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Sample records for fluorescence lifetime imaging

  1. Fluorescence lifetime imaging of coral fluorescent proteins.

    PubMed

    Cox, Guy; Matz, Mikhail; Salih, Anya

    2007-03-01

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

  2. Fluorescence Lifetime Imaging of Apoptosis

    PubMed Central

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

    2015-01-01

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

  3. 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.

  4. Combined fluorescence and phosphorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  5. Hadamard-transform fluorescence-lifetime imaging.

    PubMed

    Mizuno, Takahiko; Iwata, Tetsuo

    2016-04-18

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

  6. Fluorescence lifetime imaging in turbid media

    NASA Astrophysics Data System (ADS)

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

    1996-01-01

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

  7. High frame rate fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

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

    2003-07-01

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

  8. Fluorescence lifetime-based optical molecular imaging.

    PubMed

    Kumar, Anand T N

    2011-01-01

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

  9. Molecular Probes for Fluorescence Lifetime Imaging

    PubMed Central

    Sarder, Pinaki; Maji, Dolonchampa; Achilefu, Samuel

    2015-01-01

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

  10. Hyperspectral fluorescence lifetime imaging for optical biopsy.

    PubMed

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

    2013-09-01

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

  11. 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.

  12. Fluorescence lifetime to image epidermal ionic concentrations

    NASA Astrophysics Data System (ADS)

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

    2004-09-01

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

  13. Biological applications of fluorescence lifetime imaging beyond microscopy

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

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

  14. 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.

  15. 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

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

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

    PubMed

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

    2016-06-01

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

  18. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    PubMed

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

    2014-03-01

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

  20. Fluorescence lifetime imaging of human skin and hair

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

  1. TOPICAL REVIEW: Fluorescence lifetime imaging microscopy in life sciences

    NASA Astrophysics Data System (ADS)

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

    2010-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  3. Photon budget analysis for fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

  4. 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

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

    PubMed

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

    2015-04-01

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

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

    PubMed

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

    2015-01-01

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

  7. GPU acceleration of time-domain fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  8. 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.

  9. Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Kremers, Gert-Jan; van Munster, Erik B.; Goedhart, Joachim; Gadella, Theodorus W. J.

    2008-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is a quantitative microscopy technique for imaging nanosecond decay times of fluorophores. In the case of frequency-domain FLIM, several methods have been described to resolve the relative abundance of two fluorescent species with different fluorescence decay times. Thus far, single-frequency FLIM methods generally have been limited to quantifying two species with monoexponential decay. However, multiexponential decays are the norm rather than the exception, especially for fluorescent proteins and biological samples. Here, we describe a novel method for determining the fractional contribution in each pixel of an image of a sample containing two (multiexponentially) decaying species using single-frequency FLIM. We demonstrate that this technique allows the unmixing of binary mixtures of two spectrally identical cyan or green fluorescent proteins, each with multiexponential decay. Furthermore, because of their spectral identity, quantitative images of the relative molecular abundance of these fluorescent proteins can be generated that are independent of the microscope light path. The method is rigorously tested using samples of known composition and applied to live cell microscopy using cells expressing multiple (multiexponentially decaying) fluorescent proteins. PMID:18359789

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

    PubMed Central

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

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

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

    PubMed

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

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

  12. 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.

  13. Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics

    PubMed Central

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2005-03-01

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

  18. Applying fluorescence lifetime imaging microscopy to evaluate the efficacy of anticancer drugs

    NASA Astrophysics Data System (ADS)

    Kawanabe, Satoshi; Araki, Yoshie; Uchimura, Tomohiro; Imasaka, Totaro

    2015-06-01

    Fluorescence lifetime imaging microscopy was applied to evaluate the efficacy of anticancer drugs. A decrease in the fluorescence lifetime of the nucleus in apoptotic cancer cells stained by SYTO 13 dye was detected after treatment with antitumor antibiotics such as doxorubicin or epirubicin. It was confirmed that the change in fluorescence lifetime occurred earlier than morphological changes in the cells. We found that the fluorescence lifetime of the nucleus in the cells treated with epirubicin decreased more rapidly than that of the cells treated with doxorubicin. This implies that epirubicin was more efficacious than doxorubicin in the treatment of cancer cells. The change in fluorescence lifetime was, however, not indicated when the cells were treated with cyclophosphamide. The decrease in fluorescence lifetime was associated with the processes involving caspase activation and chromatin condensation. Therefore, this technique would provide useful information about apoptotic cells, particularly in the early stages.

