noise electronic detection subsystem. The instrument also incorporates means for self-calibration during operation, and electronic hardware and software for control, acquisition and analysis of data, and communications. The electronic circuitry is highly miniaturized and designed to minimize power demand. The instrument is housed in a package that can withstand the water pressure at the maximum depth of 300 m. A light-emitting diode excites fluorescence in the sample flow cell, which is placed at one focal point of an ellipsoidal reflector. A photomultiplier tube is placed at the other focal point. This optical arrangement enables highly efficient collection of fluorescence emitted over all polar directions. Fluorescence lifetime is measured indirectly, by use of a technique based on the

  3. Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

    2010-09-01

    The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

  4. Fluorescence lifetime imaging and its applications in cellular microenvironment measurement and auxiliary diagnosis

    NASA Astrophysics Data System (ADS)

    Luo, Teng; Levchenko, Svitlana M.; Pliss, Artem; Peng, Xiao; Yan, Wei; Prasad, Paras N.; Liu, Liwei; Qu, Junle

    2018-02-01

    We present our recent work on the applications of fluorescence lifetime imaging microscopy(FLIM), including the monitoring of macromolecule dynamic changes in the nucleolar compartments and the auxiliary diagnosis of H and E-stained sections. We demonstrated the capability of FLIM to measure protein concentration in the specific cellular compartments in live cells. We proposed to use FLIM to monitor changes in intracellular protein concentration caused by various factors e.g. cell cycle progression, drug treatment etc. In the future, FLIM technology is expected to be combined with super-resolution optical imaging. FLIM with molecular resolution will have the potential to serve as a powerful tool for discovering new phenomena and revealing new mechanisms in biomedical research, which will effectively promote the development of life science.

  5. Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations

    NASA Astrophysics Data System (ADS)

    Hosny, Neveen A.; Lee, David A.; Knight, Martin M.

    2012-01-01

    Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]2+, was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)3]2+ characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

  6. Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations.

    PubMed

    Hosny, Neveen A; Lee, David A; Knight, Martin M

    2012-01-01

    Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)(3)](2+), was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)(3)](2+) characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

  7. Differentiation of ocular fundus fluorophores by fluorescence lifetime imaging using multiple excitation and emission wavelengths

    NASA Astrophysics Data System (ADS)

    Hammer, M.; Schweitzer, D.; Schenke, S.; Becker, W.; Bergmann, A.

    2006-10-01

    Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH II provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.

  8. Dynamic nuclear protein interactions investigated using fluorescence lifetime and fluorescence fluctuation spectroscopy

    NASA Astrophysics Data System (ADS)

    Siegel, Amanda P.; Hays, Nicole M.; Day, Richard N.

    2012-03-01

    The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic interactions in pituitary specific gene regulation.

  9. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging.

    PubMed

    Yasuda, Ryohei; Harvey, Christopher D; Zhong, Haining; Sobczyk, Aleksander; van Aelst, Linda; Svoboda, Karel

    2006-02-01

    To understand the biochemical signals regulated by neural activity, it is necessary to measure protein-protein interactions and enzymatic activity in neuronal microcompartments such as axons, dendrites and their spines. We combined two-photon excitation laser scanning with fluorescence lifetime imaging to measure fluorescence resonance energy transfer at high resolutions in brain slices. We also developed sensitive fluorescent protein-based sensors for the activation of the small GTPase protein Ras with slow (FRas) and fast (FRas-F) kinetics. Using FRas-F, we found in CA1 hippocampal neurons that trains of back-propagating action potentials rapidly and reversibly activated Ras in dendrites and spines. The relationship between firing rate and Ras activation was highly nonlinear (Hill coefficient approximately 5). This steep dependence was caused by a highly cooperative interaction between calcium ions (Ca(2+)) and Ras activators. The Ras pathway therefore functions as a supersensitive threshold detector for neural activity and Ca(2+) concentration.

  10. Optically sectioned wide-field fluorescence lifetime imaging endoscopy enabled by structured illumination (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hinsdale, Taylor; Malik, Bilal H.; Rico-Jimenez, Jose J.; Jo, Javier A.; Maitland, Kristen C.

    2016-03-01

    We present a wide-field fluorescence lifetime imaging (FLIM) system with optical sectioning by structured illumination microscopy (SIM). FLIM measurements were made using a time gated ICCD camera in conjunction with a pulsed nitrogen dye laser operating at 450 nm. Intensity images were acquired at multiple time delays from a trigger initiated by a laser pulse to create a wide-field FLIM image, which was then combined with three phase SIM to provide optical sectioning. Such a mechanism has the potential to increase the reliability and accuracy of the FLIM measurements by rejecting background intensity. SIM also provides the opportunity to create volumetric FLIM images with the incorporation of scanning mechanisms for the sample plane. We present multiple embodiments of such a system: one as a free space endoscope and the other as a fiber microendoscope enabled by the introduction of a fiber bundle. Finally, we demonstrate the efficacy of such an imaging system by imaging dyes embedded in a tissue phantom.

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

    PubMed Central

    Digman, Michelle A.; Gratton, Enrico; Storti, Barbara; Beltram, Fabio

    2013-01-01

    A versatile pH-dependent fluorescent protein was applied to intracellular pH measurements by means of the phasor approach to fluorescence lifetime imaging. By this fit-less method we obtain intracellular pH maps under resting or altered physiological conditions by single-photon confocal or two-photon microscopy. PMID:22517076

  12. Simultaneous one-dimensional fluorescence lifetime measurements of OH and CO in premixed flames

    NASA Astrophysics Data System (ADS)

    Jonsson, Malin; Ehn, Andreas; Christensen, Moah; Aldén, Marcus; Bood, Joakim

    2014-04-01

    A method for simultaneous measurements of fluorescence lifetimes of two species along a line is described. The experimental setup is based on picosecond laser pulses from two tunable optical parametric generator/optical parametric amplifier systems together with a streak camera. With an appropriate optical time delay between the two laser pulses, whose wavelengths are tuned to excite two different species, laser-induced fluorescence can be both detected temporally and spatially resolved by the streak camera. Hence, our method enables one-dimensional imaging of fluorescence lifetimes of two species in the same streak camera recording. The concept is demonstrated for fluorescence lifetime measurements of CO and OH in a laminar methane/air flame on a Bunsen-type burner. Measurements were taken in flames with four different equivalence ratios, namely ϕ = 0.9, 1.0, 1.15, and 1.25. The measured one-dimensional lifetime profiles generally agree well with lifetimes calculated from quenching cross sections found in the literature and quencher concentrations predicted by the GRI 3.0 mechanism. For OH, there is a systematic deviation of approximately 30 % between calculated and measured lifetimes. It is found that this is mainly due to the adiabatic assumption regarding the flame and uncertainty in H2O quenching cross section. This emphasizes the strength of measuring the quenching rates rather than relying on models. The measurement concept might be useful for single-shot measurements of fluorescence lifetimes of several species pairs of vital importance in combustion processes, hence allowing fluorescence signals to be corrected for quenching and ultimately yield quantitative concentration profiles.

  13. Extended output phasor representation of multi-spectral fluorescence lifetime imaging microscopy

    PubMed Central

    Campos-Delgado, Daniel U.; Navarro, O. Gutiérrez; Arce-Santana, E. R.; Jo, Javier A.

    2015-01-01

    In this paper, we investigate novel low-dimensional and model-free representations for multi-spectral fluorescence lifetime imaging microscopy (m-FLIM) data. We depart from the classical definition of the phasor in the complex plane to propose the extended output phasor (EOP) and extended phasor (EP) for multi-spectral information. The frequency domain properties of the EOP and EP are analytically studied based on a multiexponential model for the impulse response of the imaged tissue. For practical implementations, the EOP is more appealing since there is no need to perform deconvolution of the instrument response from the measured m-FLIM data, as in the case of EP. Our synthetic and experimental evaluations with m-FLIM datasets of human coronary atherosclerotic plaques show that low frequency indexes have to be employed for a distinctive representation of the EOP and EP, and to reduce noise distortion. The tissue classification of the m-FLIM datasets by EOP and EP also improves with low frequency indexes, and does not present significant differences by using either phasor. PMID:26114031

  14. Ultrafast Method for the Analysis of Fluorescence Lifetime Imaging Microscopy Data Based on the Laguerre Expansion Technique

    PubMed Central

    Jo, Javier A.; Fang, Qiyin; Marcu, Laura

    2007-01-01

    We report a new deconvolution method for fluorescence lifetime imaging microscopy (FLIM) based on the Laguerre expansion technique. The performance of this method was tested on synthetic and real FLIM images. The following interesting properties of this technique were demonstrated. 1) The fluorescence intensity decay can be estimated simultaneously for all pixels, without a priori assumption of the decay functional form. 2) The computation speed is extremely fast, performing at least two orders of magnitude faster than current algorithms. 3) The estimated maps of Laguerre expansion coefficients provide a new domain for representing FLIM information. 4) The number of images required for the analysis is relatively small, allowing reduction of the acquisition time. These findings indicate that the developed Laguerre expansion technique for FLIM analysis represents a robust and extremely fast deconvolution method that enables practical applications of FLIM in medicine, biology, biochemistry, and chemistry. PMID:19444338

  15. Imaging the environment of green fluorescent protein.

    PubMed Central

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

    2002-01-01

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

  16. Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging

    PubMed Central

    Spagnol, Stephen T.; Dahl, Kris Noel

    2016-01-01

    The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes. PMID:26765322

  17. Characterization of porcine eyes based on autofluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

    2015-03-01

    Multiphoton microscopy is a non-invasive imaging technique with ideal characteristics for biological applications. In this study, we propose to characterize three major structures of the porcine eye, the cornea, crystalline lens, and retina using two-photon excitation fluorescence lifetime imaging microscopy (2PE-FLIM). Samples were imaged using a laser-scanning microscope, consisting of a broadband sub-15 femtosecond (fs) near-infrared laser. Signal detection was performed using a 16-channel photomultiplier tube (PMT) detector (PML-16PMT). Therefore, spectral analysis of the fluorescence lifetime data was possible. To ensure a correct spectral analysis of the autofluorescence lifetime data, the spectra of the individual endogenous fluorophores were acquired with the 16-channel PMT and with a spectrometer. All experiments were performed within 12h of the porcine eye enucleation. We were able to image the cornea, crystalline lens, and retina at multiple depths. Discrimination of each structure based on their autofluorescence intensity and lifetimes was possible. Furthermore, discrimination between different layers of the same structure was also possible. To the best of our knowledge, this was the first time that 2PE-FLIM was used for porcine lens imaging and layer discrimination. With this study we further demonstrated the feasibility of 2PE-FLIM to image and differentiate three of the main components of the eye and its potential as an ophthalmologic technique.

  18. Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

    PubMed Central

    Petersen, Karl J.; Peterson, Kurt C.; Muretta, Joseph M.; Higgins, Sutton E.; Gillispie, Gregory D.; Thomas, David D.

    2014-01-01

    We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5–10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications. PMID:25430092

  19. Transient Fluorescence Spectroscopy and laser induced fluorescence lifetimes of terbium doped dipicolinic acid

    NASA Astrophysics Data System (ADS)

    Makoui, Anali

    We have investigated the use of deep UV laser induced fluorescence for the sensitive detection and spectroscopic lifetime studies of terbium doped dipicolinic acid (DPA-Tb) and used this to study the optical characteristics of DPA which is a chemical surrounding most bacterial spores. Background absorption spectra, fluorescence spectra, and Excitation Emission Matrix (EEM) spectra were made of the DPA-Tb complex, using both fixed 266 nm wavelength and tunable (220 nm--280 nm) UV laser excitations. Of importance, the fluorescence lifetimes of the four main fluorescence peaks (488 nm, 543 nm, 581 nm, and 618 nm) of the DPA-Tb complex have been measured for the first time to our knowledge. The lifetimes of all the fluorescing lines have been measured as a function of DPA-Tb concentration, solvent pH, and solvent composition, including that for the weakest fluorescing line of DPA-Tb at 618 nm. In addition, a new spectroscopic lifetime measurement technique, which we call "Transient Fluorescence Spectroscopy", was developed. In this technique, a weak, quasi-CW, amplitude modulated UV laser (8.5 kHz) was used to measure the lifetimes of the fluorescence lines, and yields insight into energy transfer and excitation lifetimes within the system. This technique is especially useful when a high power laser is not either available or not suitable. In the latter case, this would be when a high power pulsed deep-UV laser could produce bleaching or destruction of the biological specimen. In addition, this technique simulated the excitation and fluorescence emission of the DPA-Tb using a 4-level energy model, and solved the dynamic transient rate equations to predict the temporal behavior of the DPA-Tb emitted fluorescence. Excellent agreement between the experiments and the simulation were found. This technique has the potential to provide a more accurate value for the fluorescence lifetime values. In addition, with the use of asymmetric excitation waveforms, the dynamic

  20. Highly sensitive detection of human papillomavirus type 16 DNA using time-resolved fluorescence microscopy and long lifetime probes

    NASA Astrophysics Data System (ADS)

    Wang, Xue F.; Periasamy, Ammasi; Wodnicki, Pawel; Siadat-Pajouh, M.; Herman, Brian

    1995-04-01

    We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using

  1. Time-resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications.

    PubMed

    Lassiter, S J; Stryjewski, W; Legendre, B L; Erdmann, R; Wahl, M; Wurm, J; Peterson, R; Middendorf, L; Soper, S A

    2000-11-01

    A compact time-resolved near-IR fluorescence imager was constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 microns) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnano-second lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in approximately 6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of approximately 0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used to construct the decay profiles. Comparison of the lifetime base calling to a single

  2. Second-harmonic generation and fluorescence lifetime imaging microscopy through a rodent mammary imaging window

    NASA Astrophysics Data System (ADS)

    Young, Pamela A.; Nazir, Muhammad; Szulczewski, Michael J.; Keely, Patricia J.; Eliceiri, Kevin W.

    2012-03-01

    Tumor-Associated Collagen Signatures (TACS) have been identified that manifest in specific ways during breast tumor progression and that correspond to patient outcome. There are also compelling metabolic changes associated with carcinoma invasion and progression. We have characterized the difference in the autofluorescent properties of metabolic co-factors, NADH and FAD, between normal and carcinoma breast cell lines. Also, we have shown in vitro that increased collagen density alters metabolic genes which are associated with glycolysis and leads to a more invasive phenotype. Establishing the relationship between collagen density, cellular metabolism, and metastasis in physiologically relevant cancer models is crucial for developing cancer therapies. To study cellular metabolism with respect to collagen density in vivo, we use multiphoton fluorescence excitation microscopy (MPM) in conjunction with a rodent mammary imaging window implanted in defined mouse cancer models. These models are ideal for the study of collagen changes in vivo, allowing determination of corresponding metabolic changes in breast cancer invasion and progression. To measure cellular metabolism, we collect fluorescence lifetime (FLIM) signatures of NADH and FAD, which are known to change based on the microenvironment of the cells. Additionally, MPM systems are capable of collecting second harmonic generation (SHG) signals which are a nonlinear optical property of collagen. Therefore, MPM, SHG, and FLIM are powerful tools with great potential for characterizing key features of breast carcinoma in vivo. Below we present the current efforts of our collaborative group to develop intravital approaches based on these imaging techniques to look at defined mouse mammary models.

  3. FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye

    PubMed Central

    Klemm, Matthias; Schweitzer, Dietrich; Peters, Sven; Sauer, Lydia; Hammer, Martin; Haueisen, Jens

    2015-01-01

    Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX’s applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation. PMID:26192624

  4. FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye.

    PubMed

    Klemm, Matthias; Schweitzer, Dietrich; Peters, Sven; Sauer, Lydia; Hammer, Martin; Haueisen, Jens

    2015-01-01

    Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX's applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation.

  5. Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins.

    PubMed

    Chanoca, Alexandra; Burkel, Brian; Kovinich, Nik; Grotewold, Erich; Eliceiri, Kevin W; Otegui, Marisa S

    2016-12-01

    Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τ m ) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τ m than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  6. Two-photon optical imaging, spectral and fluorescence lifetime analysis to discriminate urothelial carcinoma grades.

    PubMed

    Pradère, B; Poulon, F; Compérat, E; Lucas, I; Bazin, D; Doizi, S; Cussenot, O; Traxer, O; Abi Haidar, D

    2018-05-28

    In the framework of urologic oncology, mini-invasive procedures have increased in the last few decades particularly for urothelial carcinoma. One of the essential elements in the management of this disease is still the diagnosis, which strongly influences the choice of treatment. The histopathologic evaluation of the tumor grade is a keystone of diagnosis, and tumor characterization is not possible with just a macroscopic evaluation. Even today intraoperative evaluation remains difficult despite the emergence of new technologies which use exogenous fluorophore. This study assessed an optical multimodal technique based on endogenous fluorescence, combining qualitative and quantitative analysis, for the diagnostic of urothelial carcinoma. It was found that the combination of two photon fluorescence, second harmonic generation microscopy, spectral analysis and fluorescence lifetime imaging were all able to discriminate tumor from healthy tissue, and to determine the grade of tumors. Spectral analysis of fluorescence intensity and the redox ratio used as quantitative evaluations showed statistical differences between low grade and high grade tumors. These results showed that multimodal optical analysis is a promising technology for the development of an optical fiber setup designed for an intraoperative diagnosis of urothelial carcinoma in the area of endourology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Visualization of Membrane Rafts Using a Perylene Monoimide Derivative and Fluorescence Lifetime Imaging

    PubMed Central

    Margineanu, Anca; Hotta, Jun-ichi; Van der Auweraer, Mark; Ameloot, Marcel; Stefan, Alina; Beljonne, David; Engelborghs, Yves; Herrmann, Andreas; Müllen, Klaus; De Schryver, Frans C.; Hofkens, Johan

    2007-01-01

    A new membrane probe, based on the perylene imide chromophore, with excellent photophysical properties (high absorption coefficient, quantum yield (QY) ≈ 1, high photostability) and excited in the visible domain is proposed for the study of membrane rafts. Visualization of separation between the liquid-ordered (Lo) and the liquid-disordered (Ld) phases can be achieved in artificial membranes by fluorescence lifetime imaging due to the different decay times of the membrane probe in the two phases. Rafts on micrometer-scale in cell membranes due to cellular activation can also be observed by this method. The decay time of the dye in the Lo phase is higher than in organic solvents where its QY is 1. This allows proposing a (possible general) mechanism for the decay time increase in the Lo phase, based on the local field effects of the surrounding molecules. For other fluorophores with QY < 1, the suggested mechanism could also contribute, in addition to effects reducing the nonradiative decay pathways, to an increase of the fluorescence decay time in the Lo phase. PMID:17573424

  8. Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging

    PubMed Central

    Burdikova, Zuzana; Svindrych, Zdenek; Pala, Jan; Hickey, Cian D.; Wilkinson, Martin G.; Panek, Jiri; Auty, Mark A. E.; Periasamy, Ammasi; Sheehan, Jeremiah J.

    2015-01-01

    Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM). In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening. PMID:25798136

  9. Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging.

    PubMed

    Burdikova, Zuzana; Svindrych, Zdenek; Pala, Jan; Hickey, Cian D; Wilkinson, Martin G; Panek, Jiri; Auty, Mark A E; Periasamy, Ammasi; Sheehan, Jeremiah J

    2015-01-01

    Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM). In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening.

  10. Alterations in cerebral metabolism observed in living rodents using fluorescence lifetime microscopy of intrinsic NADH (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Yaseen, Mohammad A.; Sakadžić, Sava; Sutin, Jason; Wu, Weicheng; Fu, Buyin; Boas, David A.

    2017-02-01

    Monitoring cerebral energy metabolism at a cellular level is essential to improve our understanding of healthy brain function and its pathological alterations. In this study, we resolve specific alterations in cerebral metabolism utilizing minimally-invasive 2-Photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence, collected in vivo from anesthetized rats and mice. Time-resolved lifetime measurements enables distinction of different components contributing to NADH autofluorescence. These components reportedly represent different enzyme-bound formulations of NADH. Our observations from this study confirm the hypothesis that NADH FLIM can identify specific alterations in cerebral metabolism. Using time-correlated single photon counting (TCSPC) equipment and a custom-built multimodal imaging system, 2-photon fluorescence lifetime imaging (FLIM) was performed in cerebral tissue with high spatial and temporal resolution. Multi-exponential fits for NADH fluorescence lifetimes indicate 4 distinct components, or 'species.' We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in anaerobic glycolysis and aerobic oxidative metabolism. Classification models developed with experimental data correctly predict the metabolic impairments associated with bicuculline-induced focal seizures in separate experiments. Compared to traditional intensity-based NADH measurements, lifetime imaging of NADH is less susceptible to the adverse effects of overlying blood vessels. Evaluating NADH measurements will ultimately lead to a deeper understanding of cerebral energetics and its pathology-related alterations. Such knowledge will likely aid development of therapeutic strategies for neurodegenerative diseases such as Alzheimer's Disease, Parkinson's disease, and stroke.

  11. Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

    PubMed Central

    Fruhwirth, Gilbert O.; Ameer-Beg, Simon; Cook, Richard; Watson, Timothy; Ng, Tony; Festy, Frederic

    2010-01-01

    Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications. PMID:20588974

  12. Online multispectral fluorescence lifetime values estimation and overlay onto tissue white-light video frames

    NASA Astrophysics Data System (ADS)

    Gorpas, Dimitris; Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Marcu, Laura

    2016-03-01

    Fluorescence lifetime imaging has been shown to be a robust technique for biochemical and functional characterization of tissues and to present great potential for intraoperative tissue diagnosis and guidance of surgical procedures. We report a technique for real-time mapping of fluorescence parameters (i.e. lifetime values) onto the location from where the fluorescence measurements were taken. This is achieved by merging a 450 nm aiming beam generated by a diode laser with the excitation light in a single delivery/collection fiber and by continuously imaging the region of interest with a color CMOS camera. The interrogated locations are then extracted from the acquired frames via color-based segmentation of the aiming beam. Assuming a Gaussian profile of the imaged aiming beam, the segmentation results are fitted to ellipses that are dynamically scaled at the full width of three automatically estimated thresholds (50%, 75%, 90%) of the Gaussian distribution's maximum value. This enables the dynamic augmentation of the white-light video frames with the corresponding fluorescence decay parameters. A fluorescence phantom and fresh tissue samples were used to evaluate this method with motorized and hand-held scanning measurements. At 640x512 pixels resolution the area of interest augmented with fluorescence decay parameters can be imaged at an average 34 frames per second. The developed method has the potential to become a valuable tool for real-time display of optical spectroscopy data during continuous scanning applications that subsequently can be used for tissue characterization and diagnosis.

  13. Monitoring foveal sparing in geographic atrophy with fluorescence lifetime imaging ophthalmoscopy - a novel approach.

    PubMed

    Sauer, Lydia; Klemm, Matthias; Peters, Sven; Schweitzer, Dietrich; Schmidt, Johanna; Kreilkamp, Lukas; Ramm, Lisa; Meller, Daniel; Hammer, Martin

    2018-05-01

    To investigate fundus autofluorescence (FAF) lifetimes in geographic atrophy (GA) with a focus on macular pigment (MP) and foveal sparing. The study included 35 eyes from 28 patients (mean age 79.2 ± 8.0 years) with GA. A 30° retinal field, centred at the macula, was investigated using fluorescence lifetime imaging ophthalmoscopy (FLIO). The FLIO technology is based on a Heidelberg Engineering Spectralis system. Decays of FAF were detected in a short (498-560 nm, SSC) and long (560-720 nm, LSC) spectral channel. The mean fluorescence lifetime, τ m , was calculated from a three-exponential approximation of the FAF decays. Macular optical coherence tomography (OCT) scans as well as fundus photography were recorded. Review of FLIO data reveals specific patterns of significantly prolonged τ m in regions of GA (SSC 616 ± 343 ps, LSC 615 ± 154 ps) as compared to non-atrophic regions. Large τ m differences between the fovea and atrophic areas correlate with better visual acuity (VA). Shorter τ m at the fovea than within other non-atrophic regions indicates sparing, which was identified in 16 eyes. Seventy per cent of patients treated with lutein supplementation showed foveal sparing, whereas the rate among non-supplemented patients was 22%. Using FLIO, we present a novel way to detect foveal sparing, investigate MP, and analyse variability of τ m in different foveal regions (including the prognostic valuable border region) in GA. These findings support the potential utility of FLIO in monitoring disease progression. The findings also highlight the possibly protective effect of lutein supplementation, with implication in recording the presence and distributional pattern of MP. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  14. Imaging Fibrosis and Separating Collagens using Second Harmonic Generation and Phasor Approach to Fluorescence Lifetime Imaging

    PubMed Central

    Ranjit, Suman; Dvornikov, Alexander; Stakic, Milka; Hong, Suk-Hyun; Levi, Moshe; Evans, Ronald M.; Gratton, Enrico

    2015-01-01

    In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur. This is a label free approach towards fast automatable detection of fibrosis in tissue samples. FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy. Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated. PMID:26293987

  15. Demonstration of the lack of cytotoxicity of unmodified and folic acid modified graphene oxide quantum dots, and their application to fluorescence lifetime imaging of HaCaT cells.

    PubMed

    Goreham, Renee V; Schroeder, Kathryn L; Holmes, Amy; Bradley, Siobhan J; Nann, Thomas

    2018-01-24

    The authors describe the synthesis of water-soluble and fluorescent graphene oxide quantum dots via acid exfoliation of graphite nanoparticles. The resultant graphene oxide quantum dots (GoQDs) were then modified with folic acid. Folic acid receptors are overexpressed in cancer cells and hence can bind to functionalized graphene oxide quantum dots. On excitation at 305 nm, the GoQDs display green fluorescence with a peak wavelength at ~520 nm. The modified GoQDs are non-toxic to macrophage cells even after prolonged exposure and high concentrations. Fluorescence lifetime imaging and multiphoton microscopy was used (in combination) to image HeCaT cells exposed to GoQDs, resulting in a superior method for bioimaging. Graphical abstract Schematic representation of graphene oxide quantum dots, folic acid modified graphene oxide quantum dots (red), and the use of fluorescence lifetime to discriminate against green auto-fluorescence of HeCaT cells.

  16. Fluorescent biosensor for the detection of hyaluronidase: intensity-based ratiometric sensing and fluorescence lifetime-based sensing using a long lifetime azadioxatriangulenium (ADOTA) fluorophore.

    PubMed

    Chib, Rahul; Mummert, Mark; Bora, Ilkay; Laursen, Bo W; Shah, Sunil; Pendry, Robert; Gryczynski, Ignacy; Borejdo, Julian; Gryczynski, Zygmunt; Fudala, Rafal

    2016-05-01

    In this report, we have designed a rapid and sensitive, intensity-based ratiometric sensing as well as lifetime-based sensing probe for the detection of hyaluronidase activity. Hyaluronidase expression is known to be upregulated in various pathological conditions. We have developed a fluorescent probe by heavy labeling of hyaluronic acid with a new orange/red-emitting organic azadioxatriangulenium (ADOTA) fluorophore, which exhibits a long fluorescence lifetime (∼20 ns). The ADOTA fluorophore in water has a peak fluorescence lifetime of ∼20 ns and emission spectra centered at 560 nm. The heavily ADOTA-labeled hyaluronic acid (HA-ADOTA) shows a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal, and a shorter fluorescence lifetime (∼4 ns) due to efficient self-quenching and formation of aggregates. In the presence of hyaluronidase, the brightness and fluorescence lifetime of the sample increase with a blue shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA-ADOTA probe at 560 and 605 nm can be used as the sensing method for the detection of hyaluronidase. The cleavage of the hyaluronic acid macromolecule reduces the energy migration between ADOTA molecules, as well as the degree of self-quenching and aggregation. This probe can be efficiently used for both intensity-based ratiometric sensing as well as fluorescence lifetime-based sensing of hyaluronidase. The proposed method makes it a rapid and sensitive assay, useful for analyzing levels of hyaluronidase in relevant clinical samples like urine or plasma. Graphical Abstract Scheme showing cleavage of HA-ADOTA probe by hyaluronidase and the change in the emission spectrum of HA-ADOTA probe before and after cleavage by hyaluronidase.

  17. Simultaneous acquisition of trajectory and fluorescence lifetime of moving single particles

    NASA Astrophysics Data System (ADS)

    Wu, Qianqian; Qi, Jing; Lin, Danying; Yan, Wei; Hu, Rui; Peng, Xiao; Qu, Junle

    2017-02-01

    Fluorescence lifetime imaging (FLIM) has been a powerful tool in life science because it can reveal the interactions of an excited fluorescent molecule and its environment. The combination with two-photon excitation (TPE) and timecorrelated single photon counting (TCSPC) provides it the ability of optical sectioning, high time resolution and detection efficiency. In previous work, we have introduced a two-dimensional acousto-optic deflector (AOD) into TCSPC-based FLIM to achieve fast and flexible FLIM. In this work, we combined the AOD-FLIM system with a single particle tracking (SPT) setup and algorithm and developed an SPT-FLIM system. Using the system, we acquired the trajectory and fluorescence lifetime of a moving particle simultaneously and reconstructed a life-time-marked pseudocolored trajectory, which might reflect dynamic interaction between the moving particle and its local environment along its motion trail. The results indicated the potential of the technique for studying the interaction between specific moving biological macromolecules and the ambient micro-environment in live cells.

  18. Fluorescence lifetime measurements in heterogeneous scattering medium

    NASA Astrophysics Data System (ADS)

    Nishimura, Goro; Awasthi, Kamlesh; Furukawa, Daisuke

    2016-07-01

    Fluorescence lifetime in heterogeneous multiple light scattering systems is analyzed by an algorithm without solving the diffusion or radiative transfer equations. The algorithm assumes that the optical properties of medium are constant in the excitation and emission wavelength regions. If the assumption is correct and the fluorophore is a single species, the fluorescence lifetime can be determined by a set of measurements of temporal point-spread function of the excitation light and fluorescence at two different concentrations of the fluorophore. This method is not dependent on the heterogeneity of the optical properties of the medium as well as the geometry of the excitation-detection on an arbitrary shape of the sample. The algorithm was validated by an indocyanine green fluorescence in phantom measurements and demonstrated by an in vivo measurement.

  19. Visualizing Oxidative Cellular Stress Induced by Nanoparticles in the Subcytotoxic Range Using Fluorescence Lifetime Imaging.

    PubMed

    Balke, Jens; Volz, Pierre; Neumann, Falko; Brodwolf, Robert; Wolf, Alexander; Pischon, Hannah; Radbruch, Moritz; Mundhenk, Lars; Gruber, Achim D; Ma, Nan; Alexiev, Ulrike

    2018-06-01

    Nanoparticles hold a great promise in biomedical science. However, due to their unique physical and chemical properties they can lead to overproduction of intracellular reactive oxygen species (ROS). As an important mechanism of nanotoxicity, there is a great need for sensitive and high-throughput adaptable single-cell ROS detection methods. Here, fluorescence lifetime imaging microscopy (FLIM) is employed for single-cell ROS detection (FLIM-ROX) providing increased sensitivity and enabling high-throughput analysis in fixed and live cells. FLIM-ROX owes its sensitivity to the discrimination of autofluorescence from the unique fluorescence lifetime of the ROS reporter dye. The effect of subcytotoxic amounts of cationic gold nanoparticles in J774A.1 cells and primary human macrophages on ROS generation is investigated. FLIM-ROX measures very low ROS levels upon gold nanoparticle exposure, which is undetectable by the conventional method. It is demonstrated that cellular morphology changes, elevated senescence, and DNA damage link the resulting low-level oxidative stress to cellular adverse effects and thus nanotoxicity. Multiphoton FLIM-ROX enables the quantification of spatial ROS distribution in vivo, which is shown for skin tissue as a target for nanoparticle exposure. Thus, this innovative method allows identifying of low-level ROS in vitro and in vivo and, subsequently, promotes understanding of ROS-associated nanotoxicity. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Design, construction, and validation of a rotary multifunctional intravascular diagnostic catheter combining multispectral fluorescence lifetime imaging and intravascular ultrasound.

    PubMed

    Bec, Julien; Xie, Hongtao; Yankelevich, Diego R; Zhou, Feifei; Sun, Yang; Ghata, Narugopal; Aldredge, Ralph; Marcu, Laura

    2012-10-01

    We report the development and validation of an intravascular rotary catheter for bimodal interrogation of arterial pathologies. This is based on a point-spectroscopy scanning time-resolved fluorescence spectroscopy technique enabling reconstruction of fluorescence lifetime images (FLIm) and providing information on arterial intima composition and intravascular ultrasound (IVUS) providing information on arterial wall morphology. The catheter design allows for independent rotation of the ultrasonic and optical channels within an 8 Fr outer diameter catheter sheath and integrates a low volume flushing channel for blood removal in the optical pathways. In the current configuration, the two channels consist of (a) a standard 3 Fr IVUS catheter with single element transducer (40 MHz) and (b) a side-viewing fiber optic (400 μm core). Experiments conducted in tissue phantoms showed the ability of the catheter to operate in an intraluminal setting and to generate coregistered FLIm and IVUS in one pull-back scan. Current results demonstrate the feasibility of the catheter for simultaneous bimodal interrogation of arterial lumen and for generation of robust fluorescence lifetime data under IVUS guidance. These results facilitate further development of a FLIm-IVUS technique for intravascular diagnosis of atherosclerotic cardiovascular diseases including vulnerable plaques.

  1. Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry

    PubMed Central

    Houston, Jessica P.; Naivar, Mark A.; Freyer, James P.

    2010-01-01

    Frequency-domain flow cytometry techniques are combined with modifications to the digital signal processing capabilities of the Open Reconfigurable Cytometric Acquisition System (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency modulated detector signals, implementing Fourier analysis programming with ORCAS’ digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5–25 ns simulated lifetime), pulse widths ranging from 2 to 15 µs, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142° to 1.6°. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 µs and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a radiofrequency modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to ~98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively

  2. Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Uchugonova, Aisada

    2017-06-01

    The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

  3. Metabolic Mapping of Breast Cancer with Multiphoton Spectral and Lifetime Imaging

    DTIC Science & Technology

    2007-03-01

    spectral and lifetime characterization of NADH may be used to reveal metabolic changes in vivo and has potential to be used as an early diagnostic...combined spectral lifetime imaging modality will help for 5 characterization of breast cancer cells from cell culture based models to a relevant in... spectral and lifetime system and integrated into a multiphoton fluorescence excitation microscopy system 7 • Calibrated and characterized this

  4. Fluorescence lifetime evaluation of whole soils from the Amazon rainforest.

    PubMed

    Nicolodelli, Gustavo; Tadini, Amanda Maria; Nogueira, Marcelo Saito; Pratavieira, Sebastião; Mounier, Stephane; Huaman, Jose Luis Clabel; Dos Santos, Cléber Hilário; Montes, Célia Regina; Milori, Débora Marcondes Bastos Pereira

    2017-08-20

    Time-resolved fluorescence spectroscopy (TRFS) is a new tool that can be used to investigate processes of interaction between metal ions and organic matter (OM) in soils, providing a specific analysis of the structure and dynamics of macromolecules. To the best of our knowledge, there are no studies in the literature reporting the use of this technique applied to whole/non-fractionated soil samples, making it a potential method for use in future studies. This work describes the use of TRFS to evaluate the fluorescence lifetimes of OM of whole soils from the Amazon region. Analysis was made of pellets of soils from an oxisol-spodosol system, collected in São Gabriel da Cachoeira (Amazonas, Brazil). The fluorescence lifetimes in the oxisol-spodosol system were attributed to two different fluorophores. One was related to complexation of an OM fraction with metals, resulting in a shorter fluorophore lifetime. A short fluorescence lifetime (2-12 ns) could be associated with simpler structures of the OM, while a long lifetime (19-66 ns) was associated with more complex OM structures. This new TRFS technique for analysis of the fluorescence lifetime in whole soil samples complies with the principles of green chemistry.

  5. Fluorescence lifetime as a new parameter in analytical cytology measurements

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.; Deka, Chiranjit; Lehnert, Bruce E.; Crissman, Harry A.

    1996-05-01

    A phase-sensitive flow cytometer has been developed to quantify fluorescence decay lifetimes on fluorochrome-labeled cells/particles. This instrument combines flow cytometry (FCM) and frequency-domain fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved lifetime measurements, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by conventional and phase-sensitive signal detection electronics and displayed as frequency distribution histograms. In this study we describe results of fluorescence intensity and lifetime measurements on fluorescently labeled particles, cells, and chromosomes. Examples of measurements on intrinsic cellular autofluorescence, cells labeled with immunofluorescence markers for cell- surface antigens, mitochondria stains, and on cellular DNA and protein binding fluorochromes will be presented to illustrate unique differences in measured lifetimes and changes caused by fluorescence quenching. This innovative technology will be used to probe fluorochrome/molecular interactions in the microenvironment of cells/chromosomes as a new parameter and thus expand the researchers' understanding of biochemical processes and structural features at the cellular and molecular level.

  6. Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell

    PubMed Central

    Szaszák, Márta; Steven, Philipp; Shima, Kensuke; Orzekowsky-Schröder, Regina; Hüttmann, Gereon; König, Inke R.; Solbach, Werner; Rupp, Jan

    2011-01-01

    Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity. PMID:21779161

  7. Imaging a photodynamic therapy photosensitizer in vivo with a time-gated fluorescence tomography system

    NASA Astrophysics Data System (ADS)

    Mo, Weirong; Rohrbach, Daniel; Sunar, Ulas

    2012-07-01

    We report the tomographic imaging of a photodynamic therapy (PDT) photosensitizer, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) in vivo with time-domain fluorescence diffuse optical tomography (TD-FDOT). Simultaneous reconstruction of fluorescence yield and lifetime of HPPH was performed before and after PDT. The methodology was validated in phantom experiments, and depth-resolved in vivo imaging was achieved through simultaneous three-dimensional (3-D) mappings of fluorescence yield and lifetime contrasts. The tomographic images of a human head-and-neck xenograft in a mouse confirmed the preferential uptake and retention of HPPH by the tumor 24-h post-injection. HPPH-mediated PDT induced significant changes in fluorescence yield and lifetime. This pilot study demonstrates that TD-FDOT may be a good imaging modality for assessing photosensitizer distributions in deep tissue during PDT monitoring.

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

    PubMed

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

    2014-07-09

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

  9. Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

    NASA Astrophysics Data System (ADS)

    Bogh, Sidsel A.; Bora, Ilkay; Rosenberg, Martin; Thyrhaug, Erling; Laursen, Bo W.; Just Sørensen, Thomas

    2015-12-01

    Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r 0  =  0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (ϕ fl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕ fl, a ~20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.

  10. Automatic segmentation of fluorescence lifetime microscopy images of cells using multiresolution community detection--a first study.

    PubMed

    Hu, D; Sarder, P; Ronhovde, P; Orthaus, S; Achilefu, S; Nussinov, Z

    2014-01-01

    Inspired by a multiresolution community detection based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Furthermore, using the proposed method, the mean-square error in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The multiresolution community detection method appeared to perform better than a popular spectral clustering-based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in mean-square error with increasing resolution. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

  11. Three-dimensional online surface reconstruction of augmented fluorescence lifetime maps using photometric stereo (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Unger, Jakob; Lagarto, Joao; Phipps, Jennifer; Ma, Dinglong; Bec, Julien; Sorger, Jonathan; Farwell, Gregory; Bold, Richard; Marcu, Laura

    2017-02-01

    Multi-Spectral Time-Resolved Fluorescence Spectroscopy (ms-TRFS) can provide label-free real-time feedback on tissue composition and pathology during surgical procedures by resolving the fluorescence decay dynamics of the tissue. Recently, an ms-TRFS system has been developed in our group, allowing for either point-spectroscopy fluorescence lifetime measurements or dynamic raster tissue scanning by merging a 450 nm aiming beam with the pulsed fluorescence excitation light in a single fiber collection. In order to facilitate an augmented real-time display of fluorescence decay parameters, the lifetime values are back projected to the white light video. The goal of this study is to develop a 3D real-time surface reconstruction aiming for a comprehensive visualization of the decay parameters and providing an enhanced navigation for the surgeon. Using a stereo camera setup, we use a combination of image feature matching and aiming beam stereo segmentation to establish a 3D surface model of the decay parameters. After camera calibration, texture-related features are extracted for both camera images and matched providing a rough estimation of the surface. During the raster scanning, the rough estimation is successively refined in real-time by tracking the aiming beam positions using an advanced segmentation algorithm. The method is evaluated for excised breast tissue specimens showing a high precision and running in real-time with approximately 20 frames per second. The proposed method shows promising potential for intraoperative navigation, i.e. tumor margin assessment. Furthermore, it provides the basis for registering the fluorescence lifetime maps to the tissue surface adapting it to possible tissue deformations.

  12. Determination of biological activity from fluorescence-lifetime measurements in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Rudek, F.; Baselt, T.; Lempe, B.; Taudt, C.; Hartmann, P.

    2015-03-01

    The importance of fluorescence lifetime measurement as an optical analysis tool is growing. Many applications already exist in order to determine the fluorescence lifetime, but the majority of these require the addition of fluorescence-active substances to enable measurements. Every usage of such foreign materials has an associated risk. This paper investigates the use of auto-fluorescing substances in Saccharomyces cerevisiae (Baker's yeast) as a risk free alternative to fluorescence-active substance enabled measurements. The experimental setup uses a nitrogen laser with a pulse length of 350 ps and a wavelength of 337 nm. The excited sample emits light due to fluorescence of NADH/NADPH and collagen. A fast photodiode collects the light at the output of an appropriate high-pass edge-filter at 400 nm. Fluorescence lifetimes can be determined from the decay of the measurement signals, which in turn characterizes the individual materials and their surrounding environment. Information about the quantity of the fluorescence active substances can also be measured based on the received signal intensity. The correlation between the fluorescence lifetime and the metabolic state of Saccharomyces cerevisiae was investigated and is presented here.

  13. A portable time-domain LED fluorimeter for nanosecond fluorescence lifetime measurements

    NASA Astrophysics Data System (ADS)

    Wang, Hongtao; Qi, Ying; Mountziaris, T. J.; Salthouse, Christopher D.

    2014-05-01

    Fluorescence lifetime measurements are becoming increasingly important in chemical and biological research. Time-domain lifetime measurements offer fluorescence multiplexing and improved handling of interferers compared with the frequency-domain technique. In this paper, an all solid-state, filterless, and highly portable light-emitting-diode based time-domain fluorimeter (LED TDF) is reported for the measurement of nanosecond fluorescence lifetimes. LED based excitation provides more wavelengths options compared to laser diode based excitation, but the excitation is less effective due to the uncollimated beam, less optical power, and longer latency in state transition. Pulse triggering and pre-bias techniques were implemented in our LED TDF to improve the peak optical power to over 100 mW. The proposed pulsing circuit achieved an excitation light fall time of less than 2 ns. Electrical resetting technique realized a time-gated photo-detector to remove the interference of the excitation light with fluorescence. These techniques allow the LED fluorimeter to accurately measure the fluorescence lifetime of fluorescein down to concentration of 0.5 μM. In addition, all filters required in traditional instruments are eliminated for the non-attenuated excitation/emission light power. These achievements make the reported device attractive to biochemical laboratories seeking for highly portable lifetime detection devices for developing sensors based on fluorescence lifetime changes. The device was initially validated by measuring the lifetimes of three commercial fluorophores and comparing them with reported lifetime data. It was subsequently used to characterize a ZnSe quantum dot based DNA sensor.

  14. A portable time-domain LED fluorimeter for nanosecond fluorescence lifetime measurements

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Hongtao; Salthouse, Christopher D., E-mail: salthouse@ecs.umass.edu; Center for Personalized Health Monitoring, University of Massachusetts, Amherst, Massachusetts 01003

    2014-05-15

    Fluorescence lifetime measurements are becoming increasingly important in chemical and biological research. Time-domain lifetime measurements offer fluorescence multiplexing and improved handling of interferers compared with the frequency-domain technique. In this paper, an all solid-state, filterless, and highly portable light-emitting-diode based time-domain fluorimeter (LED TDF) is reported for the measurement of nanosecond fluorescence lifetimes. LED based excitation provides more wavelengths options compared to laser diode based excitation, but the excitation is less effective due to the uncollimated beam, less optical power, and longer latency in state transition. Pulse triggering and pre-bias techniques were implemented in our LED TDF to improve themore » peak optical power to over 100 mW. The proposed pulsing circuit achieved an excitation light fall time of less than 2 ns. Electrical resetting technique realized a time-gated photo-detector to remove the interference of the excitation light with fluorescence. These techniques allow the LED fluorimeter to accurately measure the fluorescence lifetime of fluorescein down to concentration of 0.5 μM. In addition, all filters required in traditional instruments are eliminated for the non-attenuated excitation/emission light power. These achievements make the reported device attractive to biochemical laboratories seeking for highly portable lifetime detection devices for developing sensors based on fluorescence lifetime changes. The device was initially validated by measuring the lifetimes of three commercial fluorophores and comparing them with reported lifetime data. It was subsequently used to characterize a ZnSe quantum dot based DNA sensor.« less

  15. Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics.

    PubMed

    Wei, Liping; Yan, Wenrong; Ho, Derek

    2017-12-04

    Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices.

  16. Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics

    PubMed Central

    Yan, Wenrong; Ho, Derek

    2017-01-01

    Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices. PMID:29207568

  17. Two-dimensional fluorescence lifetime correlation spectroscopy. 2. Application.

    PubMed

    Ishii, Kunihiko; Tahara, Tahei

    2013-10-03

    In the preceding article, we introduced the theoretical framework of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). In this article, we report the experimental implementation of 2D FLCS. In this method, two-dimensional emission-delay correlation maps are constructed from the photon data obtained with the time-correlated single photon counting (TCSPC), and then they are converted to 2D lifetime correlation maps by the inverse Laplace transform. We develop a numerical method to realize reliable transformation, employing the maximum entropy method (MEM). We apply the developed actual 2D FLCS to two real systems, a dye mixture and a DNA hairpin. For the dye mixture, we show that 2D FLCS is experimentally feasible and that it can identify different species in an inhomogeneous sample without any prior knowledge. The application to the DNA hairpin demonstrates that 2D FLCS can disclose microsecond spontaneous dynamics of biological molecules in a visually comprehensible manner, through identifying species as unique lifetime distributions. A FRET pair is attached to the both ends of the DNA hairpin, and the different structures of the DNA hairpin are distinguished as different fluorescence lifetimes in 2D FLCS. By constructing the 2D correlation maps of the fluorescence lifetime of the FRET donor, the equilibrium dynamics between the open and the closed forms of the DNA hairpin is clearly observed as the appearance of the cross peaks between the corresponding fluorescence lifetimes. This equilibrium dynamics of the DNA hairpin is clearly separated from the acceptor-missing DNA that appears as an isolated diagonal peak in the 2D maps. The present study clearly shows that newly developed 2D FLCS can disclose spontaneous structural dynamics of biological molecules with microsecond time resolution.

  18. Fluorescence lifetime assays: current advances and applications in drug discovery.

    PubMed

    Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

    2011-06-01

    Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

  19. Detection of nucleic acid-protein interactions in plant leaves using fluorescence lifetime imaging microscopy.

    PubMed

    Camborde, Laurent; Jauneau, Alain; Brière, Christian; Deslandes, Laurent; Dumas, Bernard; Gaulin, Elodie

    2017-09-01

    DNA-binding proteins (DNA-BPs) and RNA-binding proteins (RNA-BPs) have critical roles in living cells in all kingdoms of life. Various experimental approaches exist for the study of nucleic acid-protein interactions in vitro and in vivo, but the detection of such interactions at the subcellular level remains challenging. Here we describe how to detect nucleic acid-protein interactions in plant leaves by using a fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM). Proteins of interest (POI) are tagged with a GFP and transiently expressed in plant cells to serve as donor fluorophore. After sample fixation and cell wall permeabilization, leaves are treated with Sytox Orange, a nucleic acid dye that can function as a FRET acceptor. Upon close association of the GFP-tagged POI with Sytox-Orange-stained nucleic acids, a substantial decrease of the GFP lifetime due to FRET between the donor and the acceptor can be monitored. Treatment with RNase before FRET-FLIM measurements allows determination of whether the POI associates with DNA and/or RNA. A step-by-step protocol is provided for sample preparation, data acquisition and analysis. We describe how to calibrate the equipment and include a tutorial explaining the use of the FLIM software. To illustrate our approach, we provide experimental procedures to detect the interaction between plant DNA and two proteins (the AeCRN13 effector from the oomycete Aphanomyces euteiches and the AtWRKY22 defensive transcription factor from Arabidopsis). This protocol allows the detection of protein-nucleic acid interactions in plant cells and can be completed in <2 d.

  20. Application of novel low-intensity nonscanning fluorescence lifetime imaging microscopy for monitoring excited state dynamics in individual chloroplasts and living cells of photosynthetic organisms

    NASA Astrophysics Data System (ADS)

    Eckert, Hann-Jörg; Petrášek, Zdeněk; Kemnitz, Klaus

    2006-10-01

    Picosecond fluorescence lifetime imaging microscopy (FLIM) provides a most valuable tool to analyze the primary processes of photosynthesis in individual cells and chloroplasts of living cells. In order to obtain correct lifetimes of the excited states, the peak intensity of the exciting laser pulses as well as the average intensity has to be sufficiently low to avoid distortions of the kinetics by processes such as singlet-singlet annihilation, closing of the reaction centers or photoinhibition. In the present study this requirement is achieved by non-scanning wide-field FLIM based on time- and space-correlated single-photon counting (TSCSPC) using a novel microchannel plate photomultiplier with quadrant anode (QA-MCP) that allows parallel acquisition of time-resolved images under minimally invasive low-excitation conditions. The potential of the wide-field TCSPC method is demonstrated by presenting results obtained from measurements of the fluorescence dynamics in individual chloroplasts of moss leaves and living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.

  1. X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging.

    PubMed

    Malo, Gabrielle D; Pouwels, Lauren J; Wang, Meitian; Weichsel, Andrzej; Montfort, William R; Rizzo, Mark A; Piston, David W; Wachter, Rebekka M

    2007-09-04

    The crystal structure of the cyan-fluorescent Cerulean green fluorescent protein (GFP), a variant of enhanced cyan fluorescent protein (ECFP), has been determined to 2.0 A. Cerulean bears an internal fluorophore composed of an indole moiety derived from Y66W, conjugated to the GFP-like imidazolinone ring via a methylene bridge. Cerulean undergoes highly efficient fluorescence resonance energy transfer (FRET) to yellow acceptor molecules and exhibits significantly reduced excited-state heterogeneity. This feature was rationally engineered in ECFP by substituting His148 with an aspartic acid [Rizzo et al. (2004) Nat. Biotechnol. 22, 445], rendering Cerulean useful for fluorescence lifetime imaging microscopy (FLIM). The X-ray structure is consistent with a single conformation of the chromophore and surrounding residues and may therefore provide a structural rationale for the previously described monoexponential fluorescence decay. Unexpectedly, the carboxyl group of H148D is found in a buried position, directly contacting the indole nitrogen of the chromophore via a bifurcated hydrogen bond. Compared to the similarly constructed ECFP chromophore, the indole group of Cerulean is rotated around the methylene bridge to adopt a cis-coplanar conformation with respect to the imidazolinone ring, resulting in a close edge-to-edge contact of the two ring systems. The double-humped absorbance spectrum persists in single-crystal absorbance measurements, casting doubt on the idea that ground state conformational heterogeneity forms the basis of the two overlapping transitions. At low pH, a blue shift in absorbance of 10-15 nm suggests a pH-induced structural transition that proceeds with a time constant of 47 (+/-2) min and is reversible. Possible interpretations in terms of chromophore isomerization are presented.

  2. Multiphoton microscopy, fluorescence lifetime imaging and optical spectroscopy for the diagnosis of neoplasia

    NASA Astrophysics Data System (ADS)

    Skala, Melissa Caroline

    2007-12-01

    Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in

  3. Fluorescence lifetime microscopy for monitoring cell adhesion using metal induced energy transfer

    NASA Astrophysics Data System (ADS)

    Hwang, Wonsang; Seo, JinWon; Song, Jun ho; Kim, DongEun; Won, YoungJae; Choi, In-Hong; Yoo, Kyung-Hwa; Kim, Dug Young

    2018-02-01

    A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.

  4. Automatic Segmentation of Fluorescence Lifetime Microscopy Images of Cells Using Multi-Resolution Community Detection -A First Study

    PubMed Central

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

    2014-01-01

    Inspired by a multi-resolution community detection (MCD) based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Further, using the proposed method, the mean-square error (MSE) in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The MCD method appeared to perform better than a popular spectral clustering based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in MSE with increasing resolution. PMID:24251410

  5. Portable fluorescence lifetime spectroscopy system for in-situ interrogation of biological tissues

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Teixeira Rosa, Ramon Gabriel; Salvio, Ana Gabriela; Pratavieira, Sebastião; Kurachi, Cristina

    2017-12-01

    Fluorescence spectroscopy and lifetime techniques are potential methods for optical diagnosis and characterization of biological tissues with an in-situ, fast, and noninvasive interrogation. Several diseases may be diagnosed due to differences in the fluorescence spectra of targeted fluorophores, when, these spectra are similar, considering steady-state fluorescence, others may be detected by monitoring their fluorescence lifetime. Despite this complementarity, most of the current fluorescence lifetime systems are not robust and portable, and not being feasible for clinical applications. We describe the assembly of a fluorescence lifetime spectroscopy system in a suitcase, its characterization, and validation with clinical measurements of skin lesions. The assembled system is all encased and robust, maintaining its mechanical, electrical, and optical stability during transportation, and is feasible for clinical measurements. The instrument response function measured was about 300 ps, and the system is properly calibrated. At the clinical study, the system showed to be reliable, and the achieved spectroscopy results support its potential use as an auxiliary tool for skin diagnostics.

  6. In vivo and in vitro investigations of retinal fluorophores in age-related macular degeneration by fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Hammer, M.; Quick, S.; Klemm, M.; Schenke, S.; Mata, N.; Eitner, A.; Schweitzer, D.

    2009-02-01

    Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently for the observation of the age pigment lipofuscin, a precursor of age-related macular degeneration (AMD). However, a deeper understanding of the generation of single compounds contributing to the lipofuscin as well as of the role of other fluorophores such as FAD, glycated proteins, and collagen needs their discrimination by fluorescence lifetime imaging (FLIM). FLIM at the ocular fundus is performed using a scanning laser ophthalmoscope equipped with a picosecond laser source (448nm or 468nm respectively, 100ps, 80 MHz repetition rate) and dual wavelength (490-560nm and 560-7600nm) time-correlated single photon counting. A three-exponential fit of the fluorescence decay revealed associations of decay times to anatomical structures. Disease-related features are identified from alterations in decay times and-amplitudes. The in-vivo investigations in patients were paralleled by experiments in an organ culture of the porcine ocular fundus. Photo-oxidative stress was induced by exposure to blue light (467nm, 0.41 mW/mm2). Subsequent analysis (fluorescence microscopy, HPLC, LC-MS) indicated the accumulation of the pyridinium bis-retinoid A2E and its oxidation products as well as oxidized phospholipids. These compounds contribute to the tissue auto-fluorescence and may play a key role in the pathogenesis of AMD. Thus, FLIM observation at the ocular fundus in vivo enhances our knowledge on the etiology of AMD and may become a diagnostic tool.

  7. Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy.

    PubMed

    Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N; Wientjes, Emilie; van Amerongen, Herbert

    2016-09-01

    Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was achieved by separating the time-resolved fluorescence of PSI and PSII in the leaf. It is found that the PSII antenna size is larger on the abaxial side of A. thaliana leaves, presumably because chloroplasts in the spongy mesophyll are "shaded" by the palisade cells. The number of chlorophylls in PSI on the adaxial side of the A. thaliana leaf is slightly higher. The C4 plant M. x giganteus contains both mesophyll and bundle sheath cells, which have a different PSI/PSII ratio. It is shown that the time-resolved fluorescence of bundle sheath and mesophyll cells can be analysed separately. The relative number of chlorophylls, which belong to PSI (as compared to PSII) in the bundle sheath cells is at least 2.5 times higher than in mesophyll cells. FLIM is thus demonstrated to be a useful technique to study the PSI/PSII ratio and PSII antenna size in well-defined regions of plant leaves without having to isolate pigment-protein complexes. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. A 65k pixel, 150k frames-per-second camera with global gating and micro-lenses suitable for fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Burri, Samuel; Powolny, François; Bruschini, Claudio E.; Michalet, Xavier; Regazzoni, Francesco; Charbon, Edoardo

    2014-05-01

    This paper presents our work on a 65k pixel single-photon avalanche diode (SPAD) based imaging sensor realized in a 0.35μm standard CMOS process. At a resolution of 512 by 128 pixels the sensor is read out in 6.4μs to deliver over 150k monochrome frames per second. The individual pixel has a size of 24μm2 and contains the SPAD with a 12T quenching and gating circuitry along with a memory element. The gating signals are distributed across the chip through a balanced tree to minimize the signal skew between the pixels. The array of pixels is row-addressable and data is sent out of the chip on 128 lines in parallel at a frequency of 80MHz. The system is controlled by an FPGA which generates the gating and readout signals and can be used for arbitrary real-time computation on the frames from the sensor. The communication protocol between the camera and a conventional PC is USB2. The active area of the chip is 5% and can be significantly improved with the application of a micro-lens array. A micro-lens array, for use with collimated light, has been designed and its performance is reviewed in the paper. Among other high-speed phenomena the gating circuitry capable of generating illumination periods shorter than 5ns can be used for Fluorescence Lifetime Imaging (FLIM). In order to measure the lifetime of fluorophores excited by a picosecond laser, the sensor's illumination period is synchronized with the excitation laser pulses. A histogram of the photon arrival times relative to the excitation is then constructed by counting the photons arriving during the sensitive time for several positions of the illumination window. The histogram for each pixel is transferred afterwards to a computer where software routines extract the lifetime at each location with an accuracy better than 100ps. We show results for fluorescence lifetime measurements using different fluorophores with lifetimes ranging from 150ps to 5ns.

  9. Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

    NASA Astrophysics Data System (ADS)

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

    2016-03-01

    Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

  10. Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope.

    PubMed

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

    2016-03-01

    Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

  11. Portable fluorescence lifetime spectroscopy system for in-situ interrogation of biological tissues.

    PubMed

    Saito Nogueira, Marcelo; Cosci, Alessandro; Teixeira Rosa, Ramon Gabriel; Salvio, Ana Gabriela; Pratavieira, Sebastião; Kurachi, Cristina

    2017-10-01

    Fluorescence spectroscopy and lifetime techniques are potential methods for optical diagnosis and characterization of biological tissues with an in-situ, fast, and noninvasive interrogation. Several diseases may be diagnosed due to differences in the fluorescence spectra of targeted fluorophores, when, these spectra are similar, considering steady-state fluorescence, others may be detected by monitoring their fluorescence lifetime. Despite this complementarity, most of the current fluorescence lifetime systems are not robust and portable, and not being feasible for clinical applications. We describe the assembly of a fluorescence lifetime spectroscopy system in a suitcase, its characterization, and validation with clinical measurements of skin lesions. The assembled system is all encased and robust, maintaining its mechanical, electrical, and optical stability during transportation, and is feasible for clinical measurements. The instrument response function measured was about 300 ps, and the system is properly calibrated. At the clinical study, the system showed to be reliable, and the achieved spectroscopy results support its potential use as an auxiliary tool for skin diagnostics. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  12. Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts

    PubMed Central

    Cao, Ruofan; Naivar, Mark A; Wilder, Mark; Houston, Jessica P

    2014-01-01

    Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce. The fluorescence lifetime is emerging in flow cytometry and is helpful in a variety of multiparametric, single cell measurements because it is not impacted by nonlinearity that can occur with fluorescence intensity measurements. Yet time-resolved cytometry systems rely on major hardware modifications making the methodology difficult to reproduce. The motivation of this work is, by taking advantage of the dynamic nature of flow cytometry sample detection and applying digital signal processing methods, to measure fluorescence lifetimes using an unmodified flow cytometer. We collect a new lifetime-dependent parameter, referred to herein as the fluorescence-pulse-delay (FPD), and prove it is a valid representation of the average fluorescence lifetime. To verify we generated cytometric pulses in simulation, with light emitting diode (LED) pulsation, and with true fluorescence measurements of cells and microspheres. Each pulse is digitized and used in algorithms to extract an average fluorescence lifetime inherent in the signal. A range of fluorescence lifetimes is measurable with this approach including standard organic fluorophore lifetimes (∼1 to 22 ns) as well as small, simulated shifts (0.1 ns) under standard conditions (reported herein). This contribution demonstrates how digital data acquisition and signal processing can reveal time-dependent information foreshadowing the exploitation of full waveform analysis for quantification of similar photo-physical events within single cells. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:25274073

  13. Investigation of tryptophan-NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy

    NASA Astrophysics Data System (ADS)

    Jyothikumar, Vinod; Sun, Yuansheng; Periasamy, Ammasi

    2013-06-01

    A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label-free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.

  14. Development of a Multi-modal Tissue Diagnostic System Combining High Frequency Ultrasound and Photoacoustic Imaging with Lifetime Fluorescence Spectroscopy

    PubMed Central

    Sun, Yang; Stephens, Douglas N.; Park, Jesung; Sun, Yinghua; Marcu, Laura; Cannata, Jonathan M.; Shung, K. Kirk

    2010-01-01

    We report the development and validate a multi-modal tissue diagnostic technology, which combines three complementary techniques into one system including ultrasound backscatter microscopy (UBM), photoacoustic imaging (PAI), and time-resolved laser-induced fluorescence spectroscopy (TR-LIFS). UBM enables the reconstruction of the tissue microanatomy. PAI maps the optical absorption heterogeneity of the tissue associated with structure information and has the potential to provide functional imaging of the tissue. Examination of the UBM and PAI images allows for localization of regions of interest for TR-LIFS evaluation of the tissue composition. The hybrid probe consists of a single element ring transducer with concentric fiber optics for multi-modal data acquisition. Validation and characterization of the multi-modal system and ultrasonic, photoacoustic, and spectroscopic data coregistration were conducted in a physical phantom with properties of ultrasound scattering, optical absorption, and fluorescence. The UBM system with the 41 MHz ring transducer can reach the axial and lateral resolution of 30 and 65 μm, respectively. The PAI system with 532 nm excitation light from a Nd:YAG laser shows great contrast for the distribution of optical absorbers. The TR-LIFS system records the fluorescence decay with the time resolution of ~300 ps and a high sensitivity of nM concentration range. Biological phantom constructed with different types of tissues (tendon and fat) was used to demonstrate the complementary information provided by the three modalities. Fluorescence spectra and lifetimes were compared to differentiate chemical composition of tissues at the regions of interest determined by the coregistered high resolution UBM and PAI image. Current results demonstrate that the fusion of these techniques enables sequentially detection of functional, morphological, and compositional features of biological tissue, suggesting potential applications in diagnosis of tumors

  15. Development of a Multi-modal Tissue Diagnostic System Combining High Frequency Ultrasound and Photoacoustic Imaging with Lifetime Fluorescence Spectroscopy.

    PubMed

    Sun, Yang; Stephens, Douglas N; Park, Jesung; Sun, Yinghua; Marcu, Laura; Cannata, Jonathan M; Shung, K Kirk

    2008-01-01

    We report the development and validate a multi-modal tissue diagnostic technology, which combines three complementary techniques into one system including ultrasound backscatter microscopy (UBM), photoacoustic imaging (PAI), and time-resolved laser-induced fluorescence spectroscopy (TR-LIFS). UBM enables the reconstruction of the tissue microanatomy. PAI maps the optical absorption heterogeneity of the tissue associated with structure information and has the potential to provide functional imaging of the tissue. Examination of the UBM and PAI images allows for localization of regions of interest for TR-LIFS evaluation of the tissue composition. The hybrid probe consists of a single element ring transducer with concentric fiber optics for multi-modal data acquisition. Validation and characterization of the multi-modal system and ultrasonic, photoacoustic, and spectroscopic data coregistration were conducted in a physical phantom with properties of ultrasound scattering, optical absorption, and fluorescence. The UBM system with the 41 MHz ring transducer can reach the axial and lateral resolution of 30 and 65 μm, respectively. The PAI system with 532 nm excitation light from a Nd:YAG laser shows great contrast for the distribution of optical absorbers. The TR-LIFS system records the fluorescence decay with the time resolution of ~300 ps and a high sensitivity of nM concentration range. Biological phantom constructed with different types of tissues (tendon and fat) was used to demonstrate the complementary information provided by the three modalities. Fluorescence spectra and lifetimes were compared to differentiate chemical composition of tissues at the regions of interest determined by the coregistered high resolution UBM and PAI image. Current results demonstrate that the fusion of these techniques enables sequentially detection of functional, morphological, and compositional features of biological tissue, suggesting potential applications in diagnosis of tumors

  16. Substrate-based near-infrared imaging sensors enable fluorescence lifetime contrast via built-in dynamic fluorescence quenching elements.

    PubMed

    Kumar, Anand T N; Rice, William L; López, Jessica C; Gupta, Suresh; Goergen, Craig J; Bogdanov, Alexei A

    2016-04-22

    Enzymatic activity sensing in fluorescence lifetime (FLT) mode with "self-quenched" macromolecular near-infrared (NIR) sensors is a highly promising strategy for in vivo imaging of proteolysis. However, the mechanisms of FLT changes in such substrate-based NIR sensors have not yet been studied. We synthesized two types of sensors by linking the near-infrared fluorophore IRDye 800CW to macromolecular graft copolymers of methoxy polyethylene glycol and polylysine (MPEG-gPLL) with varying degrees of MPEGylation and studied their fragmentation induced by trypsin, elastase, plasmin and cathepsins (B,S,L,K). We determined that the efficiency of such NIR sensors in FLT mode depends on sensor composition. While MPEG-gPLL with a high degree of MPEGylation showed rapid (τ 1/2 =0.1-0.2 min) FLT increase (Δτ=0.25 ns) upon model proteinase-mediated hydrolysis in vivo , lower MPEGylation density resulted in no such FLT increase. Temperature-dependence of fluorescence de-quenching of NIR sensors pointed to a mixed dynamic/static-quenching mode of MPEG-gPLL-linked fluorophores. We further demonstrated that although the bulk of sensor-linked fluorophores were de-quenched due to the elimination of static quenching, proteolysis-mediated deletion of a fraction of short (8-10kD) negatively charged fragments of highly MPEGylated NIR sensor is the most likely event leading to a rapid FLT increase phenomenon in quenched NIR sensors. Therefore, the optimization of "built-in" dynamic quenching elements of macromolecular NIR sensors is a potential avenue for improving their response in FLT mode.

  17. Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

    PubMed

    Nyström, Sofie; Bäck, Marcus; Nilsson, K Peter R; Hammarström, Per

    2017-10-20

    Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

  18. Multiphoton fluorescence spectra and lifetimes of biliverdins and their protein-associated complex

    NASA Astrophysics Data System (ADS)

    Huang, Chin-Jie; Wu, Cheng-Ham; Liu, Tzu-Ming

    2012-03-01

    To investigate whether endogenous biliverdins can serve as a fluorescence metabolic marker in cancer diagnosis, we measured their multiphoton fluorescence spectra and lifetimes with femtosecond Cr:forsterite laser. Excited at 1230nm, the two-photon fluorescence of biliverdins peaks around 670nm. The corresponding lifetime (<100ps) was much shorter than those of porphyrins (~10ns), which is another commonly present metabolites in living cells. Further mixing biliverdins with proteins like fetal bovine serum (FBS), biliverdins reductase A (BVRA), or heme oxygenase-1 (HO-1), the yields of red autofluorescences didn't change a lot, but the corresponding lifetimes with HO-1 and BSA were lengthened to 200~300ps. This indicates that biliverdin can have an association with these proteins and change its lifetime. These spectral and temporal characteristics of fluorescence make biliverdin a potential marker fluorophore for hyperspectral diagnosis on the heme catabolism in human cells or tissues.

  19. Chemometric analysis for extraction of individual fluorescence spectrum and lifetimes from a target mixture

    NASA Technical Reports Server (NTRS)

    Hallidy, William H. (Inventor); Chin, Robert C. (Inventor)

    1999-01-01

    The present invention is a system for chemometric analysis for the extraction of the individual component fluorescence spectra and fluorescence lifetimes from a target mixture. The present invention combines a processor with an apparatus for generating an excitation signal to transmit at a target mixture and an apparatus for detecting the emitted signal from the target mixture. The present invention extracts the individual fluorescence spectrum and fluorescence lifetime measurements from the frequency and wavelength data acquired from the emitted signal. The present invention uses an iterative solution that first requires the initialization of several decision variables and the initial approximation determinations of intermediate matrices. The iterative solution compares the decision variables for convergence to see if further approximation determinations are necessary. If the solution converges, the present invention then determines the reduced best fit error for the analysis of the individual fluorescence lifetime and the fluorescence spectrum before extracting the individual fluorescence lifetime and fluorescence spectrum from the emitted signal of the target mixture.

  20. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  1. Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

    PubMed Central

    Paredes, Jose M.; Casares, Salvador; Ruedas-Rama, Maria J.; Fernandez, Elena; Castello, Fabio; Varela, Lorena; Orte, Angel

    2012-01-01

    Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies. PMID:22949804

  2. Automated detection of breast cancer in resected specimens with fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Phipps, Jennifer E.; Gorpas, Dimitris; Unger, Jakob; Darrow, Morgan; Bold, Richard J.; Marcu, Laura

    2018-01-01

    Re-excision rates for breast cancer lumpectomy procedures are currently nearly 25% due to surgeons relying on inaccurate or incomplete methods of evaluating specimen margins. The objective of this study was to determine if cancer could be automatically detected in breast specimens from mastectomy and lumpectomy procedures by a classification algorithm that incorporated parameters derived from fluorescence lifetime imaging (FLIm). This study generated a database of co-registered histologic sections and FLIm data from breast cancer specimens (N  =  20) and a support vector machine (SVM) classification algorithm able to automatically detect cancerous, fibrous, and adipose breast tissue. Classification accuracies were greater than 97% for automated detection of cancerous, fibrous, and adipose tissue from breast cancer specimens. The classification worked equally well for specimens scanned by hand or with a mechanical stage, demonstrating that the system could be used during surgery or on excised specimens. The ability of this technique to simply discriminate between cancerous and normal breast tissue, in particular to distinguish fibrous breast tissue from tumor, which is notoriously challenging for optical techniques, leads to the conclusion that FLIm has great potential to assess breast cancer margins. Identification of positive margins before waiting for complete histologic analysis could significantly reduce breast cancer re-excision rates.

  3. Protochlorophyll complexes with similar steady-state fluorescence characteristics can differ in fluorescence lifetimes. A model study in Triton X-100.

    PubMed

    Myśliwa-Kurdziel, Beata; Solymosi, Katalin; Kruk, Jerzy; Böddi, Béla; Strzałka, Kazimierz

    2007-03-01

    The steady-state and time-resolved fluorescence characteristics of protochlorophyll (Pchl) dissolved in neat Triton X-100 and in Triton X-100 micelles were investigated, and the fluorescence lifetimes of different Pchl spectral forms were studied. Varying the concentration of Pchl or diluting the micellar solutions either with a buffer or with a micellar solution, 631-634, 645-655, 680-692 and above 700 nm emitting Pchl complexes were prepared, the ratios of which varied from one another. The fluorescence decay of the 631-634 nm emitting (monomeric) form had a mono-exponential character with a 5.4-ns fluorescence lifetime. The long-wavelength Pchl complexes (aggregates) had two fluorescence lifetime values within a range of 1.4-3.9 ns and 0.15-0.84 ns, which showed high variability in different environments. Depending on the conditions, either mono- or double-exponential fluorescence decay was found for a fluorescence band at 680-685 nm. These data show that despite their very similar steady-state fluorescence properties, Pchl complexes can differ in fluorescence lifetimes, which may reflect different molecular structures, intrinsic geometries or different molecular interactions. This underlines the importance of complex spectroscopic analysis for a precise description of native and artificial chlorophyllous pigment forms.