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

    PubMed Central

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

    2014-01-01

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

  20. 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.

  1. 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

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

    PubMed

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

    2016-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2007-05-01

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

  5. Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    Spectrally resolved confocal microscopy and fluorescence lifetime imaging have been used to measure the polarity of lipid-rich regions in living HeLa cells stained with Nile red. The emission peak from the solvatochromic dye in lipid droplets is at a shorter wavelength than other, more polar, stained internal membranes, and this is indicative of a low polarity environment. We estimate that the dielectric constant, ɛ, is around 5 in lipid droplets and 25<ɛ<40 in other lipid-rich regions. Our spectrally resolved fluorescence lifetime imaging microscopy (FLIM) data show that intracellular Nile red exhibits complex, multiexponential fluorescence decays due to emission from a short lifetime locally excited state and a longer lifetime intramolecular charge transfer state. We measure an increase in the average fluorescence lifetime of the dye with increasing emission wavelength, as shown using phasor plots of the FLIM data. We also show using these phasor plots that the shortest lifetime decay components arise from lipid droplets. Thus, fluorescence lifetime is a viable contrast parameter for distinguishing lipid droplets from other stained lipid-rich regions. Finally, we discuss the FLIM of Nile red as a method for simultaneously mapping both polarity and relative viscosity based on fluorescence lifetime measurements.

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

    NASA Astrophysics Data System (ADS)

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

    2007-02-01

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

  7. In Situ Monitoring of the Intracellular Stability of Nanoparticles by Using Fluorescence Lifetime Imaging.

    PubMed

    Shang, Li; Yang, Linxiao; Wang, Haixia; Nienhaus, Gerd Ulrich

    2016-02-01

    FLIMaging nanoparticle degradation: semiconductor and metal nanoparticle degradation has been observed in live cells over 3 d via the change of the characteristic luminescence lifetime using fluorescence lifetime imaging microscopy (FLIM). Thus, FLIM is a simple yet robust tool to examine the intracellular stability of photoluminescent nanoparticles in live cells, tissues, and organisms. PMID:26708212

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

    PubMed Central

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

    2015-01-01

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

  9. Fluorescence-Lifetime Imaging Microscopy for Visualization of Quantum Dots’ Endocytic Pathway

    PubMed Central

    Damalakiene, Leona; Karabanovas, Vitalijus; Bagdonas, Saulius; Rotomskis, Ricardas

    2016-01-01

    Accumulation of carboxylated polyethylene glycol (PEG) CdSe/ZnSquantum dots (QDs) has been monitored in living fibroblasts using confocal microscopy for fluorescence intensity and fluorescence-lifetime imaging (FLIM). The wide range of mean photoluminescence (PL) lifetime values was observed for the intracellular QDs in different intracellular microenvironment, which revealed structural heterogeneity of endosomes and enabled the distinguishing among endosomes of different maturity.

  10. Two-photon excited fluorescence lifetime imaging microscopy for FRET study on protein interactions

    NASA Astrophysics Data System (ADS)

    Qu, Junle; Lin, Ziyang; Liu, Lixin; Guo, Xuan; Chen, Danni; Niu, Hanben

    2005-01-01

    Two-photon excited fluorescence lifetime imaging (2P-FLIM) provides a more direct and precise approach to fluorescence resonance energy transfer (FRET), which allows studying the dynamic behavior of protein-protein interactions in living cells. In this paper, we describe the combination of a Leica TCS SP2 laser scanning microscope and a time-correlated single photon counting (TCSPC) lifetime imaging module developed by Becker & Hickl for two-photon excited fluorescence lifetime imaging. This 2P-FLIM system was used for FRET study on the interaction of heat shock protein hsp27 with p38 MAP kinase in the single living cell. Results show that the reduction in donor (CFP) lifetime in the presence of acceptor (YFP) reveals interactions between the two proteins.