  4. Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

    PubMed Central

    2013-01-01

    Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell

  5. Photo-multiplier Tube Based Hybrid MRI and Frequency Domain Fluorescence Tomography System for Small Animal Imaging

    PubMed Central

    Lin, Y; Ghijsen, M T; Gao, H; Liu, N; Nalcioglu, O; Gulsen, G

    2014-01-01

    Fluorescence tomography (FT) is a promising molecular imaging technique that can spatially resolve both fluorophore concentration and lifetime parameters. However, recovered fluorophore parameters highly depend on the size and depth of the object due to the ill-posedness of the FT inverse problem. Structural a priori information from another high spatial resolution imaging modality has been demonstrated to significantly improve FT reconstruction accuracy. In this study, we have constructed a combined magnetic resonance imaging (MRI) and FT system for small animal imaging. A photo-multiplier tube (PMT) is used as the detector to acquire frequency domain FT measurements. This is the first MR-compatible time-resolved FT system that can reconstruct both fluorescence concentration and lifetime maps simultaneously. The performance of the hybrid system is evaluated with phantom studies. Two different fluorophores, Indocyanine Green (ICG) and 3-3′ Diethylthiatricarbocyanine Iodide (DTTCI), which have similar excitation and emission spectra but different lifetimes, are utilized. The fluorescence concentration and lifetime maps are both reconstructed with and without the structural a priori information obtained from MRI for comparison. We show that the hybrid system can accurately recover both fluorescence intensity and lifetime within 10% error for two 4.2 mm-diameter cylindrical objects embedded in a 38 mm-diameter cylindrical phantom when MRI structural a priori information is utilized. PMID:21753235

  6. Long lifetime near-infrared-emitting quantum dots for time-gated in vivo imaging of rare circulating cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Fragola, Alexandra; Bouccara, Sophie; Pezet, Sophie; Lequeux, Nicolas; Loriette, Vincent; Pons, Thomas

    2017-02-01

    The in vivo detection of rare circulating cells using non invasive fluorescence imaging would provide a key tool to study migration of eg. tumoral or immunological cells. Fluorescence detection is however currently limited by a lack of contrast between the small emission of isolated, fast circulating cells and the strong autofluorescence background of the surrounding tissues. We present the development of near infrared emitting quantum dots (NIR-QDs) with long fluorescence lifetime for sensitive time-gated in vivo imaging of circulating cells. These QDs are composed of low toxicity ZnCuInSe/ZnS materials and made biocompatible using a novel multidentate imidazole zwitterionic block copolymer, ensuring their long term intracellular stability. Cells of interest can thus be labeled ex vivo with QDs, injected intravenously and imaged in the near infrared range. Excitation using a pulsed laser coupled to time-gated detection enables the efficient rejection of short lifetime (≈ ns) autofluorescence background and detection of long lifetime (≈ 150 ns) fluorescence from QD-labeled cells. We demonstrate efficient in vivo imaging of single fast-flowing cells, which opens opportunities for future biological studies. [1] M. Tasso et al, "Sulfobetaine-Vinylimidazole block copolymers: a robust quantum dot surface chemistry expanding bioimaging's horizons", ACS Nano, 9(11), 2015 [2] S. Bouccara et al, "Time-gated cell imaging using long lifetime near-infrared-emitting quantum dots for autofluorescence rejection", J Biomed Optc, 19(5), 2014

  7. On the uncertainty in single molecule fluorescent lifetime and energy emission measurements

    NASA Technical Reports Server (NTRS)

    Brown, Emery N.; Zhang, Zhenhua; Mccollom, Alex D.

    1995-01-01

    Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least square methods agree and are optimal when the number of detected photons is large however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67% of those can be noise and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous poisson processes, we derive the exact joint arrival time probably density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. the ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background nose and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.

  8. On the Uncertainty in Single Molecule Fluorescent Lifetime and Energy Emission Measurements

    NASA Technical Reports Server (NTRS)

    Brown, Emery N.; Zhang, Zhenhua; McCollom, Alex D.

    1996-01-01

    Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least squares methods agree and are optimal when the number of detected photons is large, however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67 percent of those can be noise, and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous Poisson processes, we derive the exact joint arrival time probability density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. The ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background noise and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.

  9. Fluorescence lifetime correlation spectroscopy for precise concentration detection in vivo by background subtraction

    NASA Astrophysics Data System (ADS)

    Gärtner, Maria; Mütze, Jörg; Ohrt, Thomas; Schwille, Petra

    2009-07-01

    In vivo studies of single molecule dynamics by means of Fluorescence correlation spectroscopy can suffer from high background. Fluorescence lifetime correlation spectroscopy provides a tool to distinguish between signal and unwanted contributions via lifetime separation. By studying the motion of the RNA-induced silencing complex (RISC) within two compartments of a human cell, the nucleus and the cytoplasm, we observed clear differences in concentration as well as mobility of the protein complex between those two locations. Especially in the nucleus, where the fluorescence signal is very weak, a correction for background is crucial to provide reliable results of the particle number. Utilizing the fluorescent lifetime of the different contributions, we show that it is possible to distinguish between the fluorescent signal and the autofluorescent background in vivo in a single measurement.

  10. Fluorescence lifetime of normal, benign, and malignant thyroid tissues

    NASA Astrophysics Data System (ADS)

    Brandao, Mariana; Iwakura, Ricardo; Basilio, Fagne; Haleplian, Kaique; Ito, Amando; de Freitas, Luiz Carlos Conti; Bachmann, Luciano

    2015-06-01

    Fine-needle aspiration cytology is the standard technique to diagnose thyroid pathologies. However, this method results in a high percentage of inconclusive and false negatives. The use of time-resolved fluorescence techniques to detect biochemical composition and tissue structure alterations could help to develop a portable, minimally invasive, and nondestructive method to assist during surgical procedures. This study aimed to use fluorescence lifetimes to differentiate healthy and benign tissues from malignant thyroid tissue. The thyroid tissue was excited at 298-300 nm and the fluorescence decay registered at 340 and 450 nm. We observed fluorescence lifetimes at 340 nm emission of 0.80±0.26 and 3.94±0.47 ns for healthy tissue; 0.90±0.24 and 4.05±0.46 ns for benign lesions; and 1.21±0.14 and 4.63±0.25 ns for malignant lesions. For 450 nm emissions, we obtain lifetimes of 0.25±0.18 and 3.99±0.39 ns for healthy tissue, 0.24±0.17 and 4.20±0.48 ns for benign lesions, 0.33±0.32 and 4.55±0.55 ns for malignant lesions. Employing analysis of variance, we differentiate malignant lesions from benign and healthy tissues. In addition, we use quadratic discriminant analysis to distinguish malignant from benign and healthy tissues with an accuracy of 76.1%, sensitivity of 74.7%, and specificity of 83.3%. These results indicate that time-resolved fluorescence can assist medical evaluation of thyroid pathologies during surgeries.

  11. In vivo multiphoton and fluorescence lifetime imaging microscopy of the healthy and cholestatic liver

    NASA Astrophysics Data System (ADS)

    Kuznetsova, Daria S.; Dudenkova, Varvara V.; Rodimova, Svetlana A.; Bobrov, Nikolai V.; Zagainov, Vladimir E.; Zagaynova, Elena V.

    2018-02-01

    A cholestatic liver disease presents one of the most common liver diseases and can potentially progress to cirrhosis or even cholangiocarcinoma. Conventional techniques are insufficient to precisely describe the complex internal structure, heterogeneous cell populations and the dynamics of biological processes of the liver. Currently, the methods of multiphoton and fluorescence lifetime imaging microscopy are actively introducing to biomedical research. Those methods are extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. Multiphoton and FLIM microscopy do not need additional staining of samples or the incorporation of any markers to study metabolism, lipid composition, microstructure analysis, evaluation of fibrous structures. These parameters have pronounced changes in hepatocytes of liver with common pathological diseases. Thereby in this study we investigated metabolic changes in the healthy and cholestatic liver based on the fluorescence of the metabolic co-factors NAD(P)H and FAD by multiphoton microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH was presented. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.

  12. FUNDUS AUTOFLUORESCENCE LIFETIMES AND CENTRAL SEROUS CHORIORETINOPATHY

    PubMed Central

    Dysli, Chantal; Berger, Lieselotte; Wolf, Sebastian

    2017-01-01

    Purpose: To quantify retinal fluorescence lifetimes in patients with central serous chorioretinopathy (CSC) and to identify disease specific lifetime characteristics over the course of disease. Methods: Forty-seven participants were included in this study. Patients with central serous chorioretinopathy were imaged with fundus photography, fundus autofluorescence, optical coherence tomography, and fluorescence lifetime imaging ophthalmoscopy (FLIO) and compared with age-matched controls. Retinal autofluorescence was excited using a 473-nm blue laser light and emitted fluorescence light was detected in 2 distinct wavelengths channels (498–560 nm and 560–720 nm). Clinical features, mean retinal autofluorescence lifetimes, autofluorescence intensity, and corresponding optical coherence tomography (OCT) images were further analyzed. Results: Thirty-five central serous chorioretinopathy patients with a mean visual acuity of 78 ETDRS letters (range, 50–90; mean Snellen equivalent: 20/32) and 12 age-matched controls were included. In the acute stage of central serous chorioretinopathy, retinal fluorescence lifetimes were shortened by 15% and 17% in the respective wavelength channels. Multiple linear regression analysis showed that fluorescence lifetimes were significantly influenced by the disease duration (P < 0.001) and accumulation of photoreceptor outer segments (P = 0.03) but independent of the presence or absence of subretinal fluid. Prolonged central macular autofluorescence lifetimes, particularly in eyes with retinal pigment epithelial atrophy, were associated with poor visual acuity. Conclusion: This study establishes that autofluorescence lifetime changes occurring in central serous chorioretinopathy exhibit explicit patterns which can be used to estimate perturbations of the outer retinal layers with a high degree of statistical significance. PMID:28099314

  13. FUNDUS AUTOFLUORESCENCE LIFETIMES AND CENTRAL SEROUS CHORIORETINOPATHY.

    PubMed

    Dysli, Chantal; Berger, Lieselotte; Wolf, Sebastian; Zinkernagel, Martin S

    2017-11-01

    To quantify retinal fluorescence lifetimes in patients with central serous chorioretinopathy (CSC) and to identify disease specific lifetime characteristics over the course of disease. Forty-seven participants were included in this study. Patients with central serous chorioretinopathy were imaged with fundus photography, fundus autofluorescence, optical coherence tomography, and fluorescence lifetime imaging ophthalmoscopy (FLIO) and compared with age-matched controls. Retinal autofluorescence was excited using a 473-nm blue laser light and emitted fluorescence light was detected in 2 distinct wavelengths channels (498-560 nm and 560-720 nm). Clinical features, mean retinal autofluorescence lifetimes, autofluorescence intensity, and corresponding optical coherence tomography (OCT) images were further analyzed. Thirty-five central serous chorioretinopathy patients with a mean visual acuity of 78 ETDRS letters (range, 50-90; mean Snellen equivalent: 20/32) and 12 age-matched controls were included. In the acute stage of central serous chorioretinopathy, retinal fluorescence lifetimes were shortened by 15% and 17% in the respective wavelength channels. Multiple linear regression analysis showed that fluorescence lifetimes were significantly influenced by the disease duration (P < 0.001) and accumulation of photoreceptor outer segments (P = 0.03) but independent of the presence or absence of subretinal fluid. Prolonged central macular autofluorescence lifetimes, particularly in eyes with retinal pigment epithelial atrophy, were associated with poor visual acuity. This study establishes that autofluorescence lifetime changes occurring in central serous chorioretinopathy exhibit explicit patterns which can be used to estimate perturbations of the outer retinal layers with a high degree of statistical significance.

  14. Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

    NASA Astrophysics Data System (ADS)

    Just Sørensen, Thomas; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

    2013-06-01

    Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

  15. In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles.

    PubMed

    Gu, Luo; Hall, David J; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J; Howell, Stephen B; Sailor, Michael J

    2013-01-01

    Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (<10 ns) emission signals from organic chromophores or tissue autofluorescence. Here using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by >50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

  16. In Vivo Time-gated Fluorescence Imaging with Biodegradable Luminescent Porous Silicon Nanoparticles

    PubMed Central

    Gu, Luo; Hall, David J.; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J.; Howell, Stephen B.; Sailor, Michael J.

    2014-01-01

    Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (< 10 ns) emission signals from organic chromophores or tissue autofluorescence.Here, using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by > 50-fold in vitro and by > 20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed. PMID:23933660

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  18. Calibration approach for fluorescence lifetime determination for applications using time-gated detection and finite pulse width excitation.

    PubMed

    Keller, Scott B; Dudley, Jonathan A; Binzel, Katherine; Jasensky, Joshua; de Pedro, Hector Michael; Frey, Eric W; Urayama, Paul

    2008-10-15

    Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is noniterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5-12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime, which can be determined with good reproducibility (typically <100 ps) even for data with poor signal-to-noise ratios ( approximately 20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.

  19. Effect of pharmacologically induced retinal degeneration on retinal autofluorescence lifetimes in mice.

    PubMed

    Dysli, Chantal; Dysli, Muriel; Zinkernagel, Martin S; Enzmann, Volker

    2016-12-01

    Fluorescence lifetime imaging ophthalmoscopy (FLIO) was used to investigate retinal autofluorescence lifetimes in mouse models of pharmacologically induced retinal degeneration over time. Sodium iodate (NaIO 3 , 35 mg/kg intravenously) was used to induce retinal pigment epithelium (RPE) degeneration with subsequent loss of photoreceptors (PR) whereas N-methyl-N-nitrosourea (MNU, 45 mg/kg intraperitoneally) was employed for degeneration of the photoreceptor cell layer alone. All mice were measured at day 3, 7, 14, and 28 after the respective injection of NaIO 3 , MNU or NaCl (control). Fluorescence lifetime imaging was performed using a fluorescence lifetime imaging ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). Fluorescence was excited at 473 nm and fluorescence lifetimes were measured in a short and a long spectral channel (498-560 nm and 560-720 nm). Corresponding optical coherence tomography (OCT) images were consecutively acquired and histology was performed at the end of the experiments. Segmentation of OCT images and histology verified the cell type-specific degeneration process over time. Retinal autofluorescence lifetimes increased from day 3 to day 28 in mice after NaIO 3 treatment. Finally, at day 28, fluorescence lifetimes were prolonged by 8% in the short and 61% in the long spectral channel compared to control animals (p = 0.21 and p = 0.004, respectively). In mice after MNU treatment, the mean retinal autofluorescence lifetimes were already decreased at day 3 and retinal lifetimes were finally shortened by 27% in the short and 51% in the long spectral channel at day 28 (p = 0.0028). In conclusion, degeneration of the RPE with subsequent photoreceptor degeneration by NaIO 3 lead to longer mean fluorescence lifetimes of the retina compared to control mice, whereas during specific degeneration of the photoreceptor layer induced by MNU shorter lifetimes were measured. Therefore, short retinal fluorescence lifetimes may originate

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

    NASA Astrophysics Data System (ADS)

    Yankelevich, Diego R.; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Elson, Daniel S.; Marcu, Laura

    2014-03-01

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

  1. High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy.

    PubMed

    Manfredini, Marco; Arginelli, Federica; Dunsby, Christopher; French, Paul; Talbot, Clifford; König, Karsten; Pellacani, Giovanni; Ponti, Giovanni; Seidenari, Stefania

    2013-02-01

    The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view. © 2012 John Wiley & Sons A/S.

  2. Fluorescence Intensity- and Lifetime-Based Glucose Sensing Using Glucose/Galactose-Binding Protein

    PubMed Central

    Pickup, John C.; Khan, Faaizah; Zhi, Zheng-Liang; Coulter, Jonathan; Birch, David J. S.

    2013-01-01

    We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested.Fluorescence technique is one of the major solutions for achieving the continuous and noninvasive glucose sensor for diabetes. In this article, a highly sensitive nanostructured sensor is developed to detect extremely small amounts of aqueous glucose by applying fluorescence energy transfer (FRET). A one-pot method is applied to produce the dextran-fluorescein isothiocyanate (FITC)-conjugating mesoporous silica nanoparticles (MSNs), which afterward interact with the tetramethylrhodamine isothiocyanate (TRITC)-labeled concanavalin A (Con A) to form the FRET nanoparticles (FITC-dextran-Con A-TRITC@MSNs). The nanostructured glucose sensor is then formed via the self-assembly of the FRET nanoparticles on a transparent, flexible, and biocompatible substrate, e.g., poly(dimethylsiloxane). Our results indicate the diameter of the MSNs is 60 ± 5 nm. The difference in the images before and after adding 20 μl of glucose (0.10 mmol/liter) on the FRET sensor can be detected in less than 2 min by the laser confocal laser scanning microscope. The correlation between the ratio of fluorescence intensity, I(donor)/I(acceptor), of the FRET sensor and the concentration of aqueous glucose in the range of 0.04–4 mmol/liter has been investigated; a linear relationship is found. Furthermore, the durability of the nanostructured FRET sensor is evaluated for 5 days. In addition, the recorded images can be converted to digital images by

  3. Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics

    NASA Astrophysics Data System (ADS)

    Robinson, Tom; Manning, Hugh B.; Dunsby, Christopher; Neil, Mark A. A.; Baldwin, Geoff S.; de Mello, Andrew J.; French, Paul M. W.

    2010-02-01

    We have developed a rapid microfluidic mixing device to image fast kinetics. To verify the performance of the device it was simulated using computational fluid dynamics (CFD) and the results were directly compared to experimental fluorescence lifetime imaging (FLIM) measurements. The theoretical and measured mixing times of the device were found to be in agreement over a range of flow rates. This mixing device is being developed with the aim of analysing fast enzyme kinetics in the sub-millisecond time domain, which cannot be achieved with conventional macro-stopped flow devices. Here we have studied the binding of a DNA repair enzyme, uracil DNA glycosylase (UDG), to a fluorescently labelled DNA substrate. Bulk phase fluorescence measurements have been used to measure changes on binding: it was found that the fluorescence lifetime increased along with an increase in the polarisation anisotropy and rotational correlation time. Analysis of the same reaction in the microfluidic mixer by CFD enabled us to predict the mixing time of the device to be 46 μs, more than 20 times faster than current stopped-flow techniques. We also demonstrate that it is possible to image UDG-DNA interactions within the micromixer using the signal changes observed from the multidimensional spectrofluorometer.

  4. From morphology to biochemical state - intravital multiphoton fluorescence lifetime imaging of inflamed human skin

    NASA Astrophysics Data System (ADS)

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

    2016-03-01

    The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

  5. Assessing the photoaging process at sun exposed and non-exposed skin using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Kurachi, Cristina

    2016-03-01

    Photoaging is the skin premature aging due to exposure to ultraviolet light, which damage the collagen, elastin and can induce alterations on the skin cells DNA, and, then, it may evolve to precancerous lesions, which are widely investigated by fluorescence spectroscopy and lifetime. The fluorescence spectra and fluorescence lifetime analysis has been presented as a technique of great potential for biological tissue characterization at optical diagnostics. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and may contribute to a non-invasive clinical investigation of injuries such as skin lesions. These lesions and the possible areas where they may develop can be interrogated using fluorescence lifetime spectroscopy taking into account the variability of skin phototypes and the changes related to melanin, collagen and elastin, endogenous fluorophores which have emissions that spectrally overlap to the NADH and FAD emission. The objective of this study is to assess the variation on fluorescence lifetimes of normal skin at sun exposed and non-exposed areas and associate this variation to the photoaging process.

  6. Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

    2015-05-01

    Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

  7. Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

    PubMed Central

    Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

    2015-01-01

    Abstract. Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs. PMID:25688541

  8. [The analysis of sinusoidal modulated method used for measuring fluorescence lifetime].

    PubMed

    Feng, Ying; Huang, Shi-hua

    2007-12-01

    This paper has built a system with a sinusoidal modulated LED as the excitation source. Such exciter was used upon the sample Eu2 L'3 x nH2O (L' = C4H4O4). Both the excitation light and the 5Do-7F2 emission of Eu3+ ion were measured. Fluorescence lifetime, which approximate to 0.680 ms, can then be obtained from the measured excitation and fluorescence waveforms by non-linear least square curve fitting based on the principle of phase-shift measurement of fluorescence lifetime. Data processing methods considering respectively the high order harmonics in the modulation and multi-exponential decay of the fluorescence were discussed. A method of utilizing Fourier series expandedness to amendatory the result was put forward. Accordingly, the applicability for phase-shift method was expanded as well as a more exact result was acquired.

  9. Fluorescence lifetime spectroscopy in multiple-scattering environments: an application to biotechnology

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert E.; Gratton, Enrico; Fantini, Sergio

    1999-07-01

    Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in the milk, which is an application vital to the tremendous economic burden of bovine subclinical mastitis detection. Furthermore, we may in principle use the spirit of this technique as a quantitative monitor of the binding of fluorescent drugs inside tissues. This is a first step towards lifetime spectroscopy in tissues.

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

    PubMed

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

    2014-04-07

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

  11. Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning

    PubMed Central

    Silva, Susana F.; Domingues, José Paulo

    2018-01-01

    Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed. PMID:29599938

  12. Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning.

    PubMed

    Silva, Susana F; Domingues, José Paulo; Morgado, António Miguel

    2018-01-01

    Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed.

  13. Frequency domain fluorescence lifetime imaging microscopy system for detecting inflammatory cells

    NASA Astrophysics Data System (ADS)

    Yahav, Gilad; Gershanov, Sivan; Salmon-Divon, Mali; Ben-Zvi, Haim; Mircus, Gabriel; Goldenberg-Cohen, Nitza; Fixler, Dror

    2018-02-01

    Characterizing different pathological states in the cellular level with a high throughput diagnostic tool is one of the main interests today. In previously works, we demonstrated how the frequency domain (FD) fluorescence lifetime imaging microscopy (FLIM) technique could be utilized to implement that in variety of examples. Among them was to classify between different chromosomal abnormalities in patients with b-cell chronic lymphocytic leukemia (B-CLL) and between metastatic cells and inflammation cells in the cerebral spinal fluid of patients with Medulloblastoma. This research describes the use of FD-FLIM system to differentiate between patients diagnosed without any disease (controls) that showed a normal median FLT (2.65+/-0.11ns) and patients diagnosed with inflammation (viruses and bacteria) that showed a prolong median FLT and a larger distribution (3.18+/-0.44ns in viruses and 3.28+/-0.45ns). The study group of this research included 43 samples divided into 4 groups: 9 samples diagnosed with different types of bacteria, 16 samples diagnosed with different types of viruses, 12 samples diagnosed with no any bacteria or virus and 5 samples diagnosed without any disease that served as controls. Furthermore, we studied a group of patients without detection of inflammation that were sick. We found that this group was divided into two groups; one group had the same median FLT as the controls, and the other group had the same median FLT as the inflammatory patients. As a result, we believe the FD-FLIM system can suggest a faster and more accurate diagnostic technique than the methods used today. The correlations of the FLT distribution pattern with the different groups are presented.

  14. Fluorescence Lifetime Imaging of Physiological Free Cu(II) Levels in Live Cells with a Cu(II)-Selective Carbonic Anhydrase-Based Biosensor

    PubMed Central

    McCranor, Bryan J.; Szmacinski, Henryk; Zeng, Hui Hui; Stoddard, A.K.; Hurst, Tamiika; Fierke, Carol A.; Lakowicz, J.R.

    2014-01-01

    Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable. We recently developed a variant of human apocarbonic anhydrase II for sensing metal ions that exhibits 25-fold better selectivity for Cu(II) over Zn(II) than the wild type protein, enabling us to accurately measure Cu(II) in the presence of ordinary cellular (picomolar) concentrations of free zinc. We inserted a fluorescent labeled Cu(II)-specific variant of human apocarbonic anhydrase into PC-12 cells and found that the levels are indeed extremely low (in the femtomolar range). We imaged the free Cu(II) levels in living cells by means of frequency-domain fluorescence lifetime microscopy. Implications of this finding are discussed. PMID:24671220

  15. Fluorescence Lifetime Study of Cyclodextrin Complexes of Substituted Naphthalenes.

    DTIC Science & Technology

    1987-08-15

    Spectroscopy iip 17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse If necessary and identify by block number) FIELD GROUP SUB-GROUP fluorescence lifetime...measurements cyclodextrins spectroscopic techniques 19. TRACT (Continue on revere if necsary and identify by block number

  16. Fluorescence lifetime-based contrast enhancement of indocyanine green-labeled tumors

    NASA Astrophysics Data System (ADS)

    Kumar, Anand T. N.; Carp, Stefan A.; Yang, Jing; Ross, Alana; Medarova, Zdravka; Ran, Chongzhao

    2017-04-01

    Although the development of tumor-targeted fluorescent probes is a major area of investigation, it will be several years before these probes are realized for clinical use. Here, we report an approach that employs indocyanine-green (ICG), a clinically approved, nontargeted dye, in conjunction with fluorescence lifetime (FLT) detection to provide high accuracy for tumor-tissue identification in mouse models of subcutaneous human breast and brain tmors. The improved performance relies on the distinct FLTs of ICG within tumors versus tissue autofluorescence and is further aided by the well-known enhanced permeability and retention of ICG in tumors and the clearance of ICG from normal tissue several hours after intravenous injection. We demonstrate that FLT detection can provide more than 98% sensitivity and specificity, and a 10-fold reduction in error rates compared to intensity-based detection. Our studies suggest the significant potential of FLT-contrast for accurate tumor-tissue identification using ICG and other targeted probes under development, both for intraoperative imaging and for ex-vivo margin assessment of surgical specimens.

  17. 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.

  18. Interactions between natural organic ligands and trace metals studied by fluorescence lifetime and fluorescence quenching

    NASA Astrophysics Data System (ADS)

    Nouhi, Ayoub; Hajjoul, Houssam; Redon, Roland; Gagné, Jean-Pierre; Mounier, Stéphane

    2017-04-01

    Improved insight on the interactions between natural organic ligands and trace metals is of paramount importance for better understanding transport and toxicity pathways of metal ions in the environment. Fluorescence spectroscopy allows introspecting ligands-metals interactions. Time-resolved laser fluorescence spectroscopy (TRLFS) measures fluorophore lifetime probing the local molecular environment. Excitation Emission Fluorescence Matrices (EEFMs) and their statistical treatment : parallel factor analysis (PARAFAC) using PROGMEEF Matlab homemade program, can give insight on the number or nature of organic fluorophores involved in the interactions. Quenching of fluorescence by metals can occur following two processes: dynamic and static quenching (Lakowicz, 2013). In the first case, quenching is caused by physical collisions among molecules and in the second case fluorophores can form nonfluorescent complexes with quenchers. It is possible to identify the different mechanisms because each type of quenching corresponds to a different mathematical model (Lakowicz, 2013; Valeur and Berberan-Santos, 2012). In TRLFS, the study of fluorescence decay's laws induced by nanosecond pulsed laser will allow to exactly qualify the type of interaction. The crucial point of the temporal deconvolution will be the evaluation of the best fitting between the different physical models and the decays measured. From the most suitable time decay model, it will be possible to deduce the quenching which modifies the fluorescence. The aim of this study was to characterize interactions between natural organic ligands and trace metals using fluorescence tools to evaluate the fluorescence lifetime of the fluorophore, the occurrence of quenching in presence of metal, discuss its mechanism and estimate conditional stability constants if a complex organic ligand-metal is formed. This study has been done in two steps. First, we have examined the interactions between salicylic acid and copper in

  19. Remote fluorescence lifetime inspection of hermeticity of packaged food containers

    NASA Astrophysics Data System (ADS)

    Mendoza, Edgar A.; Kempen, Cornelia; Sun, Sunjian; Esterkin, Yan

    2014-09-01

    This paper describes recent progress towards the development of a remote "frequency-domain" fluorescence lifetime (SeePhase™) monitor used for the real time hermetic seal leak inspection of packaged food containers. A multitude of food goods, meets, vegetables, and beverages are typically packaged within an inert environment to reduce the risk of bacteria growth and increase the storage life of the food product. The SeePhase™ system uses a multi-parameter oxygen, carbon dioxide, and moisture sensitive patch that is placed within the hermetic sealed food package. Upon the presence of gases oxygen, carbon dioxide, or moisture inside the hermetic sealed food package, the sensor patch produces a fluorescence lifetime signature characteristic of a hermetic seal leak damage of the package.

  20. Robust Bayesian Fluorescence Lifetime Estimation, Decay Model Selection and Instrument Response Determination for Low-Intensity FLIM Imaging

    PubMed Central

    Rowley, Mark I.; Coolen, Anthonius C. C.; Vojnovic, Borivoj; Barber, Paul R.

    2016-01-01

    We present novel Bayesian methods for the analysis of exponential decay data that exploit the evidence carried by every detected decay event and enables robust extension to advanced processing. Our algorithms are presented in the context of fluorescence lifetime imaging microscopy (FLIM) and particular attention has been paid to model the time-domain system (based on time-correlated single photon counting) with unprecedented accuracy. We present estimates of decay parameters for mono- and bi-exponential systems, offering up to a factor of two improvement in accuracy compared to previous popular techniques. Results of the analysis of synthetic and experimental data are presented, and areas where the superior precision of our techniques can be exploited in Förster Resonance Energy Transfer (FRET) experiments are described. Furthermore, we demonstrate two advanced processing methods: decay model selection to choose between differing models such as mono- and bi-exponential, and the simultaneous estimation of instrument and decay parameters. PMID:27355322

  1. Characterizing non-photochemical quenching in leaves through fluorescence lifetime snapshots

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sylak-Glassman, Emily J.; Zaks, Julia; Amarnath, Kapil

    2015-03-12

    A technique is described to measure the fluorescence decay profiles of intact leaves during adaptation to high light and subsequent relaxation to dark conditions. We illustrate how to ensure that photosystem II reaction centers are closed and compare data for wild type Arabidopsis thaliana with conventional pulse-amplitude modulated (PAM) fluorescence measurements. Unlike PAM measurements, the lifetime measurements are not sensitive to photobleaching or chloroplast shielding, and the form of the fluorescence decay provides additional information to test quantitative models of excitation dynamics in intact leaves.

  2. From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients' bedside

    NASA Astrophysics Data System (ADS)

    Mess, Christian; Zens, Katharina; Gorzelanny, Christian; Metze, Dieter; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.; Huck, Volker

    2017-02-01

    Application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of skin diseases. By means of multiphoton excitation, endogenous biomolecules like NADH, collagen or elastin show autofluorescence or second harmonic generation. Thus, these molecules provide information about the subcellular morphology, epidermal architecture and physiological condition of the skin. To gain a deeper understanding of the linkage between cellular structure and physiological processes, non-invasive multiphotonbased intravital tomography (MPT) and fluorescence lifetime imaging (FLIM) were combined within the scopes of inflammatory skin, chronic wounds and drug delivery in clinical application. The optical biopsies generated via MPT were morphologically analyzed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Independent morphometric algorithms reliably showed a perinuclear accumulation in lesional skin in contrast to an even distribution in healthy skin. Confirmatively, MPT-FLIM showed an obvious metabolic shift in lesions. Moreover, detection of the onset and progression of inflammatory processes could be achieved. The feasibility of primary in vivo tracking of applied therapeutic agents further broadened our scope: We examined the permeation and subsequent distribution of agents directly visualized in patientś skin in short-term repetitive measurements. Furthermore, we performed MPT-FLIM follow-up investigations in the long-term course of therapy. Therefore, clinical MPT-FLIM application offers new insights into the pathophysiology and the individual therapeutic course of skin diseases, facilitating a better understanding of the processes of inflammation and wound healing.

  3. Multiphoton, confocal, and lifetime microscopy for molecular imaging in cartilage

    NASA Astrophysics Data System (ADS)

    Wachsmann-Hogiu, Sebastian; Krakow, Deborah; Kirilova, Veneta T.; Cohn, Daniel H.; Bertolotto, Cristina; Acuna, Dora; Fang, Qiyin; Krivorov, Nikola; Farkas, Daniel L.

    2005-03-01

    It has recently been shown that mutations in Filamin A and B genes produce a large spectrum of skeletal disorders in developing fetuses. However, high-resolution optical microscopy in cartilage growth plate using fluorescent antibody assays, which should elucidate molecular aspects of these disorders, is extremely difficult due to the high level of autofluoresce in this tissue. We apply multiphoton, confocal, lifetime and spectral microscopy to (i) image and characterize autofluorophores in chondrocytes and subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins. Taking advantage of the different fluorescence spectra of the endogenous and exogenous markers, we can significantly reduce the autofluorescence background. Preliminary results of the FRET experiments suggest no interaction between Filamin A and B proteins. However, developing of new antibodies targeting the carboxy-terminal immunoglobulin-like domain may be necessary to confirm this result.

  4. From morphology to biochemical state – intravital multiphoton fluorescence lifetime imaging of inflamed human skin

    PubMed Central

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

    2016-01-01

    The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy. PMID:27004454

  5. Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Osseiran, Sam; Roider, Elisabeth M.; Wang, Hequn; Suita, Yusuke; Murphy, Michael; Fisher, David E.; Evans, Conor L.

    2017-12-01

    Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.

  6. Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy.

    PubMed

    Osseiran, Sam; Roider, Elisabeth M; Wang, Hequn; Suita, Yusuke; Murphy, Michael; Fisher, David E; Evans, Conor L

    2017-12-01

    Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  7. 3D printed miniaturized spectral system for tissue fluorescence lifetime measurements

    NASA Astrophysics Data System (ADS)

    Zou, Luwei; Mahmoud, Mohamad; Fahs, Mehdi; Liu, Rui; Lo, Joe F.

    2016-04-01

    Various types of collagens, e.g. type I and III, represent the main load-bearing components in biological tissues. Their composition changes during processes like wound healing and fibrosis. Collagens exhibit autofluorescence when excited by ultra-violet light, distinguishable by their unique fluorescent lifetimes across a range of emission wavelengths. Therefore, we designed a miniaturized spectral-lifetime detection system for collagens as a non-invasive probe for monitoring tissue in wound healing and scarring applications. A sine modulated LED illumination was applied to enable frequency domain (FD) fluorescence lifetime measurements under different wavelengths bands, separated via a series of longpass dichroics at 387nm, 409nm and 435nm. To achieve the minute scale of optomechanics, we employed a stereolithography based 3D printer with <50 μm resolution to create a custom designed optical mount in a hand-held form factor. We examined the characteristics of the 3D printed optical system with finite element modeling to simulate the effect of thermal (LED) and mechanical (handling) strain on the optical system. Using this device, the phase shift and demodulation of collagen types were measured, where the separate spectral bands enhanced the differentiation of their lifetimes.

  8. Fluorescence lifetime, dipole orientation and bilayer polymer films

    NASA Astrophysics Data System (ADS)

    Ho, Xuan Long; Chen, Po-Jui; Woon, Wei-Yen; White, Jonathon David

    2017-10-01

    Bilayer films consisting of the optically transparent polymers, polystyrene (PS) and poly(methyl methacrylate) (PMMA) were spin-cast on glass substrates. The upper 13.5 nm layer (PS) was lightly doped with Rhodamine-6 G (RH6G) or MEH-PPV. While the fluorescence of MEH-PPV was independent of PMMA thickness, the lifetime of RH6G increased 3-fold as the underlying PMMA thickness increased from 0 to 500 nm while the collected flux decreased suggesting a reorientation of the smaller molecule's dipole with respect to the air-polymer interface with PMMA thickness. This suggests lifetime may find application for nondestructive thickness measurements of transparent films with sub-micron lateral resolution and large range.