  11. Development of a time-gated fluorescence lifetime microscope for in vivo corneal metabolic imaging

    NASA Astrophysics Data System (ADS)

    Silva, Susana F.; Batista, Ana; Castejón, Olga C.; Quadrado, Maria João.; Domingues, José Paulo; Morgado, Miguel

    2015-07-01

    Metabolic imaging can be a valuable tool in the early diagnosis of corneal diseases. Cell metabolic changes can be assessed through non-invasive optical methods due to the autofluorescence of metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Both molecules exhibit double exponential fluorescence decays, with well-separated short and long lifetime components, which are related to their protein-bound and free states. Corneal metabolism can be monitored by measuring the relative contribution of these two components. Here we report on the development of a fluorescence lifetime imaging microscope for in vivo measurement of FAD fluorescence lifetimes in corneal cells. The microscope is based on one-photon fluorescence excitation, through a pulsed blue diode laser. Fluorescence lifetime imaging is achieved using the Time-Gated technique. Structured illumination is used to improve the low axial resolution of wide-field time-gated FLIM. A Digital Micromirror Device (DMD) is used to produce the sinusoidal patterns required by structural illumination. The DMD control is integrated with the acquisition software of the imaging system which is based on an ultra-high speed gated image intensifier coupled to a CCD camera. We present preliminary results concerning optical and timing performance of the fluorescence lifetime microscope. Preliminary tests with ex-vivo bovine corneas are also described.

  12. Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime.

    PubMed

    Battisti, Antonella; Panettieri, Silvio; Abbandonato, Gerardo; Jacchetti, Emanuela; Cardarelli, Francesco; Signore, Giovanni; Beltram, Fabio; Bizzarri, Ranieri

    2013-07-01

    The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress. PMID:23780224

  13. Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

    PubMed Central

    Sun, Yuansheng; Day, Richard N; Periasamy, Ammasi

    2011-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster resonance energy transfer (FRET ). We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ~2 d. PMID:21886099

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

    PubMed

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

    2006-01-20

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

  15. 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

  16. High-speed confocal fluorescence lifetime imaging microscopy by analog mean-delay method

    NASA Astrophysics Data System (ADS)

    Won, Youngjae; Kim, Donguk; Yang, Wenzhong; Kim, Dug Y.

    2010-02-01

    We have demonstrated the high-speed confocal fluorescence lifetime imaging microscopy (FLIM) by analog mean-delay (AMD) method. The AMD method is a new signal processing technique for calculation of fluorescence lifetime and it is very suitable for the high-speed confocal FLIM with good accuracy and photon economy. We achieved the acquisition speed of 7.7 frames per second for confocal FLIM imaging. Here, the highest photon detection rate for one pixel was larger than 125 MHz and averaged photon detection rate was more than 62.5 MHz. Based on our system, we successfully obtained a sequence of confocal fluorescence lifetime images of RBL-2H3 cell labeled with Fluo-3/AM and excited by 4αPDD (TRPV channel agonist) within one second.

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  20. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes

    PubMed Central

    Estandarte, Ana Katrina; Botchway, Stanley; Lynch, Christophe; Yusuf, Mohammed; Robinson, Ian

    2016-01-01

    Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore’s fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies. PMID:27526631

  1. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes.

    PubMed

    Estandarte, Ana Katrina; Botchway, Stanley; Lynch, Christophe; Yusuf, Mohammed; Robinson, Ian

    2016-01-01

    Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore's fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies. PMID:27526631

  2. 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.

  3. Fluorescence lifetime imaging microscopy reveals quenching of fluorescein within corneal epithelium.

    PubMed

    Glasgow, Ben J

    2016-06-01

    Topical application of fluorescein results in background fluorescence of normal corneal epithelial cells. The fluorescence appears relatively weak and is often ignored clinically. The concentrations of fluorescein applied clinically exceed the threshold for self quenching. The possibility that exuberant topical concentrations of fluorescein result in quenching of fluorescence in tears and normal corneal epithelium is explored. Fluorescence lifetime measurements are sensitive to quenching and are less vulnerable to inner filter effect than steady state measurements. The types of fluorescence lifetime quenching often report informative molecular interactions. Therefore, fluorescence lifetime confocal imaging was performed in solutions, tears and corneal epithelium removed by membrane cytology following applied fluorescein. Amplitude averaged fluorescence lifetimes (τamp) were measured with time resolved single photon counting using a pulsed diode laser for excitation of fluorescein. Lifetime decays were fit to multi-exponential models with least squares analysis. Stern-Volmer plots for both intensity (I) and (τamp) were determined. Stern-Volmer plots demonstrated both dynamic and static quenching components (R(2) = 0.98 exponential fit, I0/I). Plots of τamp versus concentration of fluorescein revealed a linear relationship. Immediately after fluorescein application, quenching was evident in tears (τamp < 1 ns) versus tears sampled after 5 min (τamp = 3.7 ns). Corneal epithelium showed quenching (τamp ≤ 2 ns) from 1 to 16 min post fluorescein instillation. Clinical concentrations of fluorescein show self-quenching but rapidly dilute as tears turnover. Intracellular quenching occurs in normal corneal epithelium. Lifetime decay curves suggest complex mechanisms are involved. Quenching is a plausible explanation for the low fluorescence background observed clinically. PMID:27106141

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

    PubMed

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

    2015-07-15

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

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

    PubMed Central

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

    2016-01-01

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

  6. Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells.

    PubMed

    Shi, Jing; Zhou, Ming; Gong, Aihua; Li, Qijun; Wu, Qian; Cheng, Gary J; Yang, Mingyang; Sun, Yaocheng

    2016-02-16

    The expression level of tumor-related mRNA can reveal significant information about tumor progression and prognosis, so specific mRNA in cells provides an important approach for biological and disease studies. Here, fluorescence lifetime imaging of nanoflares in living cells was first employed to detect specific intracellular mRNA. We characterized the lifetime changes of the prepared nanoflares before and after the treatment of target mRNA and also compared the results with those of fluorescence intensity-based measurements both intracellularly and extracellularly. The nanoflares released the cy5-modified oligonucleotides and bound to the targets, resulting in a fluorescence lifetime lengthening. This work puts forward another dimension of detecting specific mRNA in cells and can also open new ways for detection of many other biomolecules. PMID:26813157

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

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. Using multiphoton fluorescence lifetime imaging to characterize liver damage and fluorescein disposition in liver in vivo

    NASA Astrophysics Data System (ADS)

    Thorling, Camilla A.; Studier, Hauke; Crawford, Darrell; Roberts, Michael S.

    2016-03-01

    Liver disease is the fifth most common cause of death and unlike many other major causes of mortality, liver disease rates are increasing rather than decreasing. There is no ideal measurement of liver disease and although biopsies are the gold standard, this only allows for a spot examination and cannot follow dynamic processes of the liver. Intravital imaging has the potential to extract detailed information over a larger sampling area continuously. The aim of this project was to investigate whether multiphoton and fluorescence lifetime imaging microscopy could detect early liver damage and to assess whether it could detect changes in metabolism of fluorescein in normal and diseased livers. Four experimental groups were used in this study: 1) control; 2) ischemia reperfusion injury; 3) steatosis and 4) steatosis with ischemia reperfusion injury. Results showed that multiphoton microscopy could visualize morphological changes such as decreased fluorescence of endogenous fluorophores and the presence of lipid droplets, characteristic of steatosis. Fluorescence lifetime imaging microscopy showed increase in NADPH in steatosis with and without ischemia reperfusion injury and could detect changes in metabolism of fluorescein to fluorescein monoglurcuronide, which was impaired in steatosis with ischemia reperfusion injury. These results concluded that the combination of multiphoton microscopy and fluorescence lifetime imaging is a promising method of assessing early stage liver damage and that it can be used to study changes in drug metabolism in the liver as an indication of liver disease and has the potential to replace the traditional static liver biopsy currently used.

  14. Cholesterol efflux monitoring in macrophage form cells by using fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Song, Young Sik; Lee, Sang Hak; Park, Byoung Hee; Kim, Soo Hyeok; Hwang, Won Sang; Kim, Dug Young

    2015-03-01

    Macrophages play a key role in atherosclerotic plaque destabilization and rupture, since they accumulate large amounts of lipid through the uptake of modified lipoproteins which results in foam cell formation. Cholesterol efflux is the process of removing cholesterol from macrophages in the subintima of the vessel wall, and efflux mechanism in a cell is one of the critical issues for the prevention of cardiovascular diseases. High density lipoproteins (HDL) stimulate cholesterol efflux from macrophage foam cells in the arterial wall. Radioisotope-labeled cholesterol analysis method is well known conventional method for observing cholesterol efflux. The major drawback of this method is its long and complicated process. Fluorescence intensity imaging schemes are replacing the radioisotope-labeled method in recent years for cholesterol efflux monitoring. Various spectroscopic methods are also adapted for cholesterol efflux imaging. Here we present a fluorescence lifetime imaging method for more quantitative observation of cholesterol efflux process in macrophages, which enables us to observe cholesterol level changes with various conditions. We used J774 macrophage cell and 25-NBD-cholesterol which is a famous cholesterol specific dye. Our lifetime imaging results clearly show cholesterol efflux rate very effectively. We believe that fluorescence lifetime analysis is new and very powerful for cholesterol imaging or monitoring.