  9. Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

    NASA Astrophysics Data System (ADS)

    Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

    2017-09-01

    Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

  10. Measurement of the Fluorescence Lifetime of Chlorophyll a In Vivo

    PubMed Central

    Singhal, G. S.; Rabinowitch, E.

    1969-01-01

    New measurements have been made of fluorescence lifetime (τ) of chlorophyll a in the algae Chlorella pyrenoidosa, Porphyridium cruentum, Anacystis nidulans, and in spinach chloroplast. τ-values of 0.6 and 0.7 nsec were obtained with green plants. Anacystis and Porphyridium gave a τ of 0.5 nsec. The previously described two stage decay of fluorescence in vivo in these organisms could not be confirmed. This observation could have been caused by a second wave of light emission from the exciting hydrogen lamp (not detected in earlier work). The lifetimes found in this study (calculated, as before, by the method of convolution integrals) were close to those found by other observers for “low” excitation intensities; the value first reported from this laboratory (1.0-1.7 nsec) may have corresponded to “high” excitation intensity. PMID:5778187

  11. Emission lifetimes of a fluorescent dye under shock compression

    DOE PAGES

    Liu, Wei-long; Bassett, Will P.; Christensen, James M.; ...

    2015-10-15

    The emission lifetimes of rhodamine 6G (R6G), were measured under shock compression to 9.1 GPa, with the dual intent of better understanding molecular photophysics in extreme environments and assessing the usefulness of fluorescence lifetime microscopy to measure spatially-dependent pressure distributions in shocked microstructured media. R6G was studied as free dye dissolved in poly-methyl methacrylate (PMMA), or dye encapsulated in silica microparticles suspended in PMMA. Thin layers of these materials in impedance-matched geometries were subjected to planar single-stage shocks created by laser-driven flyer plates. A synchronized femtosecond laser excited the dye at selected times relative to flyer plate arrival and themore » emission lifetimes were measured with a streak camera. Lifetimes decreased when shocks arrived. The lifetime decrease was attributed to a shock-induced enhancement of R6G nonradiative relaxation. At least part of the relaxation involved shock-enhanced intersystem crossing. For free dye in PMMA, the lifetime decrease during the shock was shown to be a linear function of shock pressure from 0-9 GPa, with a slope of -0.22 ns·GPa -1. Furthermore, the linear relationship makes it simple to convert lifetimes into pressures. Lifetime measurements in shocked microenvironments may be better than emission intensity measurements, since lifetimes are sensitive to the surrounding environment, but insensitive to intensity variations associated with the motion and optical properties of a dynamically changing structure.« less

  12. [Assessment of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H].

    PubMed

    Cheng, Ying; Ren, Mingming; Niu, Yanyan; Qiao, Jianhua; Aneba, S; Chorvat, D; Chorvatova, A

    2009-12-01

    The primary function of cardiac mitochondria is the production of ATP to support heart contraction. Examination of the mitochondrial redox state is therefore crucially important to sensitively detect early signs of mitochondrial function in pathophysiological conditions, such as ischemia, diabetes and heart failure. We study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply. Here NAD(P)H is studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD(P) H fluorescence is recorded in cardiac cells following excitation with 375nm UV-light and detection by spectrally-resolved time-correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and fluorescence lifetimes. Modulation of NADH production and/or mitochondrial respiration is tested to study dynamic characteristics of NAD(P) H fluorescence decay. Our results show that at least a 3-exponential decay model, with 0.4-0.7ns, 1.2-1.9ns and 8.0-13. Ons lifetime pools is necessary to describe cardiomyocyte autofluorescence (AF) within 420-560nm spectral range. Increased mitochondrial NADH production by ketone bodies enhanced the fluorescence intensity, without significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol (DNP), an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF intensity, broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects are comparable to the study of NADH fluorescence decay in vitro. In the present contribution we demonstrated that spectrally-resolved fluorescence lifetime technique provides promising new

  13. Autofluorescence Lifetimes in Geographic Atrophy in Patients With Age-Related Macular Degeneration.

    PubMed

    Dysli, Chantal; Wolf, Sebastian; Zinkernagel, Martin S

    2016-05-01

    To investigate fluorescence lifetime characteristics in patients with geographic atrophy (GA) in eyes with age-related macular degeneration and to correlate the measurements with clinical data and optical coherence tomography (OCT) findings. Patients with GA were imaged with a fluorescence lifetime imaging ophthalmoscope. Retinal autofluorescence lifetimes were measured in a short and a long spectral channel (498-560 nm and 560-720 nm). Mean retinal fluorescence lifetimes were analyzed within GA and the surrounding retina, and data were correlated with best corrected visual acuity and OCT measurements. Fluorescence lifetime maps of 41 eyes of 41 patients (80 ± 7 years) with GA were analyzed. Mean lifetimes within areas of atrophy were prolonged by 624 ± 276 ps (+152%) in the short spectral channel and 418 ± 186 ps (+83%) in the long spectral channel compared to the surrounding tissue. Autofluorescence lifetime abnormalities in GA occurred with particular patterns, similar to those seen in fundus autofluorescence intensity images. Within the fovea short mean autofluorescence lifetimes were observed, presumably representing macular pigment. Short lifetimes were preserved even in the absence of foveal sparing but were decreased in patients with advanced retinal atrophy in OCT. Short lifetimes in the fovea correlated with better best corrected visual acuity in both spectral channels. This study established that autofluorescence lifetime changes in GA present with explicit patterns. We hypothesize that the short lifetimes seen within the atrophy may be used to estimate damage induced by atrophy and to monitor disease progression in the context of natural history or interventional therapeutic studies.

  14. Relationship between the Fluorescence Lifetime of Chlorophyll 'a' and Primary Productivity within the Mississippi River Plume and Adjacent Shelf Region

    NASA Technical Reports Server (NTRS)

    Hall, Callie; Miller, Richard L.; Fernandez, Salvador M.; McKee, Brent A.

    2000-01-01

    In situ measurements of chlorophyll fluorescence intensity have been widely used to estimate phytoplankton biomass. However, because the fluorescence quantum yield of chlorophyll a in vivo can be highly variable, measurements of chlorophyll fluorescence intensity cannot be directly correlated with phytoplankton biomass and do not provide information on the physiological state of the phytoplankton under study. Conversely, lifetime-based measurements of chlorophyll fluorescence provide a framework in which photosynthetic rates of phytoplankton can be analyzed according to phytoplankton physiology. Along with the measurement of primary production and ambient nutrient concentrations within the Mississippi River plume in the northern Gulf of Mexico, phytoplankton fluorescence lifetimes were measured using a Fluorescence Lifetime Phytoplankton Analyzer (developed under a NASA Small Business Innovative Research contract to Ciencia, Inc.). Variability of fluorescence lifetimes within the plume can be used as a background from which to interpret variations in the maximum quantum yield of photochemistry. The extent to which nutrient and effluent loading in this dynamic coastal area affect the photosynthetic performance of phytoplankton will be presented as a function of phytoplankton fluorescence lifetimes.

  15. Fluorescence intensity- and lifetime-based glucose sensing using glucose/galactose-binding protein.

    PubMed

    Pickup, John C; Khan, Faaizah; Zhi, Zheng-Liang; Coulter, Jonathan; Birch, David J S

    2013-01-01

    We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested. © 2013 Diabetes Technology Society.

  16. Radiative lifetimes in B I using ultraviolet and vacuum-ultraviolet laser-induced fluorescence

    NASA Technical Reports Server (NTRS)

    O'Brian, T. R.; Lawler, J. E.

    1992-01-01

    Radiative lifetimes of the eight lowest even parity levels in the doublet system of B I are measured using time-resolved laser-induced fluorescence in the UV and VUV on an atomic beam of boron. The accurate lifetimes provide a base for improved determination of absolute transition probabilities in B I. The techniques described are broadly applicable to measurement of lifetimes of levels with transitions in the visible, UV, and VUV in almost any element.

  17. 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

  18. In vivo detection of oral epithelial cancer using endogenous fluorescence lifetime imaging: a pilot human study (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Jo, Javier A.; Hwang, Dae Yon; Palma, Jorge; Cheng, Shuna; Cuenca, Rodrigo; Malik, Bilal; Jabbour, Joey; Cheng, Lisa; Wright, John; Maitland, Kristen

    2016-03-01

    Endogenous fluorescence lifetime imaging (FLIM) provides direct access to the concomitant functional and biochemical changes accompanying tissue transition from benign to precancerous and cancerous. Since FLIM can noninvasively measure different and complementary biomarkers of precancer and cancer, we hypothesize that it will aid in clinically detecting early oral epithelial cancer. Our group has recently demonstrated the detection of benign from premalignant and malignant lesions based on endogenous multispectral FLIM in the hamster cheek-pouch model. Encouraged by these positive preliminary results, we have developed a handheld endoscope capable of acquiring multispectral FLIM images in real time from the oral mucosa. This novel FLIM endoscope is being used for imaging clinically suspicious pre-malignant and malignant lesions from patients before undergoing tissue biopsy for histopathological diagnosis of oral epithelial cancer. Our preliminary results thus far are already suggesting the potential of endogenous FLIM for distinguishing a variety of benign lesions from advanced dysplasia and squamous cell carcinoma (SCC). To the best of out knowledge, this is the first in vivo human study aiming to demonstrate the ability to predict the true malignancy of clinically suspicious lesions using endogenous FLIM. If successful, the resulting clinical tool will allow noninvasive real-time detection of epithelial precancerous and cancerous lesions in the oral mucosa and could potentially be used to assist at every step involved on the clinical management of oral cancer patients, from early screening and diagnosis, to treatment and monitoring of recurrence.

  19. Fluorescence lifetime imaging of optically levitated aerosol: a technique to quantitatively map the viscosity of suspended aerosol particles.

    PubMed

    Fitzgerald, C; Hosny, N A; Tong, H; Seville, P C; Gallimore, P J; Davidson, N M; Athanasiadis, A; Botchway, S W; Ward, A D; Kalberer, M; Kuimova, M K; Pope, F D

    2016-08-21

    We describe a technique to measure the viscosity of stably levitated single micron-sized aerosol particles. Particle levitation allows the aerosol phase to be probed in the absence of potentially artefact-causing surfaces. To achieve this feat, we combined two laser based techniques: optical trapping for aerosol particle levitation, using a counter-propagating laser beam configuration, and fluorescent lifetime imaging microscopy (FLIM) of molecular rotors for the measurement of viscosity within the particle. Unlike other techniques used to measure aerosol particle viscosity, this allows for the non-destructive probing of viscosity of aerosol particles without interference from surfaces. The well-described viscosity of sucrose aerosol, under a range of relative humidity conditions, is used to validate the technique. Furthermore we investigate a pharmaceutically-relevant mixture of sodium chloride and salbutamol sulphate under humidities representative of in vivo drug inhalation. Finally, we provide a methodology for incorporating molecular rotors into already levitated particles, thereby making the FLIM/optical trapping technique applicable to real world aerosol systems, such as atmospheric aerosols and those generated by pharmaceutical inhalers.

  20. Towards pH-sensitive imaging of small animals with photon-counting difference diffuse fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Li, Jiao; Wang, Xin; Yi, Xi; Zhang, Limin; Zhou, Zhongxing; Zhao, Huijuan; Gao, Feng

    2012-09-01

    The importance of cellular pH has been shown clearly in the study of cell activity, pathological feature, and drug metabolism. Monitoring pH changes of living cells and imaging the regions with abnormal pH-values, in vivo, could provide invaluable physiological and pathological information for the research of the cell biology, pharmacokinetics, diagnostics, and therapeutics of certain diseases such as cancer. Naturally, pH-sensitive fluorescence imaging of bulk tissues has been attracting great attentions from the realm of near infrared diffuse fluorescence tomography (DFT). Herein, the feasibility of quantifying pH-induced fluorescence changes in turbid medium is investigated using a continuous-wave difference-DFT technique that is based on the specifically designed computed tomography-analogous photon counting system and the Born normalized difference image reconstruction scheme. We have validated the methodology using two-dimensional imaging experiments on a small-animal-sized phantom, embedding an inclusion with varying pH-values. The results show that the proposed approach can accurately localize the target with a quantitative resolution to pH-sensitive variation of the fluorescent yield, and might provide a promising alternative method of pH-sensitive fluorescence imaging in addition to the fluorescence-lifetime imaging.

  1. Characterizing Fibrosis in Mouse Kidney using Label Free Fluorescence Lifetime and Second Harmonic Generation Imaging Microscopy in Unilateral Ureteral Obstruction Model

    PubMed Central

    Ranjit, Suman; Dobrinskikh, Evgenia; Montford, John; Dvornikov, Alexander; Lehman, Allison; Orlicky, David J.; Nemenoff, Raphael; Gratton, Enrico; Levi, Moshe; Furgeson, Seth

    2017-01-01

    All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. The work described here shows the development of a fast and operator-independent method to measure fibrosis. To study renal fibrosis, the unilateral ureteral obstruction (UUO) model was chosen. Mice develop a time-dependent increase in obstructed kidneys; contralateral kidneys are used as controls. After UUO, kidneys were analyzed at three time points: 7 days, 14 days, and 21 days. Fibrosis was investigated using FLIM (Fluorescence Lifetime Imaging) and SHG (Second Harmonic Generation) in the deep tissue imaging microscope called DIVER (Deep Imaging via Enhanced photon Recovery). This microscope was developed for deep tissue and SHG and THG (Third Harmonic Generation) imaging and has extraordinary sensitivity towards harmonic generation. SHG data suggests the presence of more fibrillar collagen in the diseased kidneys. The combinations of short wavelength FLIM and SHG analysis results in a robust analysis procedure independent of observer interpretation and let us create a criterion to quantify the extent of fibrosis directly from the image. The progression of fibrosis in UUO model has been studied using this new FLIM-SHG technique and it shows remarkable improvement in quantification of fibrosis compared to standard histological techniques. PMID:27555119

  2. The Use of Chlorophyll Fluorescence Lifetime to Assess Phytoplankton Physiology within a River-Dominated Environment

    NASA Technical Reports Server (NTRS)

    Hall, Callie M.; Miller, Richard L.; Redalje, Donald G.; Fernandez, Salvador M.

    2002-01-01

    Chlorophyll a fluorescence lifetime was measured for phytoplankton populations inhabiting the three physical zones surrounding the Mississippi River's terminus in the Gulf of Mexico. Observations of river discharge volume, nitrate + nitrite, silicate, phosphate, PAR (Photosynthetically Active Radiation) diffuse attenuation within the water column, salinity, temperature, SPM, and chl a concentration were used to characterize the distribution of chl fluorescence lifetime within a given region within restricted periods of time. 33 stations extending from the Mississippi River plume to the shelf break of the Louisiana coast were surveyed for analysis of chlorophyll fluorescence lifetime during two cruises conducted March 31 - April 6, 2000, and October 24 - November 1, 2000. At each station, two to three depths were chosen for fluorescence lifetime measurement to represent the vertical characteristics of the water column. Where possible, samples were taken from just below the surface and from just above and below the pycnocline. All samples collected were within the 1% light level of the water column (the euphotic zone). Upon collection, samples were transferred to amber Nalgene bottles and left in the dark for at least 15 minutes to reduce the effects of non-photochemical quenching and to insure that photosynthetic reaction centers were open. Before measurements within the phase fluorometer were begun, the instrument was allowed to warm up for no less than one hour.

  3. Lifetimes of bacteriochlorophyll fluorescence in Rhodopseudomonas viridis and Heliobacterium chlorum at low temperatures

    NASA Technical Reports Server (NTRS)

    Kleinherenbrink, F. A.; Cheng, P.; Amesz, J.; Blankenship, R. E.

    1993-01-01

    Fluorescence lifetimes of isolated membranes of Rhodopseudomonas viridis were measured in the temperature range of 77 K to 25 K. At room temperature, the main component of the fluorescence decay of bacteriochlorophyll (BChl) b had a time constant of 50 ps. In contrast to other purple bacteria, the emission at low temperature was spectrally homogeneous and showed essentially single lifetimes of 140 ps at 77 K and 180 ps at 25 K, with the primary electron donor in the oxidized state. Taking into account the relative fluorescence yields with open and closed reaction centers, we arrive at numbers of 125 ps and 215 ps, respectively, for open reaction centers. These numbers are significantly smaller than expected on the basis of measurements of the efficiency of charge separation, perhaps suggesting that the excitation decay in the absence of reaction centers is considerably faster at low temperature than at room temperature. At least four different spectral components with different lifetimes were observed at 25 K in the emission of Heliobacterium chlorum, a short-wavelength component of about 30 ps and three longer-wavelength components of about 100 ps, 300 ps, and 900 ps. This indicates a strong heterogeneity in the emitting pigment, BChl g-808. The component with the shortest lifetime does not appear to be affected by the redox state of the reaction center and might reflect energy transfer to BChl g species which are connected to the reaction center.

  4. Fluorescence lifetime dynamics of eGFP in protein aggregates with expanded polyQ

    NASA Astrophysics Data System (ADS)

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2009-02-01

    Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues (97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the process of polyQ protein aggregation per se.

  5. Water-soluble, neutral 3,5-diformyl-BODIPY with extended fluorescence lifetime in a self-healable chitosan hydrogel.

    PubMed

    Belali, Simin; Emandi, Ganapathi; Cafolla, Atillio A; O'Connell, Barry; Haffner, Benjamin; Möbius, Matthias E; Karimi, Alireza; Senge, Mathias O

    2017-11-08

    3,5-Diformyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (3,5-diformyl-BODIPY) can be used as an efficient biofunctional cross-linker to generate a new class of chitosan-based hydrogels with fluorescence resonance energy transfer (FRET) dynamics and good solubility in water. The hydrogel was fully characterized by FT-IR, UV-vis, fluorescence, FE-SEM, AFM, rheology and picosecond time-resolved spectroscopic techniques. The self-healing ability was demonstrated by rheological recovery and macroscopic and microscopic observations. The fluorescence lifetime was found to increase in aqueous solution of the BODIPY-chitosan hydrogel compared to the 3,5-diformyl-BODIPY monomer. Calculations based on experimental results such as red-shift and decreased intensity of the emission spectrum of highly dye-concentrated hydrogel in comparison to dilute hydrogels, together with changes in the fluorescence lifetime of the hydrogel at different concentration of dyes, suggest that the BDP-CS hydrogels fluorescence dynamics obey the Förster resonance energy transfer (FRET). Improvements in mechanical and photochemical properties and the acceptable values of BODIPY fluorescence lifetime in the hydrogel matrix indicate the utility of the newly synthesized hydrogels for biomedical applications.

  6. Development of a fluorimeter using laser-induced single-shot fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Eisum, Niels H.; Lynggaard-Jensen, Anders

    1990-08-01

    The developed laboratory prototype fluorimeter is the first step to a new in-situ instrument, and is based on a pulsed nitrogen laser (pumping a color dye laser and the laserbeam passing through a frequency doubler) with a pulse width less than 1 nsec. With such a short excitation pulse it is possible to measure the exponential decay of the fluorescence from the aromatic compounds and thus determine the fluorescence lifetime-curves, which are typically in the region of 5-40 nsec. The emitted fluorescence is collected simultaneously in 35 channels in the wavelength region 250-600 nm. If the fluorescence falls within the transmission areas of the interference filters in each channel the light will be collected by a plastic light guide (doped PMMA) in the actual channel and transmitted to the channels photo multiplier tube (PMT). (The use of the plastic light guide improves the sensitivity). The signal from the PMT is passed on to a 200 MHz 8-bit flash AID-converter connected to a local memory. From this local memory the digital lifetime curves from each channel are transmitted to a computer for presentation of the 3-dimensional spectrum. This spectrum has been obtained with a single laser shot.

  7. Electrogenerated Chemiluminescence and Fluorescence Lifetime Spatial Heterogeneity of Poly (2-methoxy-5-(2-ethylhexyloxy)-1, 4-phenylenevinylene) in Presence of [6,6]-phenyl-C61-butyric Acid Methyl Ester

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Geng, Hongwei; Pan, Shanlin; Hu, Dehong

    In this paper, we studied the luminescence property and fluorescence lifetime mapping of MEH-PPV/PCBM system by using electrogenerated chemiluminescence (ECL) and time-correlated single photo counting (TC-SPC) technologies. The ECL results showed that the oxidation peak of MEH-PPV near 0.7 V (vs. SCE) and ECL response of films shifted positively towards 1.2 V when in the presence of PCBM. At the same time, the oxidation peak current density of MEH-PPV increases while the ECL response decreased with the loading of PCBM in the composite films. The fluorescence lifetime images clearly show that the lifetime fluctuation is effected by different substrates andmore » MEH-PPV/PCBM ratios. Meanwhile, the lifetime of MEH-PPV decreases with the increasing of film thickness. The lifetimes of MEH-PPV films on TiO2 substrate are lower than them of films on cover slips.« less

  8. Assessment of post-implantation integration of engineered tissues using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Elahi, Sakib F.; Lee, Seung Y.; Lloyd, William R.; Chen, Leng-Chun; Kuo, Shiuhyang; Zhou, Ying; Kim, Hyungjin M.; Kennedy, Robert; Marcelo, Cynthia; Feinberg, Stephen E.; Mycek, Mary-Ann

    2018-02-01

    Clinical translation of engineered tissue constructs requires noninvasive methods to assess construct health and viability after implantation in patients. However, current practices to monitor post-implantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). As label-free fluorescence lifetime sensing can noninvasively characterize pre-implantation construct viability, we employed a handheld fluorescence lifetime spectroscopy probe to quantitatively and noninvasively assess tissue constructs that were implanted in a murine model. We designed the system to be suitable for intravital measurements: portability, localization with precise maneuverability, and rapid data acquisition. Our model tissue constructs were manufactured from primary human cells to simulate patient variability and were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess pre-implantation construct health. In vivo optical sensing assessed tissue integration of constructs at one-week and three-weeks post-implantation. At one-week post-implantation, optical parameters correlated with in vitro pre-implantation secretion levels of all three cytokines (p < 0.05). This relationship was no longer seen at three-weeks post-implantation, suggesting comparable tissue integration independent of preimplantation health. Histology confirmed re-epithelialization of these constructs independent of pre-implantation health state, supporting the lack of a correlation. These results suggest that clinical optical diagnostic tools based on label-free fluorescence lifetime sensing of endogenous tissue fluorophores could noninvasively monitor post-implantation integration of engineered tissues.

  9. 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.

  10. A CMOS Time-Resolved Fluorescence Lifetime Analysis Micro-System

    PubMed Central

    Rae, Bruce R.; Muir, Keith R.; Gong, Zheng; McKendry, Jonathan; Girkin, John M.; Gu, Erdan; Renshaw, David; Dawson, Martin D.; Henderson, Robert K.

    2009-01-01

    We describe a CMOS-based micro-system for time-resolved fluorescence lifetime analysis. It comprises a 16 × 4 array of single-photon avalanche diodes (SPADs) fabricated in 0.35 μm high-voltage CMOS technology with in-pixel time-gated photon counting circuitry and a second device incorporating an 8 × 8 AlInGaN blue micro-pixellated light-emitting diode (micro-LED) array bump-bonded to an equivalent array of LED drivers realized in a standard low-voltage 0.35 μm CMOS technology, capable of producing excitation pulses with a width of 777 ps (FWHM). This system replaces instrumentation based on lasers, photomultiplier tubes, bulk optics and discrete electronics with a PC-based micro-system. Demonstrator lifetime measurements of colloidal quantum dot and Rhodamine samples are presented. PMID:22291564

  11. Hyperspectral small animal fluorescence imaging: spectral selection imaging

    NASA Astrophysics Data System (ADS)

    Leavesley, Silas; Jiang, Yanan; Patsekin, Valery; Hall, Heidi; Vizard, Douglas; Robinson, J. Paul

    2008-02-01

    Molecular imaging is a rapidly growing area of research, fueled by needs in pharmaceutical drug-development for methods for high-throughput screening, pre-clinical and clinical screening for visualizing tumor growth and drug targeting, and a growing number of applications in the molecular biology fields. Small animal fluorescence imaging employs fluorescent probes to target molecular events in vivo, with a large number of molecular targeting probes readily available. The ease at which new targeting compounds can be developed, the short acquisition times, and the low cost (compared to microCT, MRI, or PET) makes fluorescence imaging attractive. However, small animal fluorescence imaging suffers from high optical scattering, absorption, and autofluorescence. Much of these problems can be overcome through multispectral imaging techniques, which collect images at different fluorescence emission wavelengths, followed by analysis, classification, and spectral deconvolution methods to isolate signals from fluorescence emission. We present an alternative to the current method, using hyperspectral excitation scanning (spectral selection imaging), a technique that allows excitation at any wavelength in the visible and near-infrared wavelength range. In many cases, excitation imaging may be more effective at identifying specific fluorescence signals because of the higher complexity of the fluorophore excitation spectrum. Because the excitation is filtered and not the emission, the resolution limit and image shift imposed by acousto-optic tunable filters have no effect on imager performance. We will discuss design of the imager, optimizing the imager for use in small animal fluorescence imaging, and application of spectral analysis and classification methods for identifying specific fluorescence signals.

  12. Fluorescence lifetime measurements of native and glycated human serum albumin and bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Joshi, Narahari V.; Joshi, Virgina O. d.; Contreras, Silvia; Gil, Herminia; Medina, Honorio; Siemiarczuk, Aleksander

    1999-05-01

    Nonenzymatic glycation, also known as Maillard reaction, plays an important role in the secondary complications of the diabetic pathology and aging, therefore, human serum albumin (HSA) and bovine serum albumin (BSA) were glycated by a conventional method in our laboratory using glucose as the glycating agent. Fluorescence lifetime measurements were carried out with a laser strobe fluorometer equipped with a nitrogen/dye laser and a frequency doubler as a pulsed excitation source. The samples were excited at 295 nm and the emission spectra were recorded at 345 nm. The obtained decay curves were tried for double and triple exponential functions. It has been found that the shorter lifetime increases for glycated proteins as compared with that of the native ones. For example, in the case of glycated BSA the lifetime increased from 1.36 ns to 2.30 ns. Similarly, for HSA, the lifetime increases from 1.58 ns to 2.26 ns. Meanwhile, the longer lifetime changed very slightly for both proteins (from 6.52 ns to 6.72 ns). The increase in the lifetime can be associated with the environmental effect; originated from the attachment of glucose to some lysine residues. A good example is Trp 214 which is in the cage of Lys 225, Lys 212, Lys 233, Lys 205, Lys 500, Lys 199 and Lys 195. If fluorescence lifetime technique is calibrated and properly used it could be employed for assessing glycation of proteins.

  13. Instantaneous imaging of ozone in a gliding arc discharge using photofragmentation laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Larsson, Kajsa; Hot, Dina; Gao, Jinlong; Kong, Chengdong; Li, Zhongshan; Aldén, Marcus; Bood, Joakim; Ehn, Andreas

    2018-04-01

    Ozone vapor, O3, is here visualized in a gliding arc discharge using photofragmentation laser-induced fluorescence. Ozone is imaged by first photodissociating the O3 molecule into an O radical and a vibrationally hot O2 fragment by a pump photon. Thereafter, the vibrationally excited O2 molecule absorbs a second (probe) photon that further transits the O2-molecule to an excited electronic state, and hence, fluorescence from the deexcitation process in the molecule can be detected. Both the photodissociation and excitation processes are achieved within one 248 nm KrF excimer laser pulse that is formed into a laser sheet and the fluorescence is imaged using an intensified CCD camera. The laser-induced signal in the vicinity of the plasma column formed by the gliding arc is confirmed to stem from O3 rather than plasma produced vibrationally hot O2. While both these products can be produced in plasmas a second laser pulse at 266 nm was utilized to separate the pump- from the probe-processes. Such arrangement allowed lifetime studies of vibrationally hot O2, which under these conditions were several orders of magnitude shorter than the lifetime of plasma-produced ozone.

  14. Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

    PubMed

    Rich, Ryan M; Stankowska, Dorota L; Maliwal, Badri P; Sørensen, Thomas Just; Laursen, Bo W; Krishnamoorthy, Raghu R; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal

    2013-02-01

    Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.

  15. Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

    PubMed Central

    Rich, Ryan M.; Stankowska, Dorota L.; Maliwal, Badri P.; Sørensen, Thomas Just; Laursen, Bo W.; Krishnamoorthy, Raghu R.; Gryczynski, Zygmunt; Borejdo, Julian

    2013-01-01

    Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background. PMID:23254457

  16. Probing cellular uptake and tracking of differently shaped gelatin-coated gold nanoparticles inside of ovarian cancer cells by two-photon excited photoluminescence analyzed by fluorescence lifetime imaging (FLIM).

    PubMed

    Suarasan, Sorina; Licarete, Emilia; Astilean, Simion; Craciun, Ana-Maria

    2018-06-01

    Nowadays, the non-linear optical effect of two-photon excited (TPE) fluorescence has recently grown in interest in recent years over other optical imaging method, due to improved 3D spatial resolution, deep penetrability and less photodamage of living organism owing to the excitation in near-infrared region (NIR). In parallel, gold nanoparticles (AuNPs) have gain considerable attention for NIR TPE bio-imaging applications due to their appealing ability to generate strong intrinsic photoluminescence (PL). Here, we demonstrate the capability of differently shaped gelatin-coated AuNPs to perform as reliable label-free contrast agents for the non-invasive NIR imaging of NIH:OVCAR-3 ovary cancer cells via TPE Fluorescence Lifetime Imaging Microscopy (FLIM). Examination of the spectroscopic profile of the intrinsic signals exhibited by AuNPs inside cells confirm the plasmonic nature of the emitted PL, while the evaluation of time-dependent profile of the TPE PL signal under continuous irradiation indicates the photo-stability of the signal revealing simultaneously a photo-blinking behavior. Finally, we assess the dependence of the TPE PL signal on laser excitation power and wavelength in view of contributing to a better understanding of plasmonic TPE PL in biological media towards the improvement of TPE FLIM imaging applications based on AuNPs. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Emerging biomedical applications of time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lakowicz, Joseph R.; Szmacinski, Henryk; Koen, Peter A.

    1994-07-01

    Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics, and chemical physics. Advances in laser technology, the development of long-wavelength probes, and the use of lifetime-based methods are resulting in the rapid migration of time-resolved fluorescence to the clinical chemistry lab, to the patient's bedside, to flow cytometers, to the doctor's office, and even to home health care. Additionally, time-resolved imaging is now a reality in fluorescence microscopy, and will provide chemical imaging of a variety of intracellular analytes and/or cellular phenomena. In this overview paper we attempt to describe some of the opportunities available using chemical sensing based on fluorescence lifetimes, and to predict those applications of lifetime-based sensing which are most likely in the near future.

  18. The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging.

    PubMed

    Celli, A; Sanchez, S; Behne, M; Hazlett, T; Gratton, E; Mauro, T

    2010-03-03

    Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca(2+) gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca(2+), cease proliferation, and differentiate passively in response to changes in extracellular Ca(2+). The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca(2+) gradient, and it should be broadly applicable to the study of other tissues with ionic gradients. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. In Vivo Mitochondrial Oxygen Tension Measured by a Delayed Fluorescence Lifetime Technique

    PubMed Central

    Mik, Egbert G.; Johannes, Tanja; Zuurbier, Coert J.; Heinen, Andre; Houben-Weerts, Judith H. P. M.; Balestra, Gianmarco M.; Stap, Jan; Beek, Johan F.; Ince, Can

    2008-01-01

    Mitochondrial oxygen tension (mitoPO2) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO2 in vivo exists. Here we report in vivo measurement of mitoPO2 and the recovery of mitoPO2 histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO2 in rat liver in vivo. The results demonstrate mitoPO2 values of ∼30–40 mmHg. mitoPO2 was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO2 distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications. PMID:18641065

  20. Two-photon luminescence lifetime imaging microscopy (LIM) to follow up cell metabolism and oxygen consumption during theranostic applications

    NASA Astrophysics Data System (ADS)

    Rück, A.; Breymayer, J.; Lilge, L.; Mandel, A.; Schäfer, P.; von Einem, B.; von Arnim, C.; Kalinina, S.

    2018-02-01

    A common property during tumor development is altered energy metabolism, which could lead to a switch from oxidative phosphorylation and glycolysis. The impact of this switch for theranostic applications could be significant. Interestingly altered metabolism could be correlated with a change in the fluorescence lifetimes of both NAD(P)H and FAD. However, as observed in a variety of investigations, the situation is complex and the result is influenced by parameters like oxidative stress, pH or viscosity. Besides metabolism, oxygen levels and consumption has to be taken into account in order to understand treatment responses. For this, correlated imaging of phosphorescence and fluorescence lifetime parameters has been investigated by us and used to observe metabolic markers simultaneously with oxygen concentrations. The technique is based on time correlated single photon counting to detect the fluorescence lifetime of NAD(P)H and FAD by FLIM and the phosphorescence lifetime of newly developed phosphors and photosensitizers by PLIM. For this, the photosensitizer TLD1433 from Theralase, which is based on a ruthenium (II) coordination complex, was used. TLD1433 which acts as a redox indicator was mainly found in cytoplasmatic organelles. The most important observation was that TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during photodynamic therapy. Oxygen consumption was accompanied by a change in cell metabolism, observed by simultaneous FLIM/PLIM. The combination of autofluorescence-FLIM and phosphor-PLIM in luminescence lifetime microscopy provides new insights in light induced reactions.

  1. Investigation of Fluorescence Lifetime Quenching of Ru(bpy)[subscript 3][superscript 2+] by Oxygen Using a Pulsed Light-Emitting Diode

    ERIC Educational Resources Information Center

    Rusak, David A.; James, William H., III; Ferzola, Maria J.; Stefanski, Michael J.

    2006-01-01

    An experiment related to the measurement of fluorescence lifetime for an undergraduate instrumental analysis or physical chemistry laboratory that highlights relative rates of electronic transitions in molecules and introduces students to data collection of a pulsed signal is illustrated. The experiment of the long fluorescence lifetime of…

  2. Using non-empirically tuned range-separated functionals with simulated emission bands to model fluorescence lifetimes.

    PubMed

    Wong, Z C; Fan, W Y; Chwee, T S; Sullivan, Michael B

    2017-08-09

    Fluorescence lifetimes were evaluated using TD-DFT under different approximations for the emitting molecule and various exchange-correlation functionals, such as B3LYP, BMK, CAM-B3LYP, LC-BLYP, M06, M06-2X, M11, PBE0, ωB97, ωB97X, LC-BLYP*, and ωB97X* where the range-separation parameters in the last two functionals were tuned in a non-empirical fashion. Changes in the optimised molecular geometries between the ground and electronically excited states were found to affect the quality of the calculated lifetimes significantly, while the inclusion of vibronic features led to further improvements over the assumption of a vertical electronic transition. The LC-BLYP* functional was found to return the most accurate fluorescence lifetimes with unsigned errors that are mostly within 1.5 ns of experimental values.

  3. Potential of Fluorescence Imaging Techniques To Monitor Mutagenic PAH Uptake by Microalga

    PubMed Central

    2015-01-01

    Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is one of the major environmental pollutants that causes mutagenesis and cancer. BaP has been shown to accumulate in phytoplankton and zooplankton. We have studied the localization and aggregation of BaP in Chlorella sp., a microalga that is one of the primary producers in the food chain, using fluorescence confocal microscopy and fluorescence lifetime imaging microscopy with the phasor approach to characterize the location and the aggregation of BaP in the cell. Our results show that BaP accumulates in the lipid bodies of Chlorella sp. and that there is Förster resonance energy transfer between BaP and photosystems of Chlorella sp., indicating the close proximity of the two molecular systems. The lifetime of BaP fluorescence was measured to be 14 ns in N,N-dimethylformamide, an average of 7 ns in Bold’s basal medium, and 8 ns in Chlorella cells. Number and brightness analysis suggests that BaP does not aggregate inside Chlorella sp. (average brightness = 5.330), while it aggregates in the supernatant. In Chlorella grown in sediments spiked with BaP, in 12 h the BaP uptake could be visualized using fluorescence microscopy. PMID:25020149

  4. Time-resolved laser fluorescence spectroscopy of organic ligands by europium: Fluorescence quenching and lifetime properties

    NASA Astrophysics Data System (ADS)

    Nouhi, A.; Hajjoul, H.; Redon, R.; Gagné, J. P.; Mounier, S.

    2018-03-01

    Time-resolved Laser Fluorescence Spectroscopy (TRLFS) has proved its usefulness in the fields of biophysics, life science and geochemistry to characterize the fluorescence probe molecule with its chemical environment. The purpose of this study is to demonstrate the applicability of this powerful technique combined with Steady-State (S-S) measurements. A multi-mode factor analysis, in particular CP/PARAFAC, was used to analyze the interaction between Europium (Eu) and Humic substances (HSs) extracted from Saint Lawrence Estuary in Canada. The Saint Lawrence system is a semi-enclosed water stream with connections to the Atlantic Ocean and is an excellent natural laboratory. CP/PARAFAC applied to fluorescence S-S data allows introspecting ligands-metal interactions and the one-site 1:1 modeling gives information about the stability constants. From the spectral signatures and decay lifetimes data given by TRLFS, one can deduce the fluorescence quenching which modifies the fluorescence and discuss its mechanisms. Results indicated a relatively strong binding ability between europium and humic substances samples (Log K value varies from 3.38 to 5.08 at pH 7.00). Using the Stern-Volmer plot, it has been concluded that static and dynamic quenching takes places in the case of salicylic acid and europium interaction while for HSs interaction only a static quenching is observed.

  5. Amine-Reactive Fluorene Probes: Synthesis, Optical Characterization, Bioconjugation, and Two-Photon Fluorescence Imaging

    PubMed Central

    2008-01-01

    With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially “lighting up” after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated. PMID:19090700

  6. Metal plasmon-coupled fluorescence imaging and label free coenzyme detection in cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Jian, E-mail: jian@cfs.bioment.umaryland.edu; Fu, Yi; Li, Ge

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer Metal nanoparticle for fluorescence cell imaging. Black-Right-Pointing-Pointer Non-invasive emission detection of coenzyme in cell on time-resolved confocal microscope. Black-Right-Pointing-Pointer Near-field interaction of flavin adenine dinucleotide with silver substrate. Black-Right-Pointing-Pointer Isolation of emissions by coenzymes from cellular autofluorescence on fluorescence cell imaging. -- Abstract: Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent,more » but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.« less

  7. Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

    PubMed

    Andrews, Natalie; Ramel, Marie-Christine; Kumar, Sunil; Alexandrov, Yuriy; Kelly, Douglas J; Warren, Sean C; Kerry, Louise; Lockwood, Nicola; Frolov, Antonina; Frankel, Paul; Bugeon, Laurence; McGinty, James; Dallman, Margaret J; French, Paul M W

    2016-04-01

    Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region. © 2016 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

    PubMed

    Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

    2015-12-15

    Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. New approaches to the analysis of complex samples using fluorescence lifetime techniques and organized media

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hertz, P.R.

    Fluorescence spectroscopy is a highly sensitive and selective tool for the analysis of complex systems. In order to investigate the efficacy of several steady state and dynamic techniques for the analysis of complex systems, this work focuses on two types of complex, multicomponent samples: petrolatums and coal liquids. It is shown in these studies dynamic, fluorescence lifetime-based measurements provide enhanced discrimination between complex petrolatum samples. Additionally, improved quantitative analysis of multicomponent systems is demonstrated via incorporation of organized media in coal liquid samples. This research provides the first systematic studies of (1) multifrequency phase-resolved fluorescence spectroscopy for dynamic fluorescence spectralmore » fingerprinting of complex samples, and (2) the incorporation of bile salt micellar media to improve accuracy and sensitivity for characterization of complex systems. In the petroleum studies, phase-resolved fluorescence spectroscopy is used to combine spectral and lifetime information through the measurement of phase-resolved fluorescence intensity. The intensity is collected as a function of excitation and emission wavelengths, angular modulation frequency, and detector phase angle. This multidimensional information enhances the ability to distinguish between complex samples with similar spectral characteristics. Examination of the eigenvalues and eigenvectors from factor analysis of phase-resolved and steady state excitation-emission matrices, using chemometric methods of data analysis, confirms that phase-resolved fluorescence techniques offer improved discrimination between complex samples as compared with conventional steady state methods.« less

  10. Multispectral open-air intraoperative fluorescence imaging.

    PubMed

    Behrooz, Ali; Waterman, Peter; Vasquez, Kristine O; Meganck, Jeff; Peterson, Jeffrey D; Faqir, Ilias; Kempner, Joshua

    2017-08-01

    Intraoperative fluorescence imaging informs decisions regarding surgical margins by detecting and localizing signals from fluorescent reporters, labeling targets such as malignant tissues. This guidance reduces the likelihood of undetected malignant tissue remaining after resection, eliminating the need for additional treatment or surgery. The primary challenges in performing open-air intraoperative fluorescence imaging come from the weak intensity of the fluorescence signal in the presence of strong surgical and ambient illumination, and the auto-fluorescence of non-target components, such as tissue, especially in the visible spectral window (400-650 nm). In this work, a multispectral open-air fluorescence imaging system is presented for translational image-guided intraoperative applications, which overcomes these challenges. The system is capable of imaging weak fluorescence signals with nanomolar sensitivity in the presence of surgical illumination. This is done using synchronized fluorescence excitation and image acquisition with real-time background subtraction. Additionally, the system uses a liquid crystal tunable filter for acquisition of multispectral images that are used to spectrally unmix target fluorescence from non-target auto-fluorescence. Results are validated by preclinical studies on murine models and translational canine oncology models.

  11. The fluorescence lifetime of BRI1-GFP as probe for the noninvasive determination of the membrane potential in living cells

    NASA Astrophysics Data System (ADS)

    Elgass, K.; Caesar, K.; Schleifenbaum, F.; Meixner, A. J.; Harter, K.

    2010-02-01

    As the excited state lifetime of a fluorescent molecule depends on its environment, it is possible to use it as a probe for physico-chemical parameters of the surrounding medium. Whereas this is well known for many solid guest/host systems, only few reports of quantitative, temporal resolved in vivo studies to monitor the nano-environment for a protein-coupled chromophore such as GFP are known from literature. Here we present a novel approach to determine the membrane potential of living (plant) cells based on the fluorescence lifetime (FLT) analysis of membrane-located GFP. By using confocal sample scanning microscopy (CSSM) combined with fluorescence lifetime imaging microscopy, we recently showed that the phytohormone brassinolide (BL) induces cell wall expansion and a decrease in the FLT of the BRI1-GFP in living cells of Arabidopsis thaliana seedlings. BRI1 is the dominant functional receptor for BL in Arabidopsis and locates to the plasma membrane. Although the dependence of the FLT of GFP on its physico-chemical environment such as pH-value, refractive index and pressure has been reported, the observed FLT decrease of BRI1-GFP in response to BL application could not be explained by these parameters. However, our in vivo FLT and CSSM analyses indicate that the BLinduced change in the FLT of BRI1-GFP is caused by hyperpolarisation of the plasma membrane (Em). Thus, our results indicate that BRI1-GFP serves as sensitive and non-invasive probe for recording the Em of the plasma membrane in living plant cells with high spatio-temporal resolution.

  12. Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor.

    PubMed

    Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

    2011-07-06

    Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.

  13. Fluorescence Imaging Topography Scanning System for intraoperative multimodal imaging

    PubMed Central

    Quang, Tri T.; Kim, Hye-Yeong; Bao, Forrest Sheng; Papay, Francis A.; Edwards, W. Barry; Liu, Yang

    2017-01-01

    Fluorescence imaging is a powerful technique with diverse applications in intraoperative settings. Visualization of three dimensional (3D) structures and depth assessment of lesions, however, are oftentimes limited in planar fluorescence imaging systems. In this study, a novel Fluorescence Imaging Topography Scanning (FITS) system has been developed, which offers color reflectance imaging, fluorescence imaging and surface topography scanning capabilities. The system is compact and portable, and thus suitable for deployment in the operating room without disturbing the surgical flow. For system performance, parameters including near infrared fluorescence detection limit, contrast transfer functions and topography depth resolution were characterized. The developed system was tested in chicken tissues ex vivo with simulated tumors for intraoperative imaging. We subsequently conducted in vivo multimodal imaging of sentinel lymph nodes in mice using FITS and PET/CT. The PET/CT/optical multimodal images were co-registered and conveniently presented to users to guide surgeries. Our results show that the developed system can facilitate multimodal intraoperative imaging. PMID:28437441

  14. Capture of Fluorescence Decay Times by Flow Cytometry

    PubMed Central

    Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

    2012-01-01

    In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263

  15. Novel snapshot hyperspectral imager for fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Chandler, Lynn; Chandler, Andrea; Periasamy, Ammasi

    2018-02-01

    Hyperspectral imaging has emerged as a new technique for the identification and classification of biological tissue1. Benefitting recent developments in sensor technology, the new class of hyperspectral imagers can capture entire hypercubes with single shot operation and it shows great potential for real-time imaging in biomedical sciences. This paper explores the use of a SnapShot imager in fluorescence imaging via microscope for the very first time. Utilizing the latest imaging sensor, the Snapshot imager is both compact and attachable via C-mount to any commercially available light microscope. Using this setup, fluorescence hypercubes of several cells were generated, containing both spatial and spectral information. The fluorescence images were acquired with one shot operation for all the emission range from visible to near infrared (VIS-IR). The paper will present the hypercubes obtained images from example tissues (475-630nm). This study demonstrates the potential of application in cell biology or biomedical applications for real time monitoring.

  16. Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

    PubMed

    Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

    2017-07-01

    The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

  17. Entangled-photon coincidence fluorescence imaging

    PubMed Central

    Scarcelli, Giuliano; Yun, Seok H.

    2009-01-01

    We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as “detectors” breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations. PMID:18825257

  18. Optical metabolic imaging of live tissue cultures

    NASA Astrophysics Data System (ADS)

    Walsh, Alex J.; Cook, Rebecca S.; Arteaga, Carlos L.; Skala, Melissa C.

    2013-02-01

    The fluorescence properties, both intensity and fluorescence lifetime, of NADH and FAD, two coenzymes of metabolism, are sensitive, high resolution measures of cellular metabolism. However, often in vivo measurements of tissue are not feasible. In this study, we investigate the stability over time of two-photon auto-fluorescence imaging of NADH and FAD in live-cultured tissues. Our results demonstrate that cultured tissues remain viable for at least several days post excision. Furthermore, the optical redox ratio, NADH fluorescence lifetime, and FAD fluorescence lifetime do not significantly change in the cultured tissues over time. With these findings, we demonstrate the potential of sustained tissue culture techniques for optical metabolic imaging.

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

    PubMed Central

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

    2011-01-01

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

  20. 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

  1. BSA Au clusters as a probe for enhanced fluorescence detection using multipulse excitation scheme.

    PubMed

    Raut, Sangram L; Rich, Ryan; Fudala, Rafal; Kokate, R; Kimball, J D; Borejdo, Julian; Vishwanatha, Jamboor K; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2014-01-01

    Although BSA Au clusters fluoresce in red region (λmax: 650 nm), they are of limited use due to low fluorescence quantum yield (~6%). Here we report an enhanced fluorescence imaging application of fluorescent bio-nano probe BSA Au clusters using multipulse excitation scheme. Multipulse excitation takes advantage of long fluorescence lifetime (> 1 µs) of BSA Au clusters and enhances its fluorescence intensity 15 times over short lived cellular auto-fluorescence. Moreover we have also shown that by using time gated detection strategy signal (fluorescence of BSA Au clusters) to noise (auto-fluorescence) ratio can be increased by 30 fold. Thereby with multipulse excitation long lifetime probes can be used to develop biochemical assays and perform optical imaging with zero background.

  2. Highly K+ -Selective Fluorescent Probes for Lifetime Sensing of K+ in Living Cells.

    PubMed

    Schwarze, Thomas; Mertens, Monique; Müller, Peter; Riemer, Janine; Wessig, Pablo; Holdt, Hans-Jürgen

    2017-12-06

    The new K + -selective fluorescent probes 1 and 2 were obtained by Cu I -catalyzed 1,3-dipolar azide alkyne cycloaddition (CuAAC) reactions of an alkyne-substituted [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ester fluorophore with azido-functionalized N-phenylaza-18-crown-6 ether and N-(o-isopropoxy) phenylaza-18-crown-6 ether, respectively. Probes 1 and 2 allow the detection of K + in the presence of Na + in water by fluorescence enhancement (2.2 for 1 at 2000 mm K + and 2.5 for 2 at 160 mm K + ). Fluorescence lifetime measurements in the absence and presence of K + revealed bi-exponential decay kinetics with similar lifetimes, however with different proportions changing the averaged fluorescence decay times (τ f(av) ). For 1 a decrease of τ f(av) from 12.4 to 9.3 ns and for 2 an increase from 17.8 to 21.8 ns was observed. Variation of the substituent in ortho position of the aniline unit of the N-phenylaza-18-crown-6 host permits the modulation of the K d value for a certain K + concentration. For example, substitution of H in 1 by the isopropoxy group (2) decreased the K d value from >300 mm to 10 mm. 2 was chosen for studying the efflux of K + from human red blood cells (RBC). Upon addition of the Ca 2+ ionophor ionomycin to a RBC suspension in a buffer containing Ca 2+ , the fluorescence of 2 slightly rose within 10 min, however, after 120 min a significant increase was observed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy

    PubMed Central

    Driscoll, Jonathan D.; Shih, Andy Y.; Iyengar, Satish; Field, Jeffrey J.; White, G. Allen; Squier, Jeffrey A.; Cauwenberghs, Gert

    2011-01-01

    We present a high-speed photon counter for use with two-photon microscopy. Counting pulses of photocurrent, as opposed to analog integration, maximizes the signal-to-noise ratio so long as the uncertainty in the count does not exceed the gain-noise of the photodetector. Our system extends this improvement through an estimate of the count that corrects for the censored period after detection of an emission event. The same system can be rapidly reconfigured in software for fluorescence lifetime imaging, which we illustrate by distinguishing between two spectrally similar fluorophores in an in vivo model of microstroke. PMID:21471395

  4. Fluorescence and Spectral Imaging

    PubMed Central

    DaCosta, Ralph S.; Wilson, Brian C.; Marcon, Norman E.

    2007-01-01

    Early identification of dysplasia remains a critical goal for diagnostic endoscopy since early discovery directly improves patient survival because it allows endoscopic or surgical intervention with disease localized without lymph node involvement. Clinical studies have successfully used tissue autofluorescence with conventional white light endoscopy and biopsy for detecting adenomatous colonic polyps, differentiating benign hyperplastic from adenomas with acceptable sensitivity and specificity. In Barrett's esophagus, the detection of dysplasia remains problematic because of background inflammation, whereas in the squamous esophagus, autofluorescence imaging appears to be more dependable. Point fluorescence spectroscopy, although playing a crucial role in the pioneering mechanistic development of fluorescence endoscopic imaging, does not seem to have a current function in endoscopy because of its nontargeted sampling and suboptimal sensitivity and specificity. Other point spectroscopic modalities, such as Raman spectroscopy and elastic light scattering, continue to be evaluated in clinical studies, but still suffer the significant disadvantages of being random and nonimaging. A recent addition to the fluorescence endoscopic imaging arsenal is the use of confocal fluorescence endomicroscopy, which provides real-time optical biopsy for the first time. To improve detection of dysplasia in the gastrointestinal tract, a new and exciting development has been the use of exogenous fluorescence contrast probes that specifically target a variety of disease-related cellular biomarkers using conventional fluorescent dyes and novel potent fluorescent nanocrystals (i.e., quantum dots). This is an area of great promise, but still in its infancy, and preclinical studies are currently under way. PMID:18167619

  5. A fusion-spliced near-field optical fiber probe using photonic crystal fiber for nanoscale thermometry based on fluorescence-lifetime measurement of quantum dots.

    PubMed

    Fujii, Takuro; Taguchi, Yoshihiro; Saiki, Toshiharu; Nagasaka, Yuji

    2011-01-01

    We have developed a novel nanoscale temperature-measurement method using fluorescence in the near-field called fluorescence near-field optics thermal nanoscopy (Fluor-NOTN). Fluor-NOTN enables the temperature distributions of nanoscale materials to be measured in vivo/in situ. The proposed method measures temperature by detecting the temperature dependent fluorescence lifetimes of Cd/Se quantum dots (QDs). For a high-sensitivity temperature measurement, the auto-fluorescence generated from a fiber probe should be reduced. In order to decrease the noise, we have fabricated a novel near-field optical-fiber probe by fusion-splicing a photonic crystal fiber (PCF) and a conventional single-mode fiber (SMF). The validity of the novel fiber probe was assessed experimentally by evaluating the auto-fluorescence spectra of the PCF. Due to the decrease of auto-fluorescence, a six- to ten-fold increase of S/N in the near-field fluorescence lifetime detection was achieved with the newly fabricated fusion-spliced near-field optical fiber probe. Additionally, the near-field fluorescence lifetime of the quantum dots was successfully measured by the fabricated fusion-spliced near-field optical fiber probe at room temperature, and was estimated to be 10.0 ns.

  6. Polar plot representation of time-resolved fluorescence.

    PubMed

    Eichorst, John Paul; Wen Teng, Kai; Clegg, Robert M

    2014-01-01

    Measuring changes in a molecule's fluorescence emission is a common technique to study complex biological systems such as cells and tissues. Although the steady-state fluorescence intensity is frequently used, measuring the average amount of time that a molecule spends in the excited state (the fluorescence lifetime) reveals more detailed information about its local environment. The lifetime is measured in the time domain by detecting directly the decay of fluorescence following excitation by short pulse of light. The lifetime can also be measured in the frequency domain by recording the phase and amplitude of oscillation in the emitted fluorescence of the sample in response to repetitively modulated excitation light. In either the time or frequency domain, the analysis of data to extract lifetimes can be computationally intensive. For example, a variety of iterative fitting algorithms already exist to determine lifetimes from samples that contain multiple fluorescing species. However, recently a method of analysis referred to as the polar plot (or phasor plot) is a graphical tool that projects the time-dependent features of the sample's fluorescence in either the time or frequency domain into the Cartesian plane to characterize the sample's lifetime. The coordinate transformations of the polar plot require only the raw data, and hence, there are no uncertainties from extensive corrections or time-consuming fitting in this analysis. In this chapter, the history and mathematical background of the polar plot will be presented along with examples that highlight how it can be used in both cuvette-based and imaging applications.

  7. Time-Correlated Single-Photon Counting Fluorescence Imaging of Lipid Domains In Raft-Mimicking Giant Unilamellar Vesicles

    NASA Astrophysics Data System (ADS)

    Clarke, James; Cheng, Kwan; Shindell, Orrin; Wang, Exing

    We have designed and constructed a high-throughput electrofusion chamber and an incubator to fabricate Giant Unilamellar Vesicles (GUVs) consisting of high-melting lipids, low-melting lipids, cholesterol and both ordered and disordered phase sensitive fluorescent probes (DiIC12, dehydroergosterol and BODIPY-Cholesterol). GUVs were formed in a 3 stage pulse sequence electrofusion process with voltages ranging from 50mVpp to 2.2Vpp and frequencies from 5Hz to 10Hz. Steady state and time-correlated single-photon counting (TCSPC) fluorescence lifetime (FLIM) based confocal and/or multi-photon microscopic techniques were used to characterize phase separated lipid domains in GUVs. Confocal imaging measures the probe concentration and the chemical environment of the system. TCSPC techniques determine the chemical environment through the perturbation of fluorescent lifetimes of the probes in the system. The above techniques will be applied to investigate the protein-lipid interactions involving domain formation. Specifically, the mechanisms governing lipid domain formations in the above systems that mimic the lipid rafts in cells will be explored. Murchison Fellowship at Trinity University.

  8. Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

    PubMed

    Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki

    2013-07-29

    We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

  9. Quantitative, spectrally-resolved intraoperative fluorescence imaging

    PubMed Central

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

    2012-01-01

    Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field. PMID:23152935

  10. Analytic solutions to modelling exponential and harmonic functions using Chebyshev polynomials: fitting frequency-domain lifetime images with photobleaching.

    PubMed

    Malachowski, George C; Clegg, Robert M; Redford, Glen I

    2007-12-01

    A novel approach is introduced for modelling linear dynamic systems composed of exponentials and harmonics. The method improves the speed of current numerical techniques up to 1000-fold for problems that have solutions of multiple exponentials plus harmonics and decaying components. Such signals are common in fluorescence microscopy experiments. Selective constraints of the parameters being fitted are allowed. This method, using discrete Chebyshev transforms, will correctly fit large volumes of data using a noniterative, single-pass routine that is fast enough to analyse images in real time. The method is applied to fluorescence lifetime imaging data in the frequency domain with varying degrees of photobleaching over the time of total data acquisition. The accuracy of the Chebyshev method is compared to a simple rapid discrete Fourier transform (equivalent to least-squares fitting) that does not take the photobleaching into account. The method can be extended to other linear systems composed of different functions. Simulations are performed and applications are described showing the utility of the method, in particular in the area of fluorescence microscopy.

  11. [Development of fluorescent probes for bone imaging in vivo ~Fluorescent probes for intravital imaging of osteoclast activity~.

    PubMed

    Minoshima, Masafumi; Kikuchi, Kazuya

    Fluorescent molecules are widely used as a tool to directly visualize target biomolecules in vivo. Fluorescent probes have the advantage that desired function can be rendered based on rational design. For bone-imaging fluorescent probes in vivo, they should be delivered to bone tissue upon administration. Recently, a fluorescent probe for detecting osteoclast activity was developed. The fluorescent probe has acid-sensitive fluorescence property, specific delivery to bone tissue, and durability against laser irradiation, which enabled real-time intravital imaging of bone-resorbing osteoclasts for a long period of time.

  12. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. On the heterogeneity of fluorescence lifetime of room temperature ionic liquids: onset of a journey for exploring red emitting dyes.

    PubMed

    Ghosh, Anup; Chatterjee, Tanmay; Mandal, Prasun K

    2012-06-25

    An excitation and emission wavelength dependent non-exponential fluorescence decay behaviour of room temperature ionic liquids (RTILs) has been noted. Average fluorescence lifetimes have been found to vary by a factor of three or more. Red emitting dyes dissolved in RTILs are found to follow hitherto unobserved single exponential fluorescence decay behaviour.

  14. Engineering a lifetime-based activatable probe for photoacoustic imaging

    NASA Astrophysics Data System (ADS)

    Morgounova, Ekaterina; Shao, Qi; Hackel, Benjamin; Ashkenazi, Shai

    2013-02-01

    High-resolution, high-penetration depth activatable probes are needed for in-vivo imaging of enzyme activity. In this paper, we will describe the contrast mechanism of a new photoacoustic activatable probe that changes its excitation lifetime upon activation. The excitation decay of methylene blue (MB), a chromophore commonly used in therapeutic and diagnostic applications, is probed by photoacoustic lifetime contrast imaging (PLCI). The monomer of the dye presents a high-quantum yield of intersystem-crossing and long lifetime (70 μs) whereas the dimer is statically quenched with a short lifetime (a few ns). This forms the basis of a highly sensitive contrast mechanism between monomers and dimers. Two dimerization models - one using sodium sulfate, the other using sodium dodecyl sulfate - were applied to control the monomer-to-dimer ratio in MB solutions. Preliminary results show that the photoacoustic signal of a dimer solution is efficiently suppressed (< 20 dB) due to their short lifetime compared to the monomer sample. Flash-photolysis of the same solutions reveals a 99% decrease in transient absorption confirming PLCI results. This contrast mechanism can be applied to design a MB dual-labeled activatable probe bound by an enzyme-specific cleavable peptide linker. When the probe is cleaved by its target, MB molecules will separate by molecular diffusion and recover their long excitation lifetime enabling their detection by PLCI. Our long-term goal is to investigate enzyme-specific imaging in small animals and establish pre-clinical data for translational research and implementation of the technology in clinical applications.

  15. Fluorescence lifetime spectroscopy for guided therapy of brain tumors.

    PubMed

    Butte, Pramod V; Mamelak, Adam N; Nuno, Miriam; Bannykh, Serguei I; Black, Keith L; Marcu, Laura

    2011-01-01

    This study evaluates the potential of time-resolved laser induced fluorescence spectroscopy (TR-LIFS) as intra-operative tool for the delineation of brain tumor from normal brain. Forty two patients undergoing glioma (WHO grade I-IV) surgery were enrolled in this study. A TR-LIFS prototype apparatus (gated detection, fast digitizer) was used to induce in-vivo fluorescence using a pulsed N2 laser (337 nm excitation, 0.7 ns pulse width) and to record the time-resolved spectrum (360-550 nm range, 10 nm interval). The sites of TR-LIFS measurement were validated by conventional histopathology (H&E staining). Parameters derived from the TR-LIFS data including intensity values and time-resolved intensity decay features (average fluorescence lifetime and Laguerre coefficients values) were used for tissue characterization and classification. 71 areas of tumor and normal brain were analyzed. Several parameters allowed for the differentiation of distinct tissue types. For example, normal cortex (N=35) and normal white matter (N=12) exhibit a longer-lasting fluorescence emission at 390 nm (τ390=2.12±0.10 ns) when compared with 460 nm (τ460=1.16±0.08 ns). High grade glioma (grades III and IV) samples (N=17) demonstrate emission peaks at 460 nm, with large variation at 390 nm while low grade glioma (I and II) samples (N=7) demonstrated a peak fluorescence emission at 460 nm. A linear discriminant algorithm allowed for the classification of low-grade gliomas with 100% sensitivity and 98% specificity. High-grade glioma demonstrated a high degree of heterogeneity thus reducing the discrimination accuracy of these tumors to 47% sensitivity and 94% specificity. Current findings demonstrate that TR-LIFS holds the potential to diagnose brain tumors intra-operatively and to provide a valuable tool for aiding the neurosurgeon-neuropathologist team in to rapidly distinguish between tumor and normal brain during surgery. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Quantitative oxygen concentration imaging in toluene atmospheres using Dual Imaging with Modeling Evaluation

    NASA Astrophysics Data System (ADS)

    Ehn, Andreas; Jonsson, Malin; Johansson, Olof; Aldén, Marcus; Bood, Joakim

    2013-01-01

    Fluorescence lifetimes of toluene as a function of oxygen concentration in toluene/nitrogen/oxygen mixtures have been measured at room temperature using picosecond-laser excitation of the S1-S0 transition at 266 nm. The data satisfy the Stern-Volmer relation with high accuracy, providing an updated value of the Stern-Volmer slope. A newly developed fluorescence lifetime imaging scheme, called Dual Imaging with Modeling Evaluation (DIME), is evaluated and successfully demonstrated for quantitative oxygen concentration imaging in toluene-seeded O2/N2 gas mixtures.

  17. Quantitative oxygen concentration imaging in toluene atmospheres using Dual Imaging with Modeling Evaluation

    NASA Astrophysics Data System (ADS)

    Ehn, Andreas; Jonsson, Malin; Johansson, Olof; Aldén, Marcus; Bood, Joakim

    2012-12-01

    Fluorescence lifetimes of toluene as a function of oxygen concentration in toluene/nitrogen/oxygen mixtures have been measured at room temperature using picosecond-laser excitation of the S1-S0 transition at 266 nm. The data satisfy the Stern-Volmer relation with high accuracy, providing an updated value of the Stern-Volmer slope. A newly developed fluorescence lifetime imaging scheme, called Dual Imaging with Modeling Evaluation (DIME), is evaluated and successfully demonstrated for quantitative oxygen concentration imaging in toluene-seeded O2/N2 gas mixtures.

  18. The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex

    NASA Astrophysics Data System (ADS)

    Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

    2016-02-01

    Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

  19. Fluorescence lifetime intravascular ultrasound (FLIm-IVUS) and the quest to discriminate between early and advanced lipid cores in atherosclerosis (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Phipps, Jennifer E.; Bec, Julien; Vela, Deborah; Buja, L. Maximilian; Southard, Jeffrey A.; Margulies, Kenneth B.; Marcu, Laura

    2017-02-01

    FL-IVUS combines intravascular ultrasound with fluorescence lifetime imaging to obtain morphologic and biochemical details from the arterial wall. Ultrasound measurements alone provide morphologic information (plaque burden, remodeling index and presence of calcium). Fluorescence lifetime can determine the presence of a thick fibrous cap, macrophage infiltration, and lipid cores beneath thin fibrous caps. These details are important to assess plaque vulnerability. In this study, we focused on the ability of FL-IVUS to differentiate between early and advanced lipid cores-advanced cores are vulnerable to rupture. We imaged N=12 ex vivo human coronary arteries and performed hematoxylin and eosin, Movat's pentachrome and CD68 immunohistochemistry at 500 micron intervals throughout the length of the vessels. We found only N=1 thin-capped fibroatheroma (TCFA) with an advanced necrotic core and N=7 cases of foam cell infiltration, early lipid cores or deep necrotic cores. IVUS was able to observe the increased plaque burden and calcification of the advanced and deep necrotic cores, but could not identify early lipid cores, foam cell infiltration or discriminate between deep necrotic cores and TCFA. The addition of FLIm to IVUS allowed the TCFA to be discriminated from early lipid accumulation, particularly at 542+/-50 nm (355 nm pulsed excitation): 7.6 +/- 0.5 ns compared to 6.6 +/- 0.4 ns, respectively (P<0.001 by ANOVA analysis). These differences need to be validated in a larger cohort, but exist due to specific lipid content in the necrotic core as well as increased extracellular matrix in early lesions.

  20. Fluorescence Imaging Reveals Surface Contamination

    NASA Technical Reports Server (NTRS)

    Schirato, Richard; Polichar, Raulf

    1992-01-01

    In technique to detect surface contamination, object inspected illuminated by ultraviolet light to make contaminants fluoresce; low-light-level video camera views fluorescence. Image-processing techniques quantify distribution of contaminants. If fluorescence of material expected to contaminate surface is not intense, tagged with low concentration of dye.

  1. Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

    NASA Astrophysics Data System (ADS)

    Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

    2011-07-01

    Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

  2. 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.

  3. Development of Next Generation Lifetime PSP Imaging Systems

    NASA Technical Reports Server (NTRS)

    Watkins, A. Neal; Jordan, Jeffrey D.; Leighty, Bradley D.; Ingram, JoAnne L.; Oglesby, Donald M.

    2002-01-01

    This paper describes a lifetime PSP system that has recently been developed using pulsed light-emitting diode (LED) lamps and a new interline transfer CCD camera technology. This system alleviates noise sources associated with lifetime PSP systems that use either flash-lamp or laser excitation sources and intensified CCD cameras for detection. Calibration curves have been acquired for a variety of PSP formulations using this system, and a validation test was recently completed in the Subsonic Aerodynamic Research Laboratory (SARL) at Wright-Patterson Air Force Base (WPAFB). In this test, global surface pressure distributions were recovered using both a standard intensity-based method and the new lifetime system. Results from the lifetime system agree both qualitatively and quantitatively with those measured using the intensity-based method. Finally, an advanced lifetime imaging technique capable of measuring temperature and pressure simultaneously is introduced and initial results are presented.

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

    NASA Astrophysics Data System (ADS)

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

    2002-05-01

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

  5. Multimodal imaging of vascular grafts using time-resolved fluorescence and ultrasound

    NASA Astrophysics Data System (ADS)

    Fatakdawala, Hussain; Griffiths, Leigh G.; Wong, Maelene L.; Humphrey, Sterling; Marcu, Laura

    2015-02-01

    The translation of engineered tissues into clinic requires robust monitoring of tissue development, both in vitro and in vivo. Traditional methods for the same are destructive, inefficient in time and cost and do not allow time-lapse measurements from the same sample or animal. This study reports on the ability of time-resolved fluorescence and ultrasound measurements for non-destructive characterization of explanted tissue engineered vascular grafts. Results show that TRFS and FLIm are able to assess alterations in luminal composition namely elastin, collagen and cellular (hyperplasia) content via changes in fluorescence lifetime values between normal and grafted tissue. These observations are complemented by structural changes observed in UBM pertaining to graft integration and intimal thickness over the grafted region. These results encourage the future application of a catheter-based technique that combines these imaging modalities for non-destructive characterization of vascular grafts in vivo.

  6. Multi-spectral endogenous fluorescence imaging for bacterial differentiation

    NASA Astrophysics Data System (ADS)

    Chernomyrdin, Nikita V.; Babayants, Margarita V.; Korotkov, Oleg V.; Kudrin, Konstantin G.; Rimskaya, Elena N.; Shikunova, Irina A.; Kurlov, Vladimir N.; Cherkasova, Olga P.; Komandin, Gennady A.; Reshetov, Igor V.; Zaytsev, Kirill I.

    2017-07-01

    In this paper, the multi-spectral endogenous fluorescence imaging was implemented for bacterial differentiation. The fluorescence imaging was performed using a digital camera equipped with a set of visual bandpass filters. Narrowband 365 nm ultraviolet radiation passed through a beam homogenizer was used to excite the sample fluorescence. In order to increase a signal-to-noise ratio and suppress a non-fluorescence background in images, the intensity of the UV excitation was modulated using a mechanical chopper. The principal components were introduced for differentiating the samples of bacteria based on the multi-spectral endogenous fluorescence images.

  7. Boronic acids for fluorescence imaging of carbohydrates.

    PubMed

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

    2016-02-28

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

  8. Sperm metabolism is altered during storage by female insects: evidence from two-photon autofluorescence lifetime measurements in bedbugs

    PubMed Central

    Reinhardt, Klaus; Breunig, Hans Georg; Uchugonova, Aisada; König, Karsten

    2015-01-01

    We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius, was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, τm = 1.54–1.84 ns) than in that extracted from the female sperm storage organ (tau m, τm = 1.26–2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype. PMID:26333813

  9. Longitudinal in vivo two-photon fluorescence imaging

    PubMed Central

    Crowe, Sarah E.; Ellis-Davies, Graham C.R.

    2014-01-01

    Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in 1980s, that enabled imaging both fixed and living biological tissue with three-dimensional precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to two years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. PMID:24214350

  10. 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

  11. Recent Progress in Fluorescent Imaging Probes.

    PubMed

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

    2015-09-22

    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).

  12. The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex.

    PubMed

    Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

    2016-02-05

    Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Multimodal quantitative phase and fluorescence imaging of cell apoptosis

    NASA Astrophysics Data System (ADS)

    Fu, Xinye; Zuo, Chao; Yan, Hao

    2017-06-01

    Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

  14. Study of excitation transfer in laser dye mixtures by direct measurement of fluorescence lifetime

    NASA Technical Reports Server (NTRS)

    Lin, C.; Dienes, A.

    1973-01-01

    By directly measuring the donor fluorescence lifetime as a function of acceptor concentration in the laser dye mixture Rhodamine 6G-Cresyl violet, we found that the Stern-Volmer relation is obeyed, from which the rate of excitation transfer is determined. The experimental results indicate that the dominant mechanism responsible for the efficient excitation transfer is that of resonance transfer due to long range dipole-dipole interaction.