  15. Dynamic noninvasive monitoring of renal function in vivo by fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

    Kidneys normally filter the blood of excess salts and metabolic products, such as urea, while retaining plasma proteins. In diseases such as multiple myeloma and diabetes mellitus, the renal function is compromised and protein escapes into the urine. In this study, we present the use of fluorescence lifetime imaging (FLI) to image excess serum protein in urine (proteinuria). The near-infrared fluorescent dye LS-288 has distinct lifetimes when bound to protein versus free in solution, providing contrast between the protein-rich viscera and the mostly protein-free bladder. FLI with LS-288 in mice revealed that fluorescence lifetime (FLT) differences in the bladder relative to surrounding tissues was due to the fractional contributions of the bound and unbound dye molecules. The FLT of LS-288 decreased in the case of proteinuria while fluorescence intensity was unchanged. The results show that FLI can be useful for the dynamic imaging of protein-losing nephropathy due to diabetes mellitus and other renal diseases and suggest the potential use of the FLI to distinguish tumors from fluid-filled cysts in the body.

  16. 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.

  17. Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

    NASA Astrophysics Data System (ADS)

    Dragavon, Joe; Amiri, Megdouda; Marteyn, Benoit; Sansonetti, Philipe; Shorte, Spencer

    2011-03-01

    Fluorophore concentration, the surrounding microenvironment, and photobleaching greatly influence the fluorescence intensity of a fluorophore, increasing the difficulty to directly observe micro-environmental factors such as pH and oxygen. However, the fluorescence lifetime of a fluorophore is essentially independent of both the fluorophore concentration and photobleaching, providing a viable alternative to intensity measurements. The development of fluorescence lifetime imaging (FLI) allows for the direct measurement of the microenvironment surrounding a fluorophore. Pt-porphyrin is a fluorophore whose optical properties include a very stable triplet excited state. This energy level overlaps strongly with the ground triplet state of oxygen, making the phosphorescent lifetime directly proportional to the surrounding oxygen concentration. Initial experiments using this fluorophore involved the use of individual microwells coated with the porphyrin. Cells were allowed to enter the micro-wells before being sealed to create a diffusionally isolated volume. The decrease in the extracellular oxygen concentration was observed using FLI. However, this isolation technique provides only the consumption rate but cannot indicate the subcellular oxygen distribution. To improve upon this, live macrophages are loaded with the porphyrin and the fluorescence lifetime determined using a Lambert Instruments Lifa-X FLI system. Initial results indicate that an increase in subcellular oxygen is observed upon initial exposure to invasive bacteria. A substantial decrease in oxygen is observed after about 1 hour of exposure. The cells remain in this deoxygenated state until the bacteria are removed or cell death occurs.

  18. 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.

  19. 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

  20. 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.

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

    PubMed

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

    2016-04-30

    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. PMID:27086689

  2. 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. PMID:23976789

  3. 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

  4. A wide field fluorescence lifetime imaging system using a light sheet microscope

    NASA Astrophysics Data System (ADS)

    Birch, Phil M.; Moore, Lamar; Li, Xiaofei; Phillips, Roger; Young, Rupert; Chatwin, Chris

    2016-04-01

    Fluorescence lifetime imaging microscopy (FLIM) has allowed scientists to discern information about the chemical properties of biological processes and has become a vital tool in the life sciences and medical research communities. Measuring the spatial lifetime distribution of the fluorophores as well as the intensity distribution enables users to discern vital information about the chemical environment. It however, remains challenging and often involves slow scanning. We present a new microscope system based on light sheet illumination that uses a micro channel plate (MCP) device called a Capacitive Division Imaging Readout (CDIR) which has been developed by Photek Ltd. The device uses an array of capacitors to move the charge site from the MCP to four pre-amplifiers and time-over-threshold discriminators. This camera has the ability to image photons as well as measure the arrival time, enabling high speed FLIM imaging of biological samples.