  15. Fluorescence Live Cell Imaging

    PubMed Central

    Ettinger, Andreas

    2014-01-01

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

  16. Autofluorescence lifetime imaging during transoral robotic surgery: a clinical validation study of tumor detection (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Lagarto, João. L.; Phipps, Jennifer E.; Unger, Jakob; Faller, Leta M.; Gorpas, Dimitris; Ma, Dinglong M.; Bec, Julien; Moore, Michael G.; Bewley, Arnaud F.; Yankelevich, Diego R.; Sorger, Jonathan M.; Farwell, Gregory D.; Marcu, Laura

    2017-02-01

    Autofluorescence lifetime spectroscopy is a promising non-invasive label-free tool for characterization of biological tissues and shows potential to report structural and biochemical alterations in tissue owing to pathological transformations. In particular, when combined with fiber-optic based instruments, autofluorescence lifetime measurements can enhance intraoperative diagnosis and provide guidance in surgical procedures. We investigate the potential of a fiber-optic based multi-spectral time-resolved fluorescence spectroscopy instrument to characterize the autofluorescence fingerprint associated with histologic, morphologic and metabolic changes in tissue that can provide real-time contrast between healthy and tumor regions in vivo and guide clinicians during resection of diseased areas during transoral robotic surgery. To provide immediate feedback to the surgeons, we employ tracking of an aiming beam that co-registers our point measurements with the robot camera images and allows visualization of the surgical area augmented with autofluorescence lifetime data in the surgeon's console in real-time. For each patient, autofluorescence lifetime measurements were acquired from normal, diseased and surgically altered tissue, both in vivo (pre- and post-resection) and ex vivo. Initial results indicate tumor and normal regions can be distinguished based on changes in lifetime parameters measured in vivo, when the tumor is located superficially. In particular, results show that autofluorescence lifetime of tumor is shorter than that of normal tissue (p < 0.05, n = 3). If clinical diagnostic efficacy is demonstrated throughout this on-going study, we believe that this method has the potential to become a valuable tool for real-time intraoperative diagnosis and guidance during transoral robot assisted cancer removal interventions.

  17. Fluorescent Probes for Sensing and Imaging within Specific Cellular Organelles.

    PubMed

    Zhu, Hao; Fan, Jiangli; Du, Jianjun; Peng, Xiaojun

    2016-10-18

    long fluorescence lifetimes, which are crucial for long-time tracking of biological processes, tissue and body imaging with deep penetration and low autofluorescence, and time-resolved fluorescence imaging. Research on fluorescent probes with both analyte responsiveness and organelle targetability is a new and emerging area that has attracted increasing attention over the past few years. We have extended the diversity by developing organelle-specific responsive probes capable of detecting changes in biomolecular levels (reactive oxygen species, fluoride ion, hydrogen sulfide, zinc cation, thiol-containing amino acids, and cyclooxygenase-2) and the microenvironment (viscosity, polarity, and pH). Future research should give more considerations of the "low-concern" organelles, such as the Golgi apparatus, the endoplasmic reticulum, and ribosomes. In addition, given the tiny sizes of subcellular organelles (20-1000 nm), we anticipate that clearer visulization of the cellular events within specific organelles will rely on super-resolution optical microscopy with nanoscopic-scale resolution.

  18. 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

  19. Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells.

    PubMed

    Grecco, H E; Lidke, K A; Heintzmann, R; Lidke, D S; Spagnuolo, C; Martinez, O E; Jares-Erijman, E A; Jovin, T M

    2004-11-01

    In this work, we characterized streptavidin-conjugated quantum dots (QDs) manufactured by Quantum Dot Corporation. We present data on: (1) two-photon excitation; (2) fluorescence lifetimes; (3) ensemble and single QD emission anisotropy; (4) QDs as donors for Forster resonance energy transfer (FRET); and (5) spectral conversion of QDs exposed to high-intensity illumination. We also demonstrate the utility of QDs for (1) imaging the binding and uptake of biotinylated transferrin on living cells, and (2) resolving by fluorescence lifetime imaging microscopy (FLIM) signals originating from QDs from those of spatially and spectrally overlapping visible fluorescent proteins (VFPs). (c) 2005 Wiley-Liss, Inc.

  20. Tomographic imaging of flourescence resonance energy transfer in highly light scattering media

    NASA Astrophysics Data System (ADS)

    Soloviev, Vadim Y.; McGinty, James; Tahir, Khadija B.; Laine, Romain; Stuckey, Daniel W.; Mohan, P. Surya; Hajnal, Joseph V.; Sardini, Alessandro; French, Paul M. W.; Arridge, Simon R.

    2010-02-01

    Three-dimensional localization of protein conformation changes in turbid media using Förster Resonance Energy Transfer (FRET) was investigated by tomographic fluorescence lifetime imaging (FLIM). FRET occurs when a donor fluorophore, initially in its electronic excited state, transfers energy to an acceptor fluorophore in close proximity through non-radiative dipole-dipole coupling. An acceptor effectively behaves as a quencher of the donor's fluorescence. The quenching process is accompanied by a reduction in the quantum yield and lifetime of the donor fluorophore. Therefore, FRET can be localized by imaging changes in the quantum yield and the fluorescence lifetime of the donor fluorophore. Extending FRET to diffuse optical tomography has potentially important applications such as in vivo studies in small animal. We show that FRET can be localized by reconstructing the quantum yield and lifetime distribution from time-resolved non-invasive boundary measurements of fluorescence and transmitted excitation radiation. Image reconstruction was obtained by an inverse scattering algorithm. Thus we report, to the best of our knowledge, the first tomographic FLIM-FRET imaging in turbid media. The approach is demonstrated by imaging a highly scattering cylindrical phantom concealing two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically-encoded calcium FRET sensor. A 10mM calcium chloride solution was added to one of the wells to induce a protein conformation change upon binding to TN-L15, resulting in FRET and a corresponding decrease in the donor fluorescence lifetime. The resulting fluorescence lifetime distribution, the quantum efficiency, absorption and scattering coefficients were reconstructed.

  1. Fluorescence imaging of antibiotic clofazimine encapsulated within mesoporous silica particle carriers: relevance to drug delivery and the effect on its release kinetics.

    PubMed

    Angiolini, Lorenzo; Valetti, Sabrina; Cohen, Boiko; Feiler, Adam; Douhal, Abderrazzak

    2018-05-03

    We report on the encapsulation of the antibiotic clofazimine (CLZ) within the pores of mesoporous silica particles having hydrophilic (CBET value of 137) and more hydrophobic (CBET value of 94 after calcination at 600 °C) surfaces. We studied the effect of pH on the released amount of CLZ in aqueous solutions and observed a maximum at pH 4.1 in correlation with the solubility of the drug. Less release of the drug was observed from the more hydrophobic particles which was attributed to a difference in the affinity of the drug to the carrier particles. Fluorescence lifetime imaging microscopy, emission spectra, and fluorescence lifetimes of single drug loaded particles provided detailed understanding and new knowledge of the physical form of the encapsulated drug and the distribution within the particles. The distribution of CLZ within the particles was independent of the surface chemistry of the particles. The confirmation of CLZ molecules as monomers or aggregates was revealed by controlled removal of the drug with solvent. Additionally, the observed optical "halo effect" in the fluorescent images was interpreted in terms of specific quenching of high concentration of molecules. The emission lifetime experiments suggest stronger interaction of CLZ with the more hydrophobic particles, which is relevant to its release. The results reported in this work demonstrate that tuning the hydrophilicity/hydrophobicity of mesoporous silica particles can be used as a tool to control the release without impacting their loading ability.

  2. qF-SSOP: real-time optical property corrected fluorescence imaging

    PubMed Central

    Valdes, Pablo A.; Angelo, Joseph P.; Choi, Hak Soo; Gioux, Sylvain

    2017-01-01

    Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively. PMID:28856038

  3. 5-ALA induced fluorescent image analysis of actinic keratosis

    NASA Astrophysics Data System (ADS)

    Cho, Yong-Jin; Bae, Youngwoo; Choi, Eung-Ho; Jung, Byungjo

    2010-02-01

    In this study, we quantitatively analyzed 5-ALA induced fluorescent images of actinic keratosis using digital fluorescent color and hyperspectral imaging modalities. UV-A was utilized to induce fluorescent images and actinic keratosis (AK) lesions were demarcated from surrounding the normal region with different methods. Eight subjects with AK lesion were participated in this study. In the hyperspectral imaging modality, spectral analysis method was utilized for hyperspectral cube image and AK lesions were demarcated from the normal region. Before image acquisition, we designated biopsy position for histopathology of AK lesion and surrounding normal region. Erythema index (E.I.) values on both regions were calculated from the spectral cube data. Image analysis of subjects resulted in two different groups: the first group with the higher fluorescence signal and E.I. on AK lesion than the normal region; the second group with lower fluorescence signal and without big difference in E.I. between two regions. In fluorescent color image analysis of facial AK, E.I. images were calculated on both normal and AK lesions and compared with the results of hyperspectral imaging modality. The results might indicate that the different intensity of fluorescence and E.I. among the subjects with AK might be interpreted as different phases of morphological and metabolic changes of AK lesions.

  4. Near Infrared Dyes as Lifetime Solvatochromic Probes for Micropolarity Measurements of Biological Systems

    PubMed Central

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

    2007-01-01

    The polarity of biological mediums controls a host of physiological processes such as digestion, signaling, transportation, metabolism, and excretion. With the recent widespread use of near-infrared (NIR) fluorescent dyes for biological imaging of cells and living organisms, reporting medium polarity with these dyes would provide invaluable functional information in addition to conventional optical imaging parameters. Here, we report a new approach to determine polarities of macro- and microsystems for in vitro and potential in vivo applications using NIR polymethine molecular probes. Unlike the poor solvatochromic response of NIR dyes in solvents with diverse polarity, their fluorescence lifetimes are highly sensitive, increasing by a factor of up to 8 on moving from polar to nonpolar mediums. We also established a correlation between fluorescence lifetime and solvent orientation polarizability and developed a lifetime polarity index for determining the polarity of complex systems, including micelles and albumin binding sites. Because of the importance of medium polarity in molecular, cellular, and biochemical processes and the significance of reduced autofluorescence and deep tissue penetration of light in the NIR region, the findings reported herein represent an important advance toward using NIR molecular probes to measure the polarity of complex biological systems in vitro and in vivo. PMID:17573433

  5. Uncertainty analysis for fluorescence tomography with Monte Carlo method

    NASA Astrophysics Data System (ADS)

    Reinbacher-Köstinger, Alice; Freiberger, Manuel; Scharfetter, Hermann

    2011-07-01

    Fluorescence tomography seeks to image an inaccessible fluorophore distribution inside an object like a small animal by injecting light at the boundary and measuring the light emitted by the fluorophore. Optical parameters (e.g. the conversion efficiency or the fluorescence life-time) of certain fluorophores depend on physiologically interesting quantities like the pH value or the oxygen concentration in the tissue, which allows functional rather than just anatomical imaging. To reconstruct the concentration and the life-time from the boundary measurements, a nonlinear inverse problem has to be solved. It is, however, difficult to estimate the uncertainty of the reconstructed parameters in case of iterative algorithms and a large number of degrees of freedom. Uncertainties in fluorescence tomography applications arise from model inaccuracies, discretization errors, data noise and a priori errors. Thus, a Markov chain Monte Carlo method (MCMC) was used to consider all these uncertainty factors exploiting Bayesian formulation of conditional probabilities. A 2-D simulation experiment was carried out for a circular object with two inclusions. Both inclusions had a 2-D Gaussian distribution of the concentration and constant life-time inside of a representative area of the inclusion. Forward calculations were done with the diffusion approximation of Boltzmann's transport equation. The reconstruction results show that the percent estimation error of the lifetime parameter is by a factor of approximately 10 lower than that of the concentration. This finding suggests that lifetime imaging may provide more accurate information than concentration imaging only. The results must be interpreted with caution, however, because the chosen simulation setup represents a special case and a more detailed analysis remains to be done in future to clarify if the findings can be generalized.

  6. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

    NASA Astrophysics Data System (ADS)

    Stringari, Chiara; Sierra, Robert; Donovan, Peter J.; Gratton, Enrico

    2012-04-01

    We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation.

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

    PubMed

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

    2015-11-13

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

  8. Intravital Fluorescence Excitation in Whole-Animal Optical Imaging.

    PubMed

    Nooshabadi, Fatemeh; Yang, Hee-Jeong; 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.

  9. 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…

  10. Advanced Imaging Approaches to Characterize Stromal and Metabolic Changes in In Vivo Mammary Tumor Models

    DTIC Science & Technology

    2013-03-01

    characterization and toward future intravital studies. Preliminary fluorescence lifetime images were also collected intravitally through a mammary imaging window...intend to use this characterization to understand shifts in fluorescence lifetime collected by intravital imaging using a mammary imaging window...collected intravitally through a mammary imaging window implanted in a female, PyVT positive, Col1a1 heterozygote, mouse (Figure 7). A paper has

  11. 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.

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

    PubMed

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

    2009-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-06-01

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

  14. Ultra-sensitive fluorescent imaging-biosensing using biological photonic crystals

    NASA Astrophysics Data System (ADS)

    Squire, Kenny; Kong, Xianming; Wu, Bo; Rorrer, Gregory; Wang, Alan X.

    2018-02-01

    Optical biosensing is a growing area of research known for its low limits of detection. Among optical sensing techniques, fluorescence detection is among the most established and prevalent. Fluorescence imaging is an optical biosensing modality that exploits the sensitivity of fluorescence in an easy-to-use process. Fluorescence imaging allows a user to place a sample on a sensor and use an imager, such as a camera, to collect the results. The image can then be processed to determine the presence of the analyte. Fluorescence imaging is appealing because it can be performed with as little as a light source, a camera and a data processor thus being ideal for nontrained personnel without any expensive equipment. Fluorescence imaging sensors generally employ an immunoassay procedure to selectively trap analytes such as antigens or antibodies. When the analyte is present, the sensor fluoresces thus transducing the chemical reaction into an optical signal capable of imaging. Enhancement of this fluorescence leads to an enhancement in the detection capabilities of the sensor. Diatoms are unicellular algae with a biosilica shell called a frustule. The frustule is porous with periodic nanopores making them biological photonic crystals. Additionally, the porous nature of the frustule allows for large surface area capable of multiple analyte binding sites. In this paper, we fabricate a diatom based ultra-sensitive fluorescence imaging biosensor capable of detecting the antibody mouse immunoglobulin down to a concentration of 1 nM. The measured signal has an enhancement of 6× when compared to sensors fabricated without diatoms.

  15. 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

  16. 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

  17. 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.

  18. Imaging autofluorescence temporal signatures of the human ocular fundus in vivo

    NASA Astrophysics Data System (ADS)

    Papour, Asael; Taylor, Zachary; Stafsudd, Oscar; Tsui, Irena; Grundfest, Warren

    2015-11-01

    We demonstrate real-time in vivo fundus imaging capabilities of our fluorescence lifetime imaging technology for the first time. This implementation of lifetime imaging uses light emitting diodes to capture full-field images capable of showing direct tissue contrast without executing curve fitting or lifetime calculations. Preliminary results of fundus images are presented, investigating autofluorescence imaging potential of various retina biomarkers for early detection of macular diseases.

  19. Segmented frequency-domain fluorescence lifetime measurements: minimizing the effects of photobleaching within a multi-component system.

    PubMed

    Marwani, Hadi M; Lowry, Mark; Keating, Patrick; Warner, Isiah M; Cook, Robert L

    2007-11-01

    This study introduces a newly developed frequency segmentation and recombination method for frequency-domain fluorescence lifetime measurements to address the effects of changing fractional contributions over time and minimize the effects of photobleaching within multi-component systems. Frequency segmentation and recombination experiments were evaluated using a two component system consisting of fluorescein and rhodamine B. Comparison of experimental data collected in traditional and segmented fashion with simulated data, generated using different changing fractional contributions, demonstrated the validity of the technique. Frequency segmentation and recombination was also applied to a more complex system consisting of pyrene with Suwannee River fulvic acid reference and was shown to improve recovered lifetimes and fractional intensity contributions. It was observed that photobleaching in both systems led to errors in recovered lifetimes which can complicate the interpretation of lifetime results. Results showed clear evidence that the frequency segmentation and recombination method reduced errors resulting from a changing fractional contribution in a multi-component system, and allowed photobleaching issues to be addressed by commercially available instrumentation.

  20. 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

  1. Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy.

    PubMed

    Zhu, Yongjian; Choe, Chun-Sik; Ahlberg, Sebastian; Meinke, Martina C; Alexiev, Ulrike; Lademann, Juergen; Darvin, Maxim E

    2015-05-01

    In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1 ± 2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6 ± 8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

  2. 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

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

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

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

  5. Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent.

    PubMed

    Schwenck, Johannes; Maier, Florian C; Kneilling, Manfred; Wiehr, Stefan; Fuchs, Kerstin

    2017-05-08

    This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models. Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

  6. Candida, fluorescent stain (image)

    MedlinePlus

    ... a fluorescent stain of Candida. Candida is a yeast (fungus) that causes mild disease, but in immunocompromised individuals it may cause life-threatening illness. (Image courtesy of the Centers for ...

  7. 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

  8. 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.

  9. 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

  10. Fluorescent Microscopy Enhancement Using Imaging

    NASA Astrophysics Data System (ADS)

    Conrad, Morgan P.; Reck tenwald, Diether J.; Woodhouse, Bryan S.

    1986-06-01

    To enhance our capabilities for observing fluorescent stains in biological systems, we are developing a low cost imaging system based around an IBM AT microcomputer and a commercial image capture board compatible with a standard RS-170 format video camera. The image is digitized in real time with 256 grey levels, while being displayed and also stored in memory. The software allows for interactive processing of the data, such as histogram equalization or pseudocolor enhancement of the display. The entire image, or a quadrant thereof, can be averaged over time to improve the signal to noise ratio. Images may be stored to disk for later use or comparison. The camera may be selected for better response in the UV or near IR. Combined with signal averaging, this increases the sensitivity relative to that of the human eye, while still allowing for the fluorescence distribution on either the surface or internal cytoskeletal structure to be observed.

  11. Ultrashort fluorescence lifetimes of hydrogen-bonded base pairs of guanosine and cytidine in solution.

    PubMed

    Schwalb, Nina K; Michalak, Thomas; Temps, Friedrich

    2009-12-24

    The optically excited electronic states of hydrogen-bonded homo- and heterodimers of guanosine (G) and deoxycytidine (C) were investigated by femtosecond fluorescence up-conversion spectroscopy. The base pairs were prepared in CHCl(3) solution by employing tert-butyldimethylsilyl (TBDMS) groups at the OH positions of the ribose (G) or deoxyribose (C) moieties to enhance the solubilities of the nucleosides in organic solvents. The H-bonded complexes that were obtained were characterized by FTIR spectroscopy. Fluorescence lifetime measurements were performed following electronic excitation at a series of UV wavelengths from lambda(pump) = 294 nm, close to the electronic origins of the bases, to lambda(pump) = 262 nm, where significant excess vibronic energy is deposited in the molecules, at nucleoside concentrations of c(0) = 0.1 and 1.0 mM. The experimental results revealed the existence of an ultrafast deactivation pathway for the optically prepared electronically excited state(s) of the G.C Watson-Crick base pair, which was found to have a lifetime of tau(GC) = 0.30(3) ps (with 2sigma error limits) irrespective of the pump wavelength. A similar short decay time, tau(GG) = 0.32(2) ps, was observed for the respective excited G.G homodimer. In contrast, the excited G monomer displayed a significantly longer-lived and wavelength-dependent deactivation, requiring three time constants, between 0.43(6) ps < or = tau(G,1) < or = 1.2(1) ps, 4.2(8) ps < or = tau(G,2) < or = 8(1) ps, and tau(G,3) = 195(32) ps. Self-complexation of C, on the other hand, led to a longer-lived excited state with a lifetime estimated between 1 ps < or = tau(CC) < or = 10 ps, compared to the dominant initial subpicosecond decay time of the C monomer of tau(C,1) = 0.80(4) ps.

  12. Non-invasive assessment of the liver using imaging

    NASA Astrophysics Data System (ADS)

    Thorling Thompson, Camilla; Wang, Haolu; Liu, Xin; Liang, Xiaowen; Crawford, Darrell H.; Roberts, Michael S.

    2016-12-01

    Chronic liver disease causes 2,000 deaths in Australia per year and early diagnosis is crucial to avoid progression to cirrhosis and end stage liver disease. There is no ideal method to evaluate liver function. Blood tests and liver biopsies provide spot examinations and are unable to track changes in function quickly. Therefore better techniques are needed. Non-invasive imaging has the potential to extract increased information over a large sampling area, continuously tracking dynamic changes in liver function. This project aimed to study the ability of three imaging techniques, multiphoton and fluorescence lifetime imaging microscopy, infrared thermography and photoacoustic imaging, in measuring liver function. Collagen deposition was obvious in multiphoton and fluorescence lifetime imaging in fibrosis and cirrhosis and comparable to conventional histology. Infrared thermography revealed a significantly increased liver temperature in hepatocellular carcinoma. In addition, multiphoton and fluorescence lifetime imaging and photoacoustic imaging could both track uptake and excretion of indocyanine green in rat liver. These results prove that non-invasive imaging can extract crucial information about the liver continuously over time and has the potential to be translated into clinic in the assessment of liver disease.

  13. Comparitive study of fluorescence lifetime quenching of rhodamine 6G by MoS2 and Au-MoS2

    NASA Astrophysics Data System (ADS)

    Shakya, Jyoti; Kasana, Parath; Mohanty, T.

    2018-04-01

    Time resolved fluorescence study of Rhodamine 6G (R6G) in the presence of Molybdenum disulfide (MoS2) nanosheets and gold doped MoS2 (Au-MoS2) have been carried out and discussed. We have analyzed the fluorescence decay curves of R6G and it is observed that Au-MoS2 is a better fluorescence lifetime quencher as compare to MoS2 nanosheets. Also, the energy transfer efficiency and energy transfer rate from R6G to MoS2 and Au-MoS2 has been calculated and found higher for Au-MoS2.

  14. A study on a portable fluorescence imaging system

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

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

  15. Quantitative fluorescence microscopy and image deconvolution.

    PubMed

    Swedlow, Jason R

    2013-01-01

    Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used

  16. 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.

  17. 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.

  18. 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

  19. Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

    PubMed

    Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

    2017-05-01

    A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

  20. 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.

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

    PubMed

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

    2004-12-01

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

  2. Highly luminescent, biocompatible ytterbium(iii) complexes as near-infrared fluorophores for living cell imaging.

    PubMed

    Ning, Yingying; Tang, Juan; Liu, Yi-Wei; Jing, Jing; Sun, Yuansheng; Zhang, Jun-Long

    2018-04-21

    Herein, we report the design and synthesis of biocompatible Yb 3+ complexes for near-infrared (NIR) living cell imaging. Upon excitation at either the visible (Soret band) or red region (Q band), these β-fluorinated Yb 3+ complexes display high NIR luminescence (quantum yields up to 23% and 13% in dimethyl sulfoxide and water, respectively) and have higher stabilities and prolonged decay lifetimes (up to 249 μs) compared to the β-non-fluorinated counterparts. This renders the β-fluorinated Yb 3+ complexes as a new class of biological optical probes in both steady-state imaging and time-resolved fluorescence lifetime imaging (FLIM). NIR confocal fluorescence images showed strong and specific intracellular Yb 3+ luminescence signals when the biocompatible Yb 3+ complexes were uptaken into the living cells. Importantly, FLIM measurements showed an intracellular lifetime distribution between 100 and 200 μs, allowing an effective discrimination from cell autofluorescence, and afforded high signal-to-noise ratios as firstly demonstrated in the NIR region. These results demonstrated the prospects of NIR lanthanide complexes as biological probes for NIR steady-state fluorescence and time-resolved fluorescence lifetime imaging.

  3. Tissue oxygen monitoring by photoacoustic lifetime imaging (PALI) and its application to image-guided photodynamic therapy (PDT)

    NASA Astrophysics Data System (ADS)

    Shao, Qi; Morgounova, Ekaterina; Ashkenazi, Shai

    2015-03-01

    The oxygen partial pressure (pO2), which results from the balance between oxygen delivery and its consumption, is a key component of the physiological state of a tissue. Images of oxygen distribution can provide essential information for identifying hypoxic tissue and optimizing cancer treatment. Previously, we have reported a noninvasive in vivo imaging modality based on photoacoustic lifetime. The technique maps the excited triplet state of oxygen-sensitive dye, thus reflects the spatial and temporal distribution of tissue oxygen. We have applied PALI on tumor on small animals to identify hypoxia area. We also showed that PALI is able monitor changes of tissue oxygen, in an acute ischemia and breathing modulation model. Here we present our work on developing a treatment/imaging modality (PDT-PALI) that integrates PDT and a combined ultrasound/photoacoustic imaging system. The system provides real-time feedback of three essential parameters namely: tissue oxygen, light penetration in tumor location, and distribution of photosensitizer. Tissue oxygen imaging is performed by applying PALI, which relies on photoacoustic probing of oxygen-dependent, excitation lifetime of Methylene Blue (MB) photosensitizer. Lifetime information can also be used to generate image showing the distribution of photosensitizer. The level and penetration depth of PDT illumination can be deduced from photoacoustic imaging at the same wavelength. All images will be combined with ultrasound B-mode images for anatomical reference.

  4. Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II

    NASA Astrophysics Data System (ADS)

    Nemtseva, Elena V.; Lashchuk, Olesya O.; Gerasimova, Marina A.; Melnik, Tatiana N.; Nagibina, Galina S.; Melnik, Bogdan S.

    2018-01-01

    In most cases, intermediate states of multistage folding proteins are not ‘visible’ under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

  5. Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II.

    PubMed

    Nemtseva, Elena V; Lashchuk, Olesya O; Gerasimova, Marina A; Melnik, Tatiana N; Nagibina, Galina S; Melnik, Bogdan S

    2017-12-21

    In most cases, intermediate states of multistage folding proteins are not 'visible' under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

  6. Clinical application of indocyanine green (ICG) fluorescent imaging of hepatoblastoma.

    PubMed

    Yamamichi, Taku; Oue, Takaharu; Yonekura, Takeo; Owari, Mitsugu; Nakahata, Kengo; Umeda, Satoshi; Nara, Keigo; Ueno, Takehisa; Uehara, Shuichiro; Usui, Noriaki

    2015-05-01

    Although the usefulness of intraoperative indocyanine green (ICG) fluorescent imaging for the resection of hepatocellular carcinoma has been reported, its usefulness for the resection of hepatoblastoma remains unclear. This study clarifies the feasibility of intraoperative ICG fluorescent imaging for the resection of hepatoblastoma. In three hepatoblastoma patients, a primary tumor, recurrent tumor, and lung metastatic lesions were intraoperatively examined using a near-infrared fluorescence imaging system after the preoperative administration of ICG. ICG fluorescent imaging was useful for the surgical navigation in hepatoblastoma patients. In the first case, the primary hepatoblastoma exhibited intense fluorescence during right hepatectomy, but no fluorescence was detected in the residual liver. In the second case, a recurrent tumor exhibited fluorescence between the residual liver and diaphragm. A complete resection of the residual liver, with a partial resection of the diaphragm, followed by liver transplantation was performed. In the third case with multiple lung metastases, each metastatic lesion showed positive fluorescence, and all were completely resected. These fluorescence-positive lesions were pathologically proven to be viable hepatoblastoma cells. Intraoperative ICG fluorescence imaging for patients with hepatoblastoma was feasible and useful for identifying small viable lesions and confirming that no remnant tumor remained after resection. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Compact solid-state CMOS single-photon detector array for in vivo NIR fluorescence lifetime oncology measurements.

    PubMed

    Homulle, H A R; Powolny, F; Stegehuis, P L; Dijkstra, J; Li, D-U; Homicsko, K; Rimoldi, D; Muehlethaler, K; Prior, J O; Sinisi, R; Dubikovskaya, E; Charbon, E; Bruschini, C

    2016-05-01

    In near infrared fluorescence-guided surgical oncology, it is challenging to distinguish healthy from cancerous tissue. One promising research avenue consists in the analysis of the exogenous fluorophores' lifetime, which are however in the (sub-)nanosecond range. We have integrated a single-photon pixel array, based on standard CMOS SPADs (single-photon avalanche diodes), in a compact, time-gated measurement system, named FluoCam. In vivo measurements were carried out with indocyanine green (ICG)-modified derivatives targeting the αvβ 3 integrin, initially on a genetically engineered mouse model of melanoma injected with ICG conjugated with tetrameric cyclic pentapeptide (ICG-E[c(RGD f K)4]), then on mice carrying tumour xenografts of U87-MG (a human primary glioblastoma cell line) injected with monomeric ICG-c(RGD f K). Measurements on tumor, muscle and tail locations allowed us to demonstrate the feasibility of in vivo lifetime measurements with the FluoCam, to determine the characteristic lifetimes (around 500 ps) and subtle lifetime differences between bound and unbound ICG-modified fluorophores (10% level), as well as to estimate the available photon fluxes under realistic conditions.

  8. Temporal and spatial binning of TCSPC data to improve signal-to-noise ratio and imaging speed

    NASA Astrophysics Data System (ADS)

    Walsh, Alex J.; Beier, Hope T.

    2016-03-01

    Time-correlated single photon counting (TCSPC) is the most robust method for fluorescence lifetime imaging using laser scanning microscopes. However, TCSPC is inherently slow making it ineffective to capture rapid events due to the single photon product per laser pulse causing extensive acquisition time limitations and the requirement of low fluorescence emission efficiency to avoid bias of measurement towards short lifetimes. Furthermore, thousands of photons per pixel are required for traditional instrument response deconvolution and fluorescence lifetime exponential decay estimation. Instrument response deconvolution and fluorescence exponential decay estimation can be performed in several ways including iterative least squares minimization and Laguerre deconvolution. This paper compares the limitations and accuracy of these fluorescence decay analysis techniques to accurately estimate double exponential decays across many data characteristics including various lifetime values, lifetime component weights, signal-to-noise ratios, and number of photons detected. Furthermore, techniques to improve data fitting, including binning data temporally and spatially, are evaluated as methods to improve decay fits and reduce image acquisition time. Simulation results demonstrate that binning temporally to 36 or 42 time bins, improves accuracy of fits for low photon count data. Such a technique reduces the required number of photons for accurate component estimation if lifetime values are known, such as for commercial fluorescent dyes and FRET experiments, and improve imaging speed 10-fold.

  9. Hyperspectral imaging fluorescence excitation scanning for colon cancer detection

    PubMed Central

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

    2016-01-01

    Abstract. Optical spectroscopy and hyperspectral imaging have shown the potential to discriminate between cancerous and noncancerous tissue with high sensitivity and specificity. However, to date, these techniques have not been effectively translated to real-time endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents 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 goal of this study was to evaluate the initial feasibility of using fluorescence excitation-scanning hyperspectral imaging for measuring changes in fluorescence excitation spectrum concurrent with colonic adenocarcinoma using a small pre-pilot-scale sample size. Ex vivo analysis was performed using resected pairs of colorectal adenocarcinoma and normal mucosa. Adenocarcinoma was confirmed by histologic evaluation of hematoxylin and eosin (H&E) permanent sections. Specimens were imaged using a custom hyperspectral imaging fluorescence excitation-scanning microscope system. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation range of 390 to 450 nm that could be the basis for wavelength-dependent detection of colorectal cancers. Hence, excitation-scanning hyperspectral imaging may offer an alternative approach for discriminating adenocarcinoma from surrounding normal colonic mucosa, but further studies will be required to evaluate the accuracy of this approach using a larger patient cohort. PMID:27792808

  10. Hyperspectral imaging fluorescence excitation scanning for colon cancer detection

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

    Optical spectroscopy and hyperspectral imaging have shown the potential to discriminate between cancerous and noncancerous tissue with high sensitivity and specificity. However, to date, these techniques have not been effectively translated to real-time endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents 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 goal of this study was to evaluate the initial feasibility of using fluorescence excitation-scanning hyperspectral imaging for measuring changes in fluorescence excitation spectrum concurrent with colonic adenocarcinoma using a small pre-pilot-scale sample size. Ex vivo analysis was performed using resected pairs of colorectal adenocarcinoma and normal mucosa. Adenocarcinoma was confirmed by histologic evaluation of hematoxylin and eosin (H&E) permanent sections. Specimens were imaged using a custom hyperspectral imaging fluorescence excitation-scanning microscope system. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation range of 390 to 450 nm that could be the basis for wavelength-dependent detection of colorectal cancers. Hence, excitation-scanning hyperspectral imaging may offer an alternative approach for discriminating adenocarcinoma from surrounding normal colonic mucosa, but further studies will be required to evaluate the accuracy of this approach using a larger patient cohort.

  11. Highly sensitive strategy for Hg2+ detection in environmental water samples using long lifetime fluorescence quantum dots and gold nanoparticles.

    PubMed

    Huang, Dawei; Niu, Chenggang; Ruan, Min; Wang, Xiaoyu; Zeng, Guangming; Deng, Canhui

    2013-05-07

    The authors herein described a time-gated fluorescence resonance energy transfer (TGFRET) sensing strategy employing water-soluble long lifetime fluorescence quantum dots and gold nanoparticles to detect trace Hg(2+) ions in aqueous solution. The water-soluble long lifetime fluorescence quantum dots and gold nanoparticles were functionalized by two complementary ssDNA, except for four deliberately designed T-T mismatches. The quantum dot acted as the energy-transfer donor, and the gold nanoparticle acted as the energy-transfer acceptor. When Hg(2+) ions were present in the aqueous solution, DNA hybridization will occur because of the formation of T-Hg(2+)-T complexes. As a result, the quantum dots and gold nanoparticles are brought into close proximity, which made the energy transfer occur from quantum dots to gold nanoparticles, leading to the fluorescence intensity of quantum dots to decrease obviously. The decrement fluorescence intensity is proportional to the concentration of Hg(2+) ions. Under the optimum conditions, the sensing system exhibits the same liner range from 1 × 10(-9) to 1 × 10(-8) M for Hg(2+) ions, with the detection limits of 0.49 nM in buffer and 0.87 nM in tap water samples. This sensor was also used to detect Hg(2+) ions from samples of tap water, river water, and lake water spiked with Hg(2+) ions, and the results showed good agreement with the found values determined by an atomic fluorescence spectrometer. In comparison to some reported colorimetric and fluorescent sensors, the proposed method displays the advantage of higher sensitivity. The TGFRET sensor also exhibits excellent selectivity and can provide promising potential for Hg(2+) ion detection.

  12. Fluorescence multispectral imaging-based diagnostic system for atherosclerosis.

    PubMed

    Ho, Cassandra Su Lyn; Horiuchi, Toshikatsu; Taniguchi, Hiroaki; Umetsu, Araya; Hagisawa, Kohsuke; Iwaya, Keiichi; Nakai, Kanji; Azmi, Amalina; Zulaziz, Natasha; Azhim, Azran; Shinomiya, Nariyoshi; Morimoto, Yuji

    2016-08-20

    Composition of atherosclerotic arterial walls is rich in lipids such as cholesterol, unlike normal arterial walls. In this study, we aimed to utilize this difference to diagnose atherosclerosis via multispectral fluorescence imaging, which allows for identification of fluorescence originating from the substance in the arterial wall. The inner surface of extracted arteries (rabbit abdominal aorta, human coronary artery) was illuminated by 405 nm excitation light and multispectral fluorescence images were obtained. Pathological examination of human coronary artery samples were carried out and thickness of arteries were calculated by measuring combined media and intima thickness. The fluorescence spectra in atherosclerotic sites were different from those in normal sites. Multiple regions of interest (ROI) were selected within each sample and a ratio between two fluorescence intensity differences (where each intensity difference is calculated between an identifier wavelength and a base wavelength) from each ROI was determined, allowing for discrimination of atherosclerotic sites. Fluorescence intensity and thickness of artery were found to be significantly correlated. These results indicate that multispectral fluorescence imaging provides qualitative and quantitative evaluations of atherosclerosis and is therefore a viable method of diagnosing the disease.