  5. Quantitative diagnosis of cervical neoplasia using fluorescence lifetime imaging on haematoxylin and eosin stained tissue sections.

    PubMed

    Gu, Jun; Fu, Chit Yaw; Ng, Beng Koon; Gulam Razul, Sirajudeen so; Lim, Soo Kim

    2014-07-01

    The use of conventional fluorescence microscopy for characterizing tissue pathological states is limited by overlapping spectra and the dependence on excitation power and fluorophore concentration. Fluorescence lifetime imaging microscopy (FLIM) can overcome these limitations due to its insensitivity to fluorophore concentration, excitation power and spectral similarity. This study investigates the diagnosis of early cervical cancer using FLIM and a neural network extreme learning machine classifier. A concurrently high sensitivity and specificity of 92.8% and 80.2%, respectively, were achieved. The results suggest that the proposed technique can be used to supplement the traditional histopathological examination of early cervical cancer. PMID:23281280

  6. 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.

  7. 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

  8. 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.

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

    PubMed

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

    2007-10-01

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

  10. Deep-tissue multiphoton fluorescence lifetime microscopy for intravital imaging of protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Fruhwirth, G. O.; Matthews, D. R.; Brock, A.; Keppler, M.; Vojnovic, B.; Ng, T.; Ameer-Beg, S.

    2009-02-01

    Fluorescent lifetime imaging microscopy (FLIM) has proven to be a valuable tool in beating the Rayleigh criterion for light microscopy by measuring Förster resonance energy transfer (FRET) between two fluorophores. Applying multiphoton FLIM, we previously showed in a human breast cancer cell line that recycling of a membrane receptorgreen fluorescent protein fusion is enhanced concomitantly with the formation of a receptor:protein kinase C α complex in the endosomal compartment. We have extended this established technique to probe direct protein-protein interactions also in vivo. Therefore, we used various expressible fluorescent tags fused to membrane receptor molecules in order to generate stable two-colour breast carcinoma cell lines via controlled retroviral infection. We used these cell lines for establishing a xenograft tumour model in immune-compromised Nude mice. Using this animal model in conjunction with scanning Ti:Sapphire laser-based two-photon excitation, we established deep-tissue multiphoton FLIM in vivo. For the first time, this novel technique enables us to directly assess donor fluorescence lifetime changes in vivo and we show the application of this method for intravital imaging of direct protein-protein interactions.

  11. Multimodal in vivo imaging of oral cancer using fluorescence lifetime, photoacoustic and ultrasound techniques

    PubMed Central

    Fatakdawala, Hussain; Poti, Shannon; Zhou, Feifei; Sun, Yang; Bec, Julien; Liu, Jing; Yankelevich, Diego R.; Tinling, Steven P.; Gandour-Edwards, Regina F.; Farwell, D. Gregory; Marcu, Laura

    2013-01-01

    This work reports a multimodal system for label-free tissue diagnosis combining fluorescence lifetime imaging (FLIm), ultrasound backscatter microscopy (UBM), and photoacoustic imaging (PAI). This system provides complementary biochemical, structural and functional features allowing for enhanced in vivo detection of oral carcinoma. Results from a hamster oral carcinoma model (normal, precancer and carcinoma) are presented demonstrating the ability of FLIm to delineate biochemical composition at the tissue surface, UBM and related radiofrequency parameters to identify disruptions in the tissue microarchitecture and PAI to map optical absorption associated with specific tissue morphology and physiology. PMID:24049693

  12. Deconvolution of fluorescence lifetime imaging microscopy by a library of exponentials.

    PubMed

    Campos-Delgado, Daniel U; Navarro, O Gutierrez; Arce-Santana, E R; Walsh, Alex J; Skala, Melissa C; Jo, Javier A

    2015-09-01

    Fluorescence lifetime microscopy imaging (FLIM) is an optic technique that allows a quantitative characterization of the fluorescent components of a sample. However, for an accurate interpretation of FLIM, an initial processing step is required to deconvolve the instrument response of the system from the measured fluorescence decays. In this paper, we present a novel strategy for the deconvolution of FLIM data based on a library of exponentials. Our approach searches for the scaling coefficients of the library by non-negative least squares approximations plus Thikonov/l(2) or l(1) regularization terms. The parameters of the library are given by the lower and upper bounds in the characteristic lifetimes of the exponential functions and the size of the library, where we observe that this last variable is not a limiting factor in the resulting fitting accuracy. We compare our proposal to nonlinear least squares and global non-linear least squares estimations with a multi-exponential model, and also to constrained Laguerre-base expansions, where we visualize an advantage of our proposal based on Thikonov/l(2) regularization in terms of estimation accuracy, computational time, and tuning strategy. Our validation strategy considers synthetic datasets subject to both shot and Gaussian noise and samples with different lifetime maps, and experimental FLIM data of ex-vivo atherosclerotic plaques and human breast cancer cells. PMID:26368470

  13. Fluorescence lifetime imaging for the characterization of the biochemical composition of atherosclerotic plaques

    NASA Astrophysics Data System (ADS)

    Phipps, Jennifer; Sun, Yinghua; Saroufeem, Ramez; Hatami, Nisa; Fishbein, Michael C.; Marcu, Laura

    2011-09-01

    This study investigates the ability of a flexible fiberoptic-based fluorescence lifetime imaging microscopy (FLIM) technique to resolve biochemical features in plaque fibrotic cap associated with plaque instability and based solely on fluorescence decay characteristics. Autofluorescence of atherosclerotic human aorta (11 autopsy samples) was measured at 48 locations through two filters, F377: 377/50 and F460: 460/60 nm (center wavelength/bandwidth). The fluorescence decay dynamic was described by average lifetime (τ) and four Laguerre coefficients (LECs) retrieved through a Laguerre deconvolution technique. FLIM-derived parameters discriminated between four groups [elastin-rich (ER), elastin and macrophage-rich (E+M), collagen-rich (CR), and lipid-rich (LR)]. For example, τF377 discriminated ER from CR (R = 0.84); τF460 discriminated E+M from CR and ER (R = 0.60 and 0.54, respectively); LEC-1F377 discriminated CR from LR and E+M (R = 0.69 and 0.77, respectively); P < 0.05 for all correlations. Linear discriminant analysis was used to classify this data set with specificity >87% (all cases) and sensitivity as high as 86%. Current results demonstrate for the first time that clinically relevant features (e.g., ratios of lipid versus collagen versus elastin) can be evaluated with a flexible-fiber based FLIM technique without the need for fluorescence intensity information or contrast agents.

  14. Optimizing Laguerre expansion based deconvolution methods for analysing bi-exponential fluorescence lifetime images.

    PubMed

    Zhang, Yongliang; Chen, Yu; Li, David Day-Uei

    2016-06-27

    Fast deconvolution is an essential step to calibrate instrument responses in big fluorescence lifetime imaging microscopy (FLIM) image analysis. This paper examined a computationally effective least squares deconvolution method based on Laguerre expansion (LSD-LE), recently developed for clinical diagnosis applications, and proposed new criteria for selecting Laguerre basis functions (LBFs) without considering the mutual orthonormalities between LBFs. Compared with the previously reported LSD-LE, the improved LSD-LE allows to use a higher laser repetition rate, reducing the acquisition time per measurement. Moreover, we extended it, for the first time, to analyze bi-exponential fluorescence decays for more general FLIM-FRET applications. The proposed method was tested on both synthesized bi-exponential and realistic FLIM data for studying the endocytosis of gold nanorods in Hek293 cells. Compared with the previously reported constrained LSD-LE, it shows promising results. PMID:27410552

  15. 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.

  16. Monitor RNA synthesis in live cell nuclei by using two-photon excited fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Peng, Xiao; Lin, Danying; Wang, Yan; Qi, Jing; Yan, Wei; Qu, Junle

    2015-03-01

    Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

  17. Statistical properties of amplitude and decay parameter estimators for fluorescence lifetime imaging.

    PubMed

    Kim, Jeongtae; Seok, Jiyeong

    2013-03-11

    We analyze the statistical properties of the maximum likelihood estimator, least squares estimator, and Pearson's χ(2)-based and Neyman's χ(2)-based estimators for the estimation of decay constants and amplitudes for fluorescence lifetime imaging. Our analysis is based on the linearization of the gradient of the objective functions around true parameters. The analysis shows that only the maximum likelihood estimator based on the Poisson likelihood function yields unbiased and efficient estimation. All other estimators yield either biased or inefficient estimations. We validate our analysis by using simulations. PMID:23482174

  18. A versatile fluorescence lifetime imaging system for scanning large