  13. 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.

  14. Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

    PubMed

    Kim, Younghoon; Kim, Sung Hoon; Tanyeri, Melikhan; Katzenellenbogen, John A; Schroeder, Charles M

    2013-04-02

    Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. Fluorescence image excited by a scanning UV-LED light

    NASA Astrophysics Data System (ADS)

    Tsai, Hsin-Yi; Chen, Yi-Ju; Huang, Kuo-Cheng

    2013-03-01

    An optical scanning system using UV-LED light to induced fluorescence technology can enhance a fluorescence image significantly in a short period. It has several advantages such as lower power consumption, no scattering effect in skins, and multilayer images can be obtained to analyze skin disease. From the experiment results, the light intensity increases with increase spot size and decrease scanning speed, but the image resolution is oppositely. Moreover, the system could be widely used in clinical diagnosis and photodynamic therapy for skin disease because even the irradiated time of fluorescence substance is short but it will provide accurately positioning of fluorescence object.

  16. Self-interference fluorescence microscopy with three-phase detection for depth-resolved confocal epi-fluorescence imaging.

    PubMed

    Braaf, Boy; de Boer, Johannes F

    2017-03-20

    Three-dimensional confocal fluorescence imaging of in vivo tissues is challenging due to sample motion and limited imaging speeds. In this paper a novel method is therefore presented for scanning confocal epi-fluorescence microscopy with instantaneous depth-sensing based on self-interference fluorescence microscopy (SIFM). A tabletop epi-fluorescence SIFM setup was constructed with an annular phase plate in the emission path to create a spectral self-interference signal that is phase-dependent on the axial position of a fluorescent sample. A Mach-Zehnder interferometer based on a 3 × 3 fiber-coupler was developed for a sensitive phase analysis of the SIFM signal with three photon-counter detectors instead of a spectrometer. The Mach-Zehnder interferometer created three intensity signals that alternately oscillated as a function of the SIFM spectral phase and therefore encoded directly for the axial sample position. Controlled axial translation of fluorescent microsphere layers showed a linear dependence of the SIFM spectral phase with sample depth over axial image ranges of 500 µm and 80 µm (3.9 × Rayleigh range) for 4 × and 10 × microscope objectives respectively. In addition, SIFM was in good agreement with optical coherence tomography depth measurements on a sample with indocyanine green dye filled capillaries placed at multiple depths. High-resolution SIFM imaging applications are demonstrated for fluorescence angiography on a dye-filled capillary blood vessel phantom and for autofluorescence imaging on an ex vivo fly eye.

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

    PubMed Central

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

    2009-01-01

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

  18. Mid-Infrared Lifetime Imaging for Viability Evaluation of Lettuce Seeds Based on Time-Dependent Thermal Decay Characterization

    PubMed Central

    Kim, Ghiseok; Kim, Geon Hee; Ahn, Chi-Kook; Yoo, Yoonkyu; Cho, Byoung-Kwan

    2013-01-01

    An infrared lifetime thermal imaging technique for the measurement of lettuce seed viability was evaluated. Thermal emission signals from mid-infrared images of healthy seeds and seeds aged for 24, 48, and 72 h were obtained and reconstructed using regression analysis. The emission signals were fitted with a two-term exponential model that had two amplitudes and two time variables as lifetime parameters. The lifetime thermal decay parameters were significantly different for seeds with different aging times. Single-seed viability was visualized using thermal lifetime images constructed from the calculated lifetime parameter values. The time-dependent thermal signal decay characteristics, along with the decay amplitude and delay time images, can be used to distinguish aged lettuce seeds from normal seeds. PMID:23529120

  19. Small-Animal Imaging Using Diffuse Fluorescence Tomography.

    PubMed

    Davis, Scott C; Tichauer, Kenneth M

    2016-01-01

    Diffuse fluorescence tomography (DFT) has been developed to image the spatial distribution of fluorescence-tagged tracers in living tissue. This capability facilitates the recovery of any number of functional parameters, including enzymatic activity, receptor density, blood flow, and gene expression. However, deploying DFT effectively is complex and often requires years of know-how, especially for newer mutlimodal systems that combine DFT with conventional imaging systems. In this chapter, we step through the process of using MRI-DFT imaging of a receptor-targeted tracer in small animals.

  20. Anomalous fluorescence enhancement and fluorescence quenching of graphene quantum dots by single walled carbon nanotubes.

    PubMed

    Das, Ruma; Rajender, Gone; Giri, P K

    2018-02-07

    We explore the mechanism of the fluorescence enhancement and fluorescence quenching effect of single walled carbon nanotubes (SWCNTs) on highly fluorescent graphene quantum dots (GQDs) over a wide range of concentrations of SWCNTs. At very low concentrations of SWCNTs, the fluorescence intensity of the GQDs is enhanced, while at higher concentrations, systematic quenching of fluorescence is observed. The nature of the Stern-Volmer plot for the latter case was found to be non-linear indicating a combined effect of dynamic and static quenching. The contribution of the dynamic quenching component was assessed through the fluorescence lifetime measurements. The contribution of static quenching is confirmed from the red shift of the fluorescence spectra of the GQDs after addition of SWCNTs. The fluorescence intensity is first enhanced at very low concentration due to improved dispersion and higher absorption by GQDs, while at higher concentration, the fluorescence of GQDs is quenched due to the complex formation and associated reduction of the radiative sites of the GQDs, which is confirmed from time-resolved fluorescence measurements. Laser confocal microscopy imaging provides direct evidence of the enhancement and quenching of fluorescence at low and high concentrations of SWCNTs, respectively. This study provides an important insight into tuning the fluorescence of GQDs and understanding the interaction between GQDs and different CNTs, which is important for bio-imaging and drug delivery applications.

  1. 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.

  2. 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

  3. Image Restoration for Fluorescence Planar Imaging with Diffusion Model

    PubMed Central

    Gong, Yuzhu; Li, Yang

    2017-01-01

    Fluorescence planar imaging (FPI) is failure to capture high resolution images of deep fluorochromes due to photon diffusion. This paper presents an image restoration method to deal with this kind of blurring. The scheme of this method is conceived based on a reconstruction method in fluorescence molecular tomography (FMT) with diffusion model. A new unknown parameter is defined through introducing the first mean value theorem for definite integrals. System matrix converting this unknown parameter to the blurry image is constructed with the elements of depth conversion matrices related to a chosen plane named focal plane. Results of phantom and mouse experiments show that the proposed method is capable of reducing the blurring of FPI image caused by photon diffusion when the depth of focal plane is chosen within a proper interval around the true depth of fluorochrome. This method will be helpful to the estimation of the size of deep fluorochrome. PMID:29279843

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

    NASA Astrophysics Data System (ADS)

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

    2003-07-01

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

  5. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Time-resolved fluorescence microscopy to study biologically related applications using sol-gel derived and cellular media

    NASA Astrophysics Data System (ADS)

    Toury, Marion; Chandler, Lin; Allison, Archie; Campbell, David; McLoskey, David; Holmes-Smith, A. Sheila; Hungerford, Graham

    2011-03-01

    Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA Scientific DeltaDiode and DynaMyc).

  7. 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.

  8. Indocyanine green fluorescence imaging in hepatobiliary surgery.

    PubMed

    Majlesara, Ali; Golriz, Mohammad; Hafezi, Mohammadreza; Saffari, Arash; Stenau, Esther; Maier-Hein, Lena; Müller-Stich, Beat P; Mehrabi, Arianeb

    2017-03-01

    Indocyanine green (ICG) is a fluorescent dye that has been widely used for fluorescence imaging during hepatobiliary surgery. ICG is injected intravenously, selectively taken up by the liver, and then secreted into the bile. The catabolism and fluorescence properties of ICG permit a wide range of visualization methods in hepatobiliary surgery. We have characterized the applications of ICG during hepatobiliary surgery into: 1) liver mapping, 2) cholangiography, 3) tumor visualization, and 4) partial liver graft evaluation. In this literature review, we summarize the current understanding of ICG use during hepatobiliary surgery. Intra-operative ICG fluorescence imaging is a safe, simple, and feasible method that improves the visualization of hepatobiliary anatomy and liver tumors. Intravenous administration of ICG is not toxic and avoids the drawbacks of conventional imaging. In addition, it reduces post-operative complications without any known side effects. ICG fluorescence imaging provides a safe and reliable contrast for extra-hepatic cholangiography when detecting intra-hepatic bile leakage following liver resection. In addition, liver tumors can be visualized and well-differentiated hepatocellular carcinoma tumors can be accurately identified. Moreover, vascular reconstruction and outflow can be evaluated following partial liver transplantation. However, since tissue penetration is limited to 5-10mm, deeper tissue cannot be visualized using this method. Many instances of false positive or negative results have been reported, therefore further characterization is required. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. 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.

  10. Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 2. Multi-frequency phase and modulation analysis evidences a loosely connected PSII pigment-protein complex.

    PubMed

    Moise, Nicolae; Moya, Ismaël

    2004-06-28

    We report the first direct decomposition of the fluorescence lifetime heterogeneity during multiphasic fluorescence induction in dark-adapted leaves by multi-frequency phase and modulation fluorometry (PMF). A very fast component, assigned to photosystem I (PSI), was found to be constant in lifetime and yield, whereas the two slow components, which are strongly affected by the closure of the reaction centers by light, were assigned to PSII. Based on a modified "reversible radical pair" kinetic model with three compartments, we showed that a loosely connected pigment complex, which is assumed to be the CP47 complex, plays a specific role with respect to the structure and function of the PSII: (i) it explains the heterogeneity of PSII fluorescence lifetime as a compartmentation of excitation energy in the antenna, (ii) it is the site of a conformational change in the first second of illumination, and (iii) it is involved in the mechanisms of nonphotochemical quenching (NPQ). On the basis of the multi-frequency PMF analysis, we reconciled two apparently antagonistic aspects of chlorophyll a fluorescence in vivo: it is heterogeneous with respect to the kinetic structure (several lifetime components) and homogeneous with respect to average quantities (quasi-linear mean tau-Phi relationship).

  11. Mechanical Damage Detection of Indonesia Local Citrus Based on Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    Siregar, T. H.; Ahmad, U.; Sutrisno; Maddu, A.

    2018-05-01

    Citrus experienced physical damage in peel will produce essential oils that contain polymethoxylated flavone. Polymethoxylated flavone is fluorescence substance; thus can be detected by fluorescence imaging. This study aims to study the fluorescence spectra characteristic and to determine the damage region in citrus peel based on fluorescence image. Pulung citrus from Batu district, East Java, as a famous citrus production area in Indonesia, was used in the experiment. It was observed that the image processing could detect the mechanical damage region. Fluorescence imaging can be used to classify the citrus into two categories, sound and defect citruses.

  12. 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.

  13. FastFLIM, the all-in-one engine for measuring photoluminescence lifetime of 100 picoseconds to 100 milliseconds

    NASA Astrophysics Data System (ADS)

    Sun, Yuansheng; Coskun, Ulas; Liao, Shih-Chu Jeff; Barbieri, Beniamino

    2018-02-01

    Photoluminescence (PL) refers to light emission initiated by any form of photon excitation. PL spectroscopy and microscopy imaging has been widely applied in material, chemical and life sciences. Measuring its lifetime yields a new dimension of the PL imaging and opens new opportunities for many PL applications. In solar cell research, quantification of the PL lifetime has become an important evaluation for the characteristics of the Perovskite thin film. Depending upon the PL process (fluorescence, phosphorescence, photon upconversion, etc.), the PL lifetimes to be measured can vary in a wide timescale range (e.g. from sub-nanoseconds to microseconds or even milliseconds) - it is challenging to cover this wide range of lifetime measurements by a single technique efficiently. Here, we present a novel digital frequency domain (DFD) technique named FastFLIM, capable of measuring the PL lifetime from 100 ps to 100 ms at the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear leastsquare fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the PL lifetime microscopy imaging.

  14. Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.

    PubMed

    Hontani, Yusaku; Shcherbakova, Daria M; Baloban, Mikhail; Zhu, Jingyi; Verkhusha, Vladislav V; Kennis, John T M

    2016-11-18

    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.

  15. 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

  16. Fluorescence Lifetime and UV-Vis Spectroscopy to Evaluate the Interactions Between Quercetin and Its Yeast Microcapsule.

    PubMed

    Pham-Hoang, Bao-Ngoc; Winckler, Pascale; Waché, Yves

    2018-01-01

    Quercetin is a fragile bioactive compound. Several works have tried to preserve it by encapsulation but the form of encapsulation (mono- or supra-molecular structure, tautomeric form), though important for stability and bioavailability, remains unknown. The present work aims at developing a fluorescence lifetime technique to evaluate the structure of quercetin during encapsulation in a vector capsule that has already proven efficiency, yeast cells. Molecular stabilization was observed during a 4-month storage period. The time-correlated single-photon counting (TCSPC) technique was used to evaluate the interaction between quercetin molecules and the yeast capsule. The various tautomeric forms, as identified by UV-Vis spectroscopy, result in various lifetimes in TCSPC, although they varied also with the buffer environment. Quercetin in buffer exhibited a three-to-four longer long-time after 24 h (changing from 6-7 to 18-23 ns), suggesting an aggregation of molecules. In yeast microcapsules, the long-time population exhibited a longer lifetime (around 27 ns) from the beginning and concerned about 20% of molecules compared to dispersed quercetin. This shows that lifetime analysis can show the monomolecular instability of quercetin in buffer and the presence of interactions between quercetin molecules and their microcapsules. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. [Development of a near-infrared fluorescence imaging system based on fluorescence properties of methylene blue].

    PubMed

    Huang, Lu-Mao; DU, Pei-Yan; Chen, Lan; Zhang, Sa; Zhou, Di-Fu; Chen, Chun-Lin; Xin, Xue-Gang

    2018-04-20

    To develop a near-infrared fluorescence imaging system based on the fluorescence properties of methylene blue. According to the optical properties of methylene blue, we used a custom-made specific LED light source and an interference filter, a CCD camera and other relevant components to construct the near-infrared fluorescence imaging system. We tested the signal-to-background ratio (SBR) of this imaging system for detecting methylene blue under different experimental conditions and analyzed the SBR in urine samples collected from 15 Wistar rats with intravenous injection of methylene blue at the doses of 0, 1.4, 1.6, 1.8, or 2.0 0 mg/kg methylene blue. The SBR of this imaging system for detecting methylene blue was affected by the concentration of methylene blue and the distance from the sample (P<0.05). In the urine samples from Wistar rats, the SBR varied with the the injection dose, and the rats injected with 1.6 mg/kg methylene blue showed the highest SBR (8.71∓0.20) in the urine (P<0.05). This near-infrared fluorescence imaging system is useful for fluorescence detection of methylene blue and can be used for real-time recognition of ureters during abdominal surgery.

  18. 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

  19. Carbon dots in zeolites: A new class of thermally activated delayed fluorescence materials with ultralong lifetimes

    PubMed Central

    Liu, Jiancong; Wang, Ning; Yu, Yue; Yan, Yan; Zhang, Hongyue; Li, Jiyang; Yu, Jihong

    2017-01-01

    Thermally activated delayed fluorescence (TADF) materials are inspiring intensive research in optoelectronic applications. To date, most of the TADF materials are limited to metal-organic complexes and organic molecules with lifetimes of several microseconds/milliseconds that are sensitive to oxygen. We report a facial and general “dots-in-zeolites” strategy to in situ confine carbon dots (CDs) in zeolitic matrices during hydrothermal/solvothermal crystallization to generate high-efficient TADF materials with ultralong lifetimes. The resultant CDs@zeolite composites exhibit high quantum yields up to 52.14% and ultralong lifetimes up to 350 ms at ambient temperature and atmosphere. This intriguing TADF phenomenon is due to the fact that nanoconfined space of zeolites can efficiently stabilize the triplet states of CDs, thus enabling the reverse intersystem crossing process for TADF. Meanwhile, zeolite frameworks can also hinder oxygen quenching to present TADF behavior at air atmosphere. This design concept introduces a new perspective to develop materials with unique TADF performance and various novel delayed fluorescence–based applications. PMID:28560347

  20. Quantum dots versus organic fluorophores in fluorescent deep-tissue imaging--merits and demerits.

    PubMed

    Bakalova, Rumiana; Zhelev, Zhivko; Gadjeva, Veselina

    2008-12-01

    The use of fluorescence in deep-tissue imaging is rapidly expanding in last several years. The progress in fluorescent molecular probes and fluorescent imaging techniques gives an opportunity to detect single cells and even molecular targets in live organisms. The highly sensitive and high-speed fluorescent molecular sensors and detection devices allow the application of fluorescence in functional imaging. With the development of novel bright fluorophores based on nanotechnologies and 3D fluorescence scanners with high spatial and temporal resolution, the fluorescent imaging has a potential to become an alternative of the other non-invasive imaging techniques as magnetic resonance imaging, positron-emission tomography, X-ray, computing tomography. The fluorescent imaging has also a potential to give a real map of human anatomy and physiology. The current review outlines the advantages of fluorescent nanoparticles over conventional organic dyes in deep-tissue imaging in vivo and defines the major requirements to the "perfect fluorophore". The analysis proceeds from the basic principles of fluorescence and major characteristics of fluorophores, light-tissue interactions, and major limitations of fluorescent deep-tissue imaging. The article is addressed to a broad readership - from specialists in this field to university students.

  1. Long lifetime generation IV image intensifiers with unfilmed microchannel plate

    NASA Astrophysics Data System (ADS)

    Estrera, Joseph P.; Bender, Edward J.; Giordana, A.; Glesener, John W.; Iosue, Mike J.; Lin, P. P.; Sinor, Timothy W.

    2000-11-01

    Current Generation II Gallium Arsenide (GaAs) image intensifier tube technology requires that the tube microchannel plate (MCP) component have a thin dielectric coating on the side facing the tube's photocathode component. This protective coating substantially reduces the release from the MCP of ions and neutral species, particularly when the image intensifier is operated. The prevention of MCP outgassing is necessary in order ot prevent the poisoning of the Cs:O surface on the GaAs photocathode. Many authors have experimented with omitting the MCP coating. Such experiments have consistently led to an intensifier with a significantly reduced lifetime, due to contamination of the Cs:O layer on the photocathode. Unfortunately the MCP film acts as a scattering cneter to electron transport within the intensifier and effectively reduces the photoelectron detection efficiency. Substantial enhancement of the image intensifier operating parameters is the motivation for the removal of the MCP film. Removal of the MCP film promises to simplify MCP fabrication and enhance the intensifier parameters related to Electro-Optical performance and image quality. This paper presents results showing for the first time that it is possible to fabricate a long lifetime image intensifier with a single unfilmed MCP and achieve improved imaging and performance characteristics.

  2. 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

  3. Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

    PubMed Central

    Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

    2012-01-01

    There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

  4. Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

    NASA Astrophysics Data System (ADS)

    Horilova, Julia; Cunderlikova, Beata; Marcek Chorvatova, Alzbeta

    2015-05-01

    Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

  5. Applications of two-photon fluorescence microscopy in deep-tissue imaging

    NASA Astrophysics Data System (ADS)

    Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.

    2000-07-01

    Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

  6. A low-cost method for visible fluorescence imaging.

    PubMed

    Tarver, Crissy L; Pusey, Marc

    2017-12-01

    A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

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

    NASA Astrophysics Data System (ADS)

    McWade, Melanie A.

    2016-03-01

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

  8. Temporal binning of time-correlated single photon counting data improves exponential decay fits and imaging speed

    PubMed Central

    Walsh, Alex J.; Sharick, Joe T.; Skala, Melissa C.; Beier, Hope T.

    2016-01-01

    Time-correlated single photon counting (TCSPC) enables acquisition of fluorescence lifetime decays with high temporal resolution within the fluorescence decay. However, many thousands of photons per pixel are required for accurate lifetime decay curve representation, instrument response deconvolution, and lifetime estimation, particularly for two-component lifetimes. TCSPC imaging speed is inherently limited due to the single photon per laser pulse nature and low fluorescence event efficiencies (<10%) required to reduce bias towards short lifetimes. Here, simulated fluorescence lifetime decays are analyzed by SPCImage and SLIM Curve software to determine the limiting lifetime parameters and photon requirements of fluorescence lifetime decays that can be accurately fit. Data analysis techniques to improve fitting accuracy for low photon count data were evaluated. Temporal binning of the decays from 256 time bins to 42 time bins significantly (p<0.0001) improved fit accuracy in SPCImage and enabled accurate fits with low photon counts (as low as 700 photons/decay), a 6-fold reduction in required photons and therefore improvement in imaging speed. Additionally, reducing the number of free parameters in the fitting algorithm by fixing the lifetimes to known values significantly reduced the lifetime component error from 27.3% to 3.2% in SPCImage (p<0.0001) and from 50.6% to 4.2% in SLIM Curve (p<0.0001). Analysis of nicotinamide adenine dinucleotide–lactate dehydrogenase (NADH-LDH) solutions confirmed temporal binning of TCSPC data and a reduced number of free parameters improves exponential decay fit accuracy in SPCImage. Altogether, temporal binning (in SPCImage) and reduced free parameters are data analysis techniques that enable accurate lifetime estimation from low photon count data and enable TCSPC imaging speeds up to 6x and 300x faster, respectively, than traditional TCSPC analysis. PMID:27446663

  9. Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

    PubMed Central

    Scolaro, Loretta; Lorenser, Dirk; Madore, Wendy-Julie; Kirk, Rodney W.; Kramer, Anne S.; Yeoh, George C.; Godbout, Nicolas; Sampson, David D.; Boudoux, Caroline; McLaughlin, Robert A.

    2015-01-01

    Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue. PMID:26137379

  10. Novel techniques with multiphoton microscopy: Deep-brain imaging with microprisms, neurometabolism of epilepsy, and counterfeit paper money detection

    NASA Astrophysics Data System (ADS)

    Chia, Thomas H.

    Multiphoton microscopy is a laser-scanning fluorescence imaging method with extraordinary potential. We describe three innovative multiphoton microscopy techniques across various disciplines. Traditional in vivo fluorescence microscopy of the mammalian brain has a limited penetration depth (<400 microm). We present a method of imaging 1 mm deep into mouse neocortex by using a glass microprism to relay the excitation and emission light. This technique enables simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution was sufficient to resolve dendritic spines on layer V GFP neurons. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. Multiphoton fluorescence lifetime imaging (FLIM) of NADH reveals information on neurometabolism. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic coenzyme, has a lifetime dependent on enzymatic binding. A novel NADH FLIM algorithm is presented that produces images showing spatially distinct NADH fluorescence lifetimes in mammalian brain slices. This program provides advantages over traditional FLIM processing of multi-component lifetime data. We applied this technique to a GFP-GFAP pilocarpine mouse model of temporal lobe epilepsy. Results indicated significant changes in the neurometabolism of astrocytes and neuropil in the cell and dendritic layers of the hippocampus when compared to control tissue. Data obtained with NADH FLIM were subsequently interpreted based on the abnormal activity reported in epileptic tissue. Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. This allows for detection of counterfeit paper money because of its significant differences in fluorescence lifetime when compared to genuine paper money. We used

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

    PubMed

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

    2015-11-07

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

  12. Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

    PubMed

    Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

    2012-10-17

    There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Fluorescence lifetimes of anthracycline drugs in phospholipid bilayers determined by frequency-domain fluorometry

    NASA Astrophysics Data System (ADS)

    Burke, Thomas G.; Malak, Henryk M.; Doroshow, James H.

    1990-05-01

    Time-resolved fluorescence intensity decay data from anthracycline anticancer drugs present in model membranes were obtained using a gigahertz frequency-domain fluorometer [Lakowicz et al. (1986) Rev. Sci. Instrum. 57, 2499-2506]. Exciting light of 290 nm, modulated at multiple frequencies from 8 MHz to 400 MHz, was used to study the interactions of Adriamycin, daunomycin and related antibiotics with small unilamellar vesicles composed of dimyristoylphosphatidylcholine (DMPC) at 28°C. Fluorescence decay data for drug molecules free in solution as well as bound to membranes were best fit by exponentials requiring two terms rather than by single exponential decays. For example, one-component analysis of the decay data for Adriamycin free in phosphate buffered saline (PBS) solution resulted in a reduced x2 value of 140 ((tau) = 0.88 ns), while a two-component fit resulted in a substantially smaller reduced x2 value of 2.6 ((tau)1 = 1.13 ns, (alpha)1 = 0.60, (tau)2 = 0.30 ns). Upon association with membranes, each of the anthracyclines studied displayed a larger r1 value while the r2 value remained the same or increased (for example, DMPC-bound Adriamycin showed r1 = 1.68 ns , a1 = 0 . 64 , r2 = 0 . 33 ns) . Analyses of the fluorescence emission decays of anthracyclines were also made assuming each decay is composed of a single Lorentzian distribution of lifetimes. Data taken on Adriamycin in PBS, when fit using one continuous component, displayed (tau), (alpha), w, and reduced x2 values of 0.68 ns, 1, 0.60 ns, and 9.1, respectively. The distribution became quite broad upon drug association with membrane (DMPCbound Adriamycin: (tau) = 0.75 ns, (alpha) = 1, w = 2.24 ns, x2 = 13). For each anthracycline studied, continuous component fits showed significant broadening in the distributions upon drug association with membrane. Relatively large shifts in lifetime values were observed for the carminomycin and 4-demethoxydaunomycin analogues upon binding model lipid membranes

  14. Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

    PubMed Central

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

    2013-01-01

    Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

  15. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

  17. Photon counting phosphorescence lifetime imaging with TimepixCam

    DOE PAGES

    Hirvonen, Liisa M.; Fisher-Levine, Merlin; Suhling, Klaus; ...

    2017-01-12

    TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window, and read out by a Timepix ASIC. The 256 x 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting (TCSPC) imaging. We have characterised the photon detection capabilities of this detector system, and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200more » μm diameter polystyrene beads.« less

  18. Photon counting phosphorescence lifetime imaging with TimepixCam.

    PubMed

    Hirvonen, Liisa M; Fisher-Levine, Merlin; Suhling, Klaus; Nomerotski, Andrei

    2017-01-01

    TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window and read out by a Timepix Application Specific Integrated Circuit. The 256 × 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting imaging. We have characterised the photon detection capabilities of this detector system and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200 μm diameter polystyrene beads.

  19. Photon counting phosphorescence lifetime imaging with TimepixCam

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hirvonen, Liisa M.; Fisher-Levine, Merlin; Suhling, Klaus

    TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window, and read out by a Timepix ASIC. The 256 x 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting (TCSPC) imaging. We have characterised the photon detection capabilities of this detector system, and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200more » μm diameter polystyrene beads.« less

  20. Photon counting phosphorescence lifetime imaging with TimepixCam

    NASA Astrophysics Data System (ADS)

    Hirvonen, Liisa M.; Fisher-Levine, Merlin; Suhling, Klaus; Nomerotski, Andrei

    2017-01-01

    TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window and read out by a Timepix Application Specific Integrated Circuit. The 256 × 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting imaging. We have characterised the photon detection capabilities of this detector system and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200 μm diameter polystyrene beads.

  1. Single Cell Fluorescence Imaging Using Metal Plasmon-Coupled Probe

    PubMed Central

    Zhang, Jian; Fu, Yi; Lakowicz, Joseph R.

    2009-01-01

    This work constitutes the first fluorescent imaging of cells using metal plasmon-coupled probes (PCPs) at single cell resolution. N-(2-Mercapto-propionyl)glycine-coated silver nanoparticles were synthesized by reduction of silver nitrate using sodium borohyride and then succinimidylated via ligand exchange. Alexa Fluor 647-labeled concanavalin A (con A) was chemically bound to the silver particles to make the fluorescent metal plasmon-coupled probes. The fluorescence images were collected using a scanning confocal microscopy. The fluorescence intensity was observed to enhance 7-fold when binding the labeled con A on a single silver particle. PCPs were conjugated on HEK 293 A cells. Imaging results demonstrate that cells labeled by PCPs were 20-fold brighter than those by free labeled con A. PMID:17375898

  2. 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.

  3. Non-invasive Photoacoustic and Fluorescence Sentinel Lymph Node Identification using Dye-loaded Perfluorocarbon Nanoparticles

    PubMed Central

    Akers, Walter J.; Kim, Chulhong; Berezin, Mikhail; Guo, Kevin; Fuhrhop, Ralph; Lanza, Gregory M.; Fischer, Georg M.; Daltrozzo, Ewald; Zumbusch, Andreas; Cai, Xin; Wang, Lihong V.; Achilefu, Samuel

    2010-01-01

    The contrast mechanisms used for photoacoustic tomography (PAT) and fluorescence imaging differ in subtle but significant ways. Design of contrast agents for each or both modalities requires an understanding of the spectral characteristics as well as intra- and intermolecular interactions that occur during formulation. We found that fluorescence quenching that occurs in the formulation of near infrared (NIR) fluorescent dyes in nanoparticles results in enhanced contrast for PAT. The ability of the new PAT method to utilize strongly absorbing chromophores for signal generation allowed us to convert a highly fluorescent dye into an exceptionally high PA contrast material. Spectroscopic characterization of the developed NIR dye-loaded perfluorocarbon-based nanoparticles for combined fluorescence and PA imaging revealed distinct dye-dependent photophysical behavior. We demonstrate that the enhanced contrast allows detection of regional lymph nodes of rats in vivo with time-domain optical and photoacoustic imaging methods. The results further show that the use of fluorescence lifetime (FLT) imaging, which is less dependent on fluorescence intensity, provides a strategic approach to bridge the disparate contrast reporting mechanisms of fluorescence and PA imaging methods. PMID:21171567

  4. Intravital phosphorescence lifetime imaging of the renal cortex accurately measures renal hypoxia.

    PubMed

    Hirakawa, Yosuke; Mizukami, Kiichi; Yoshihara, Toshitada; Takahashi, Ippei; Khulan, Purevsuren; Honda, Tomoko; Mimura, Imari; Tanaka, Tetsuhiro; Tobita, Seiji; Nangaku, Masaomi

    2018-06-01

    Renal tubulointerstitial hypoxia is recognized as a final common pathway of chronic kidney disease and is considered a promising drug target. However, hypoxia in the tubules is not well examined because of limited detection methods. Here, we devised a method to visualize renal tubular oxygen tension with spatial resolution at a cellular level using the cell-penetrating phosphorescent probe, BTPDM1 (an iridium-based cationic lipophilic dye), and confocal phosphorescence lifetime imaging microscopy to precisely assess renal hypoxia. Imaging with BTPDM1 revealed an oxygen gradient between S1 and S2 segments in mouse kidney. We also demonstrated that our microscopy system can detect subtle changes of hypoxemia and reoxygenation, and the acquired phosphorescence lifetime can be converted to partial pressure of oxygen. This new method allows, for the first time, visualization of intravital oxygen gradients at the renal surface with high spatial resolution. Thus, the confocal phosphorescence lifetime imaging microscopy platform, combined with BTPDM1, will promote an accurate understanding of tissue hypoxia, including renal hypoxia. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  5. 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

  6. Optofluidic fluorescent imaging cytometry on a cell phone.

    PubMed

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

    2011-09-01

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

  7. 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.

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

    PubMed

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

    2014-11-07

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

  9. Preparation and characterization of alginate based-fluorescent magnetic nanoparticles for fluorescence/magnetic resonance multimodal imaging applications

    NASA Astrophysics Data System (ADS)

    Kwon, Yong-Su; Choi, Kee-Bong; Lim, Hyungjun; Lee, Sunghwi; Lee, Jae-Jong

    2018-06-01

    Simple and versatile methodologies have been reported that customize the surface of superparamagnetic iron oxide (SPIO) nanoparticles and impart additional fluorescence capabilities to these contrast agents. Herein, we present the rational design, synthesis, characterization, and biological applications of a new magnetic-based fluorescent probe. The dual modality imaging protocol was developed by labeling fluorophore with alginate natural polymers that have excellent biocompatibility and biodegradability, and using gelification method to form nanocomposites containing SPIO. The formation of alginate-based fluorescent magnetic (AFM) nanoparticles was observed in spherical and elliptical forms with a diameter of less than 500 nm by a transmission electron microscope (TEM). The fluorescent wavelength band in the range of 560 nm was also confirmed in the UV–visible spectrophotometer. In this study, we demonstrate that the multi-tasking design of AFM nanoparticles provides an ideal platform for building balanced dual-image probes of magnetic resonance imaging and optical imaging.

  10. Genetically encoded sensors and fluorescence microscopy for anticancer research

    NASA Astrophysics Data System (ADS)

    Zagaynova, Elena V.; Shirmanova, Marina V.; Sergeeva, Tatiana F.; Klementieva, Natalia V.; Mishin, Alexander S.; Gavrina, Alena I.; Zlobovskay, Olga A.; Furman, Olga E.; Dudenkova, Varvara V.; Perelman, Gregory S.; Lukina, Maria M.; Lukyanov, Konstantin A.

    2017-02-01

    Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

  11. AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

    PubMed

    Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J

    2015-04-01

    A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

  12. 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

  13. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  15. "siRNA traffic lights": arabino-configured 2'-anchors for fluorescent dyes are key for dual color readout in cell imaging.

    PubMed

    Steinmeyer, Jeannine; Walter, Heidi-Kristin; Bichelberger, Mathilde A; Schneider, Violetta; Kubař, Tomáš; Rönicke, Franziska; Olshausen, Bettina; Nienhaus, Karin; Nienhaus, Gerd Ulrich; Schepers, Ute; Elstner, Marcus; Wagenknecht, Hans-Achim

    2018-05-23

    Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.

  16. A multi-analytical investigation of semi-conductor pigments with time-resolved spectroscopy and imaging

    NASA Astrophysics Data System (ADS)

    Nevin, A.; Cesaratto, A.; D'Andrea, C.; Valentini, Gianluca; Comelli, D.

    2013-05-01

    We present the non-invasive study of historical and modern Zn- and Cd-based pigments with time-resolved fluorescence spectroscopy, fluorescence multispectral imaging and fluorescence lifetime imaging (FLIM). Zinc oxide and Zinc sulphide are semiconductors which have been used as white pigments in paintings, and the luminescence of these pigments from trapped states is strongly dependent on the presence of impurities and crystal defects. Cadmium sulphoselenide pigments vary in hue from yellow to deep red based on their composition, and are another class of semiconductor pigments which emit both in the visible and the near infrared. The Fluorescence lifetime of historical and modern pigments has been measured using both an Optical Multichannel Analyser (OMA) coupled with a Nd:YAG nslaser, and a streak camera coupled with a ps-laser for spectrally-resolved fluorescence lifetime measurements. For Znbased pigments we have also employed Fluorescence Lifetime Imaging (FLIM) for the measurement of luminescence. A case study of FLIM applied to the analysis of the painting by Vincent Van Gogh on paper - "Les Bretonnes et le pardon de Pont-Aven" (1888) is presented. Through the integration of complementary, portable and non-invasive spectroscopic techniques, new insights into the optical properties of Zn- and Cd-based pigments have been gained which will inform future analysis of late 19th] and early 20th C. paintings.

  17. Proton-induced x-ray fluorescence CT imaging

    PubMed Central

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

    2015-01-01

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

  18. Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy

    PubMed Central

    Barretto, Robert P. J.; Ko, Tony H.; Jung, Juergen C.; Wang, Tammy J.; Capps, George; Waters, Allison C.; Ziv, Yaniv; Attardo, Alessio; Recht, Lawrence; Schnitzer, Mark J.

    2013-01-01

    The combination of intravital microscopy and animal models of disease has propelled studies of disease mechanisms and treatments. However, many disorders afflict tissues inaccessible to light microscopy in live subjects. Here we introduce cellular-level time-lapse imaging deep within the live mammalian brain by one- and two-photon fluorescence microendoscopy over multiple weeks. Bilateral imaging sites allowed longitudinal comparisons within individual subjects, including of normal and diseased tissues. Using this approach we tracked CA1 hippocampal pyramidal neuron dendrites in adult mice, revealing these dendrites' extreme stability (>8,000 day mean lifetime) and rare examples of their structural alterations. To illustrate disease studies, we tracked deep lying gliomas by observing tumor growth, visualizing three-dimensional vasculature structure, and determining microcirculatory speeds. Average erythrocyte speeds in gliomas declined markedly as the disease advanced, notwithstanding significant increases in capillary diameters. Time-lapse microendoscopy will be applicable to studies of numerous disorders, including neurovascular, neurological, cancerous, and trauma-induced conditions. PMID:21240263

  19. New solutions for standardization, monitoring and quality management of fluorescence-based imaging systems (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Royon, Arnaud; Papon, Gautier

    2016-03-01

    Fluorescence microscopes have become ubiquitous in life sciences laboratories, including those focused on pharmaceuticals, diagnosis, and forensics. For the past few years, the need for both performance guarantees and quantifiable results has driven development in this area. However, the lack of appropriate standards and reference materials makes it difficult or impossible to compare the results of two fluorescence microscopes, or to measure performance fluctuations of one microscope over time. Therefore, the operation of fluorescence microscopes is not monitored as often as their use warrants - an issue that is recognized by both systems manufacturers and national metrology institutes. We have developed a new process that enables the etching of long-term stable fluorescent patterns with sub-micrometer sizes in three dimensions inside glass. In this paper, we present, based on this new process, a fluorescent multi-dimensional ruler and a dedicated software that are suitable for monitoring and quality management of fluorescence-based imaging systems (wide-field, confocal, multiphoton, high content machines). In addition to fluorescence, the same patterns exhibit bright- and dark-field contrast, DIC, and phase contrast, which make them also relevant to monitor these types of microscopes. Non-exhaustively, this new solution enables the measurement of: The stage repositioning accuracy; The illumination and detection homogeneities; The field flatness; The detectors' characteristics; The lateral and axial spatial resolutions; The spectral response (spectrum, intensity and lifetime) of the system. Thanks to the stability of the patterns, microscope performance assessment can be carried out as well in a daily basis as in the long term.

  20. Development of ultrasound-assisted fluorescence imaging of indocyanine green.

    PubMed

    Morikawa, Hiroyasu; Toyota, Shin; Wada, Kenji; Uchida-Kobayashi, Sawako; Kawada, Norifumi; Horinaka, Hiromichi

    2017-01-01

    Indocyanine green (ICG) accumulation in hepatocellular carcinoma means tumors can be located by fluorescence. However, because of light scattering, it is difficult to detect ICG fluorescence from outside the body. We propose a new fluorescence imaging method that detects changes in the intensity of ICG fluorescence by ultrasound-induced temperature changes. ICG fluorescence intensity decreases as the temperature rises. Therefore, it should theoretically be possible to detect tissue distribution of ICG using ultrasound to heat tissue, moving the point of ultrasound transmission, and monitoring changes in fluorescence intensity. A new probe was adapted for clinical application. It consisted of excitation light from a laser, fluorescence sensing through a light pipe, and heating by ultrasound. We applied the probe to bovine liver to image the accumulation of ICG. ICG emits fluorescence (820 nm) upon light irradiation (783 nm). With a rise in temperature, the fluorescence intensity of ICG decreased by 0.85 %/°C. The distribution of fluorescent ICG was detected using an ultrasonic warming method in a new integrated probe. Modulating fluorescence by changing the temperature using ultrasound can determine where ICG accumulates at a depth, highlighting its potential as a means to locate hepatocellular carcinoma.

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

    PubMed

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

    2016-09-01

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

  2. Catheter-based time-gated near-infrared fluorescence/OCT imaging system

    NASA Astrophysics Data System (ADS)

    Lu, Yuankang; Abran, Maxime; Cloutier, Guy; Lesage, Frédéric

    2018-02-01

    We developed a new dual-modality intravascular imaging system based on fast time-gated fluorescence intensity imaging and spectral domain optical coherence tomography (SD-OCT) for the purpose of interventional detection of atherosclerosis. A pulsed supercontinuum laser was used for fluorescence and OCT imaging. A double-clad fiber (DCF)- based side-firing catheter was designed and fabricated to have a 23 μm spot size at a 2.2 mm working distance for OCT imaging. Its single-mode core is used for OCT, while its inner cladding transports fluorescence excitation light and collects fluorescent photons. The combination of OCT and fluorescence imaging was achieved by using a DCF coupler. For fluorescence detection, we used a time-gated technique with a novel single-photon avalanche diode (SPAD) working in an ultra-fast gating mode. A custom-made delay chip was integrated in the system to adjust the delay between the excitation laser pulse and the SPAD gate-ON window. This technique allowed to detect fluorescent photons of interest while rejecting most of the background photons, thus leading to a significantly improved signal to noise ratio (SNR). Experiments were carried out in turbid media mimicking tissue with an indocyanine green (ICG) inclusion (1 mM and 100 μM) to compare the time-gated technique and the conventional continuous detection technique. The gating technique increased twofold depth sensitivity, and tenfold SNR at large distances. The dual-modality imaging capacity of our system was also validated with a silicone-based tissue-mimicking phantom.

  3. Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates.

    PubMed

    Cordina, Nicole M; Sayyadi, Nima; Parker, Lindsay M; Everest-Dass, Arun; Brown, Louise J; Packer, Nicolle H

    2018-03-14

    Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

  4. KrF laser-induced OH fluorescence imaging in a supersonic combustion tunnel

    NASA Technical Reports Server (NTRS)

    Quagliaroli, T. M.; Laufer, G.; Hollo, S. D.; Krauss, R. H.; Whitehurst, R. B., III; Mcdaniel, J. C., Jr.

    1992-01-01

    Planar fluorescence images of OH in a continuous-flow, electrical-resistively heated, high enthalpy, hydrogen-air combustion tunnel, induced by a tunable KrF laser, were recorded. These images were compared to previously recorded fluorescence images induced by a doubled-dye laser under similar conditions. Images induced by the doubled-dye laser system demonstrated a severe distortion caused by absorption and fluorescence trapping. By contrast, images of the fluorescence induced by the tunable KrF laser retained the symmetry properties of the flow. Based on signal-to-noise ratio measurements the yield of the fluorescence induced by the doubled-dye laser is larger than the fluorescence yield induced by the KrF laser. The measurements in the present facility of OH fluorescence induced by the KrF laser were limited by the photon-statistical noise. Based 2 on this result, doubled-dye laser systems are recommended for OH imaging in small and OH lean (less than 10 exp 15/cu cm) facilities. KrF lasers should be selected otherwise.

  5. Sprayable enzyme-activatable fluorescent probes: kinetic mapping using dynamic fluorescence imaging can help detecting tiny cancer foci (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kobayashi, Hisataka

    2017-02-01

    Optical fluorescence-guided imaging is increasingly used to guide surgery and endoscopic procedures. Sprayable enzyme-activatable probes are particularly useful because of high target-to-background ratios that increase sensitivity for tiny cancer foci. However, green fluorescent activatable probes suffers from interference from autofluorescence found in biological tissue. Dynamic imaging followed by the kinetic analysis could be detected local enzyme activity and used to differentiate specific fluorescence arising from an activated probe in a tumor from autofluorescence in background tissues especially when low concentrations of the dye are applied to detect tiny cancer foci. Serial fluorescence imaging was performed using various concentrations of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) which was sprayed on the peritoneal surface with tiny implants of SHIN3-dsRed ovarian cancer tumors. Temporal differences in signal between specific green fluorescence in cancer foci and non-specific autofluorescence in background tissue was measured and processed into three kinetic maps reflecting maximum fluorescence signal (MF), wash-in rate (WIR), and area under the curve (AUC), respectively. Especially at lower concentrations, kinetic maps derived from dynamic fluorescence imaging were clearly superior to unprocessed images for detection small cancer foci.

  6. Fluorescence decay data analysis correcting for detector pulse pile-up at very high count rates

    NASA Astrophysics Data System (ADS)

    Patting, Matthias; Reisch, Paja; Sackrow, Marcus; Dowler, Rhys; Koenig, Marcelle; Wahl, Michael

    2018-03-01

    Using time-correlated single photon counting for the purpose of fluorescence lifetime measurements is usually limited in speed due to pile-up. With modern instrumentation, this limitation can be lifted significantly, but some artifacts due to frequent merging of closely spaced detector pulses (detector pulse pile-up) remain an issue to be addressed. We propose a data analysis method correcting for this type of artifact and the resulting systematic errors. It physically models the photon losses due to detector pulse pile-up and incorporates the loss in the decay fit model employed to obtain fluorescence lifetimes and relative amplitudes of the decay components. Comparison of results with and without this correction shows a significant reduction of systematic errors at count rates approaching the excitation rate. This allows quantitatively accurate fluorescence lifetime imaging at very high frame rates.

  7. Fluorescence imaging as a diagnostic of M-band x-ray drive condition in hohlraum with fluorescent Si targets

    NASA Astrophysics Data System (ADS)

    Li, Qi; Hu, Zhimin; Yao, Li; Huang, Chengwu; Yuan, Zheng; Zhao, Yang; Xiong, Gang; Qing, Bo; Lv, Min; Zhu, Tuo; Deng, Bo; Li, Jin; Wei, Minxi; Zhan, Xiayu; Li, Jun; Yang, Yimeng; Su, Chunxiao; Yang, Guohong; Zhang, Jiyan; Li, Sanwei; Yang, Jiamin; Ding, Yongkun

    2017-01-01

    Fluorescence imaging of surrogate Si-doped CH targets has been used to provide a measurement for drive condition of high-energy x-ray (i.e. M-band x-ray) drive symmetry upon the capsule in hohlraum on Shenguang-II laser facility. A series of experiments dedicated to the study of photo-pumping and fluorescence effect in Si-plasma are presented. To investigate the feasibility of fluorescence imaging in Si-plasma, an silicon plasma in Si-foil target is pre-formed at ground state by the soft x-ray from a half-hohlraum, which is then photo-pumped by the K-shell lines from a spatially distinct laser-produced Si-plasma. The resonant Si photon pump is used to improve the fluorescence signal and cause visible image in the Si-foil. Preliminary fluorescence imaging of Si-ball target is performed in both Si-doped and pure Au hohlraum. The usual capsule at the center of the hohlraum is replaced with a solid Si-doped CH-ball (Si-ball). Since the fluorescence is proportional to the photon pump upon the Si-plasma, high-energy x-ray drive symmetry is equal to the fluorescence distribution of the Si-ball.

  8. 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.

  9. Synthesis and characterization of citrate-based fluorescent small molecules and biodegradable polymers.

    PubMed

    Xie, Zhiwei; Kim, Jimin P; Cai, Qing; Zhang, Yi; Guo, Jinshan; Dhami, Ranjodh S; Li, Li; Kong, Bin; Su, Yixue; Schug, Kevin A; Yang, Jian

    2017-03-01

    Novel citric acid based photoluminescent dyes and biodegradable polymers are synthesized via a facile "one-pot" reaction. A comprehensive understanding of the fluorescence mechanisms of the resulting citric acid-based fluorophores is reported. Two distinct types of fluorophores are identified: a thiozolopyridine family with high quantum yield, long lifetime, and exceptional photostability, and a dioxopyridine family with relatively lower quantum yield, multiple lifetimes, and solvent-dependent band shifting behavior. Applications in molecular labeling and cell imaging were demonstrated. The above discoveries contribute to the field of fluorescence chemistry and have laid a solid foundation for further development of new fluorophores and materials that show promise in a diversity of fluorescence-based applications. Photoluminescent materials are pivotal for fluorescence based imaging, labeling and sensing applications. Understanding their fluorescence mechanism is challenging and imperative. We develop a new class of citric acid-derived fluorescent materials in forms of polymers and small molecular dyes by a one-step solvent free reaction. We discovered two different classes of citric acid-derived fluorophores. A two-ring thiozolopyridine structure demonstrates strong fluorescence and exceptional resistance to photo-bleaching. A one-ring dioxopyridine exhibits relative weak fluorescence but with intriguing excitation and solvent-dependent emission wavelength shifting. Our methodology of synthesizing citric acid-derived fluorophores and the understanding on their luminescence are instrumental to the design and production of a large number of new photoluminescent materials for biological and biomedical applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  10. Scanning fluorescent microthermal imaging apparatus and method

    DOEpatents

    Barton, Daniel L.; Tangyunyong, Paiboon

    1998-01-01

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

  11. Low-cost fluorescence microscopy for point-of-care cell imaging

    NASA Astrophysics Data System (ADS)

    Lochhead, Michael J.; Ives, Jeff; Givens, Monique; Delaney, Marie; Moll, Kevin; Myatt, Christopher J.

    2010-02-01

    Fluorescence microscopy has long been a standard tool in laboratory medicine. Implementation of fluorescence microscopy for near-patient diagnostics, however, has been limited due to cost and complexity associated with traditional fluorescence microscopy techniques. There is a particular need for robust, low-cost imaging in high disease burden areas in the developing world, where access to central laboratory facilities and trained staff is limited. Here we describe a point-of-care assay that combines a disposable plastic cartridge with an extremely low cost fluorescence imaging instrument. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements. A two-color cell imager is presented, with magnification optimized for enumeration of immunostained human T cells. To demonstrate the system, peripheral blood mononuclear cells were stained with fluorescently labeled anti-human-CD4 and anti-human-CD3 antibodies. Registered images were used to generate fractional CD4+ and CD3+ staining and enumeration results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.

  12. Toward intravascular morphological and biochemical imaging of atherosclerosis with optical coherence tomography (OCT) and fluorescence lifetime imaging (FLIM) (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Chen, Xi; Kim, Wihan; Serafino, Michael; Walton, Brian; Jo, Javier A.; Applegate, Brian E.

    2017-02-01

    We have shown in an ex vivo human coronary artery study that the biochemical information derived from FLIM interpreted in the context of the morphological information from OCT enables a detailed classification of human coronary plaques associated with atherosclerosis. The identification of lipid-rich plaques prone to erosion or rupture and associated with sudden coronary events can impact current clinical practice as well as future development of targeted therapies for "vulnerable" plaques. In order to realize clinical translation of intravascular OCT/FLIM we have had to develop several key technologies. A multimodal catheter endoscope capable of delivering near UV excitation for FLIM and shortwave IR for OCT has been fabricated using a ball lens design with a double clad fiber. The OCT illumination and the FLIM excitation propogate down the inner core while the large outer multimode core captures the fluorescence emission. To enable intravascular pullback imaging with this endoscope we have developed an ultra-wideband fiber optic rotary joint using the same double clad fiber. The rotary joint is based on a lensless design where two cleaved fibers, one fixed and one rotating, are brought into close proximity but not touching. Using water as the lubricant enabled operation over the near UV-shortwave IR range. Transmission over this bandwidth has been measured to be near 100% at rotational frequencies up to 147 Hz. The entire system has been assembled and placed on a mobile cart suitable for cath lab based imaging. System development, performance, and early ex vivo imaging results will be discussed.

  13. Dental fluorosis in populations from Chiang Mai, Thailand with different fluoride exposures - Paper 2: The ability of fluorescence imaging to detect differences in fluorosis prevalence and severity for different fluoride intakes from water

    PubMed Central

    2012-01-01

    Background To assess the ability of fluorescence imaging to detect a dose response relationship between fluorosis severity and different levels of fluoride in water supplies compared to remote photographic scoring in selected populations participating in an observational, epidemiological survey in Chiang Mai, Thailand. Methods Subjects were male and female lifetime residents aged 8-13 years. For each child the fluoride content of cooking water samples (CWS) was assessed to create categorical intervals of water fluoride concentration. Fluorescence images were taken of the maxillary central incisors and analyzed for dental fluorosis using two different software techniques. Output metrics for the fluorescence imaging techniques were compared to TF scores from blinded photographic scores obtained from the survey. Results Data from 553 subjects were available. Both software analysis techniques demonstrated significant correlations with the photographic scores. The metrics for area effected by fluorosis and the overall fluorescence loss had the strongest association with the photographic TF score (Spearman’s rho 0.664 and 0.652 respectively). Both software techniques performed well for comparison of repeat fluorescence images with ICC values of 0.95 and 0.85 respectively. Conclusions This study supports the potential use of fluorescence imaging for the objective quantification of dental fluorosis. Fluorescence imaging was able to discriminate between populations with different fluoride exposures on a comparable level to remote photographic scoring with acceptable levels of repeatability. PMID:22908997

  14. Shortwave infrared fluorescence imaging with the clinically approved near-infrared dye indocyanine green.

    PubMed

    Carr, Jessica A; Franke, Daniel; Caram, Justin R; Perkinson, Collin F; Saif, Mari; Askoxylakis, Vasileios; Datta, Meenal; Fukumura, Dai; Jain, Rakesh K; Bawendi, Moungi G; Bruns, Oliver T

    2018-04-24

    Fluorescence imaging is a method of real-time molecular tracking in vivo that has enabled many clinical technologies. Imaging in the shortwave IR (SWIR; 1,000-2,000 nm) promises higher contrast, sensitivity, and penetration depths compared with conventional visible and near-IR (NIR) fluorescence imaging. However, adoption of SWIR imaging in clinical settings has been limited, partially due to the absence of US Food and Drug Administration (FDA)-approved fluorophores with peak emission in the SWIR. Here, we show that commercially available NIR dyes, including the FDA-approved contrast agent indocyanine green (ICG), exhibit optical properties suitable for in vivo SWIR fluorescence imaging. Even though their emission spectra peak in the NIR, these dyes outperform commercial SWIR fluorophores and can be imaged in the SWIR, even beyond 1,500 nm. We show real-time fluorescence imaging using ICG at clinically relevant doses, including intravital microscopy, noninvasive imaging in blood and lymph vessels, and imaging of hepatobiliary clearance, and show increased contrast compared with NIR fluorescence imaging. Furthermore, we show tumor-targeted SWIR imaging with IRDye 800CW-labeled trastuzumab, an NIR dye being tested in multiple clinical trials. Our findings suggest that high-contrast SWIR fluorescence imaging can be implemented alongside existing imaging modalities by switching the detection of conventional NIR fluorescence systems from silicon-based NIR cameras to emerging indium gallium arsenide-based SWIR cameras. Using ICG in particular opens the possibility of translating SWIR fluorescence imaging to human clinical applications. Indeed, our findings suggest that emerging SWIR-fluorescent in vivo contrast agents should be benchmarked against the SWIR emission of ICG in blood.

  15. Application of indocyanine green-fluorescence imaging to full-thickness cholecystectomy.

    PubMed

    Morita, Kiyomi; Ishizawa, Takeaki; Tani, Keigo; Harada, Nobuhiro; Shimizu, Atsushi; Yamamoto, Satoshi; Takemura, Nobuyuki; Kaneko, Junichi; Aoki, Taku; Sakamoto, Yoshihiro; Sugawara, Yasuhiko; Hasegawa, Kiyoshi; Kokudo, Norihiro

    2014-05-01

    Fluorescence imaging using indocyanine green (ICG) has recently been applied to laparoscopic surgery to identify cancerous tissues, lymph nodes, and vascular anatomy. Here we report the application of ICG-fluorescence imaging to visualize the boundary between the liver and subserosal tissues of the gallbladder during laparoscopic full-thickness cholecystectomy. A patient with a potentially malignant gallbladder lesion was administered 2.5-mg intravenous ICG just before laparoscopic full-thickness cholecystectomy. Intraoperative fluorescence imaging enabled the real-time delineation of both extrahepatic bile duct anatomy and hepatic parenchyma throughout the procedure, which resulted in complete removal of subserosal tissues between liver and gallbladder. Safe and feasible ICG-fluorescence imaging can be widely applied to laparoscopic hepatobiliary surgery by utilizing a biliary excretion property of ICG. © 2014 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and Wiley Publishing Asia Pty Ltd.

  16. Before In Vivo Imaging: Evaluation of Fluorescent Probes Using Fluorescence Microscopy, Multiplate Reader, and Cytotoxicity Assays.

    PubMed

    Zhang, Shaojuan

    2016-01-01

    Fluorescent probes are widely utilized for noninvasive fluorescence imaging. Continuing efforts have been made in developing novel fluorescent probes with improved fluorescence quantum yield, enhanced target-specificity, and lower cytotoxicity. Before such probes are administrated into a living system, it is essential to evaluate the subcellular uptake, targeting specificity, and cytotoxicity in vitro. In this chapter, we briefly outline common methods used to evaluate fluorescent probes using fluorescence microscopy, multiplate reader, and cytotoxicity assay.

  17. Photoacoustic lifetime imaging for direct in vivo tissue oxygen monitoring

    PubMed Central

    Shao, Qi; Ashkenazi, Shai

    2015-01-01

    Abstract. Measuring the partial pressure of oxygen (pO2) in tissue may provide physicians with essential information about the physiological state of tissue. However, currently available methods for measuring or imaging tissue pO2 have significant limitations, preventing them from being widely used in clinics. Recently, we have reported a direct and noninvasive in vivo imaging modality based on the photoacoustic lifetime which overcomes certain drawbacks of the existing methods. The technique maps the excited triplet state of oxygen-sensitive dye, thus reflecting the spatial and temporal distributions of tissue oxygen. Here, we present two studies which apply photoacoustic lifetime imaging (PALI) to monitor changes of tissue oxygen induced by external modulations. The first study modulates tissue oxygen by controlling the percentage of oxygen a normal mouse inhales. We demonstrate that PALI is able to reflect the change in oxygen level with respect to normal, oxygen-rich, and oxygen-poor breathing conditions. The second study involves an acute ischemia model using a thin thread tied around the hindlimb of a normal mouse to reduce the blood flow. PALI images were acquired before, during, and after the restriction. The drop of tissue pO2 and recovery from hypoxia due to reperfusion were tracked and observed by PALI. PMID:25748857

  18. Preassembled Fluorescent Multivalent Probes for the Imaging of Anionic Membranes.

    PubMed

    Roland, Felicia M; Peck, Evan M; Rice, Douglas R; Smith, Bradley D

    2017-04-19

    A new self-assembly process known as Synthavidin (synthetic avidin) technology was used to prepare targeted probes for near-infrared fluorescence imaging of anionic membranes and cell surfaces, a hallmark of many different types of disease. The probes were preassembled by threading a tetralactam macrocycle with six appended zinc-dipicolylamine (ZnDPA) targeting units onto a linear scaffold with one or two squaraine docking stations to produce hexavalent or dodecavalent fluorescent probes. A series of liposome titration experiments showed that multivalency promoted stronger membrane binding by the dodecavalent probe. In addition, the dodecavalent probe exhibited turn-on fluorescence due to probe unfolding during fluorescence microscopy at the membrane surface. However, the dodecavalent probe also had a higher tendency to self-aggregate after membrane binding, leading to probe self-quenching under certain conditions. This self-quenching effect was apparent during fluorescence microscopy experiments that recorded low fluorescence intensity from anionic dead and dying mammalian cells that were saturated with the dodecavalent probe. Conversely, probe self-quenching was not a factor with anionic microbial surfaces, where there was intense fluorescence staining by the dodecavalent probe. A successful set of rat tumor imaging experiments confirmed that the preassembled probes have sufficient mechanical stability for effective in vivo imaging. The results demonstrate the feasibility of this general class of preassembled fluorescent probes for multivalent targeting, but fluorescence imaging performance depends on the specific physical attributes of the biomarker target, such as the spatial distance between different copies of the biomarker and the propensity of the probe-biomarker complex to self-aggregate.

  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. In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography

    PubMed Central

    Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

    2016-01-01

    Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT®). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors. PMID:27809256

  1. In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography.

    PubMed

    Genevois, Coralie; Loiseau, Hugues; Couillaud, Franck

    2016-10-31

    Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT ® ). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors.

  2. 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.

  3. 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.

  4. Fluorescence imaging spectrometer optical design

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  5. Time-resolved autofluorescence imaging of human donor retina tissue from donors with significant extramacular drusen.

    PubMed

    Schweitzer, Dietrich; Gaillard, Elizabeth R; Dillon, James; Mullins, Robert F; Russell, Stephen; Hoffmann, Birgit; Peters, Sven; Hammer, Martin; Biskup, Christoph

    2012-06-08

    Time and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen. Tissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The repetition rate was 76 MHz and the pulse width was 170 femtoseconds (fs). The time-resolved autofluorescence was recorded simultaneously in 16 spectral channels (445-605 nm) and bi-exponentially fitted. RPE can be discriminated clearly from Bruch's membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected. The demonstrated differentiation of fluorescent structures by their fluorescence decay time is important for interpretation of in vivo measurements by the new fluorescence lifetime imaging (FLIM) ophthalmoscopy on healthy subjects as well as on patients.

  6. Fluorescence-enhanced optical tomography and nuclear imaging system for small animals

    NASA Astrophysics Data System (ADS)

    Tan, I.-Chih; Lu, Yujie; Darne, Chinmay; Rasmussen, John C.; Zhu, Banghe; Azhdarinia, Ali; Yan, Shikui; Smith, Anne M.; Sevick-Muraca, Eva M.

    2012-03-01

    Near-infrared (NIR) fluorescence is an alternative modality for molecular imaging that has been demonstrated in animals and recently in humans. Fluorescence-enhanced optical tomography (FEOT) using continuous wave or frequency domain photon migration techniques could be used to provide quantitative molecular imaging in vivo if it could be validated against "gold-standard," nuclear imaging modalities, using dual-labeled imaging agents. Unfortunately, developed FEOT systems are not suitable for incorporation with CT/PET/SPECT scanners because they utilize benchtop devices and require a large footprint. In this work, we developed a miniaturized fluorescence imaging system installed in the gantry of the Siemens Inveon PET/CT scanner to enable NIR transillumination measurements. The system consists of a CCD camera equipped with NIR sensitive intensifier, a diode laser controlled by a single board compact controller, a 2-axis galvanometer, and RF circuit modules for homodyne detection of the phase and amplitude of fluorescence signals. The performance of the FEOT system was tested and characterized. A mouse-shaped solid phantom of uniform optical properties with a fluorescent inclusion was scanned using CT, and NIR fluorescence images at several projections were collected. The method of high-order approximation to the radioactive transfer equation was then used to reconstruct the optical images. Dual-labeled agents were also used on a tumor bearing mouse to validate the results of the FEOT against PET/CT image. The results showed that the location of the fluorophore obtained from the FEOT matches the location of tumor obtained from the PET/CT images. Besides validation of FEOT, this hybrid system could allow multimodal molecular imaging (FEOT/PET/CT) for small animal imaging.

  7. 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.

  8. Speckle correlation resolution enhancement of wide-field fluorescence imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Yilmaz, Hasan

    2016-03-01

    Structured illumination enables high-resolution fluorescence imaging of nanostructures [1]. We demonstrate a new high-resolution fluorescence imaging method that uses a scattering layer with a high-index substrate as a solid immersion lens [2]. Random scattering of coherent light enables a speckle pattern with a very fine structure that illuminates the fluorescent nanospheres on the back surface of the high-index substrate. The speckle pattern is raster-scanned over the fluorescent nanospheres using a speckle correlation effect known as the optical memory effect. A series of standard-resolution fluorescence images per each speckle pattern displacement are recorded by an electron-multiplying CCD camera using a commercial microscope objective. We have developed a new phase-retrieval algorithm to reconstruct a high-resolution, wide-field image from several standard-resolution wide-field images. We have introduced phase information of Fourier components of standard-resolution images as a new constraint in our algorithm which discards ambiguities therefore ensures convergence to a unique solution. We demonstrate two-dimensional fluorescence images of a collection of nanospheres with a deconvolved Abbe resolution of 116 nm and a field of view of 10 µm × 10 µm. Our method is robust against optical aberrations and stage drifts, therefore excellent for imaging nanostructures under ambient conditions. [1] M. G. L. Gustafsson, J. Microsc. 198, 82-87 (2000). [2] H. Yilmaz, E. G. van Putten, J. Bertolotti, A. Lagendijk, W. L. Vos, and A. P. Mosk, Optica 2, 424-429 (2015).

  9. Open source tools for fluorescent imaging.

    PubMed

    Hamilton, Nicholas A

    2012-01-01

    As microscopy becomes increasingly automated and imaging expands in the spatial and time dimensions, quantitative analysis tools for fluorescent imaging are becoming critical to remove both bottlenecks in throughput as well as fully extract and exploit the information contained in the imaging. In recent years there has been a flurry of activity in the development of bio-image analysis tools and methods with the result that there are now many high-quality, well-documented, and well-supported open source bio-image analysis projects with large user bases that cover essentially every aspect from image capture to publication. These open source solutions are now providing a viable alternative to commercial solutions. More importantly, they are forming an interoperable and interconnected network of tools that allow data and analysis methods to be shared between many of the major projects. Just as researchers build on, transmit, and verify knowledge through publication, open source analysis methods and software are creating a foundation that can be built upon, transmitted, and verified. Here we describe many of the major projects, their capabilities, and features. We also give an overview of the current state of open source software for fluorescent microscopy analysis and the many reasons to use and develop open source methods. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. The nature of multiphoton fluorescence from red blood cells

    NASA Astrophysics Data System (ADS)

    Saytashev, Ilyas; Murphy, Michael; Osseiran, Sam; Spence, Dana M.; Evans, Conor L.; Dantus, Marcos

    2016-03-01

    We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC's, but longer more intense pulses lead to their destruction.

  11. Estrogen receptor-targeted optical imaging of breast cancer cells with near-infrared fluorescent dye

    NASA Astrophysics Data System (ADS)

    Jose, Iven; Deodhar, Kodand; Chiplunkar, Shuba V.; Patkar, Meena

    2010-02-01

    Molecular imaging provides the in vivo characterization of cellular molecular events involved in normal and pathologic processes. With the advent of optical molecular imaging, specific molecules, proteins and genes may be tagged with a luminescent reporter and visualized in small animals. This powerful new tool has pushed in vivo optical imaging to the forefront as it allows for direct determination of drug bio-distribution and uptake kinetics as well as an indicator of biochemical activity and drug efficacy. Although optical imaging encompasses diverse techniques and makes use of various wavelengths of light, a great deal of excitement in molecular research lies in the use of tomographic and fluorescence techniques to image living tissues with near-infrared (NIR) light. Nonionizing, noninvasive near-infrared optical imaging has great potential to become promising alternative for breast cancer detection. Fluorescence spectroscopy studies of human tissue suggest that a variety of lesions show distinct fluorescence spectra compared to those of normal tissue. It has also been shown that exogenous dyes exhibit selective uptake in neoplastic lesions and may offer the best contrast for optical imaging. Use of exogenous agents would provide fluorescent markers, which could serve to detect embedded tumors in the breast. In particular, the ability to monitor the fluorescent yield and lifetime may also enable biochemical specificity if the fluorophore is sensitive to a specific metabolite, such as oxygen. As a first step, we have synthesized and characterized one such NIR fluorescent dye conjugate, which could potentially be used to detect estrogen receptors (ER)[2] . The conjugate was synthesized by ester formation between 17-β estradiol and a hydrophilic derivative of indocyanine green (ICG) cyanine dye, bis-1, 1-(4-sulfobutyl) indotricarbocyanine-5- carboxylic acid, sodium salt. The ester formed was found to have an extra binding ability with the receptor cites as

  12. Fluorescence imaging host pathogen interactions: fifteen years benefit of hindsight….

    PubMed

    Aulner, Nathalie; Danckaert, Anne; Fernandes, Julien; Nicola, Marie-Anne; Roux, Pascal; Salles, Audrey; Tinevez, Jean-Yves; Shorte, Spencer L

    2018-03-19

    We consider in review current state-of-the-art fluorescence microscopy for investigating the host-pathogen interface. Our perspective is honed from years with literally thousands of microbiologists using the variety of imaging technologies available within our dedicated BSL2/BSL3 optical imaging research service facilities at the Institut Pasteur Paris founded from scratch in 2001. During fifteen years learning from the success and failures of introducing different fluorescence imaging technologies, methods, and technical development strategies we provide here a synopsis review of our experience to date and a synthesis of how we see the future in perspective for fluorescence imaging at the host-pathogen interface. Copyright © 2018. Published by Elsevier Ltd.

  13. High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime

    PubMed Central

    Kurokawa, Hiromi; Ito, Hidehiro; Inoue, Mai; Tabata, Kenji; Sato, Yoshifumi; Yamagata, Kazuya; Kizaka-Kondoh, Shinae; Kadonosono, Tetsuya; Yano, Shigenobu; Inoue, Masahiro; Kamachi, Toshiaki

    2015-01-01

    Optical methods using phosphorescence quenching by oxygen are suitable for sequential monitoring and non-invasive measurements for oxygen concentration (OC) imaging within cells. Phosphorescence intensity measurement is widely used with phosphorescent dyes. These dyes are ubiquitously but heterogeneously distributed inside the whole cell. The distribution of phosphorescent dye is a major disadvantage in phosphorescence intensity measurement. We established OC imaging system for a single cell using phosphorescence lifetime and a laser scanning confocal microscope. This system had improved spatial resolution and reduced the measurement time with the high repetition rate of the laser. By the combination of ubiquitously distributed phosphorescent dye with this lifetime imaging microscope, we can visualize the OC inside the whole cell and spheroid. This system uses reversible phosphorescence quenching by oxygen, so it can measure successive OC changes from normoxia to anoxia. Lower regions of OC inside the cell colocalized with mitochondria. The time-dependent OC change in an insulin-producing cell line MIN6 by the glucose stimulation was successfully visualized. Assessing the detailed distribution and dynamics of OC inside cells achieved by the presented system will be useful to understanding a physiological and pathological oxygen metabolism. PMID:26065366

  14. 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.

  15. 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.

  16. 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

  17. In Vivo Deep Tissue Fluorescence and Magnetic Imaging Employing Hybrid Nanostructures.

    PubMed

    Ortgies, Dirk H; de la Cueva, Leonor; Del Rosal, Blanca; Sanz-Rodríguez, Francisco; Fernández, Nuria; Iglesias-de la Cruz, M Carmen; Salas, Gorka; Cabrera, David; Teran, Francisco J; Jaque, Daniel; Martín Rodríguez, Emma

    2016-01-20

    Breakthroughs in nanotechnology have made it possible to integrate different nanoparticles in one single hybrid nanostructure (HNS), constituting multifunctional nanosized sensors, carriers, and probes with great potential in the life sciences. In addition, such nanostructures could also offer therapeutic capabilities to achieve a wider variety of multifunctionalities. In this work, the encapsulation of both magnetic and infrared emitting nanoparticles into a polymeric matrix leads to a magnetic-fluorescent HNS with multimodal magnetic-fluorescent imaging abilities. The magnetic-fluorescent HNS are capable of simultaneous magnetic resonance imaging and deep tissue infrared fluorescence imaging, overcoming the tissue penetration limits of classical visible-light based optical imaging as reported here in living mice. Additionally, their applicability for magnetic heating in potential hyperthermia treatments is assessed.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1997-12-01

    A fiber optic pH-sensor capable of both visualizing corrosion sites and measuring local chemical concentrations is applied to real-time corrosion monitoring. The imaging fiber`s distal face containing an immobilized pH-sensitive fluorescent dye is brought into contact with metal surfaces submerged in aqueous buffers and fluorescence images are acquired as a function of time. The observed changes in fluorescence due to increases in pH at cathodic sites and decreases in pH at anodic sites are indicative of localized corrosion rates.

  19. Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems.

    PubMed

    Yan, Yuling; Marriott, M Emma; Petchprayoon, Chutima; Marriott, Gerard

    2011-02-01

    Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.

  20. Nondestructive assessment of collagen hydrogel cross-linking using time-resolved autofluorescence imaging

    NASA Astrophysics Data System (ADS)

    Sherlock, Benjamin E.; Harvestine, Jenna N.; Mitra, Debika; Haudenschild, Anne; Hu, Jerry; Athanasiou, Kyriacos A.; Leach, J. Kent; Marcu, Laura

    2018-03-01

    We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable.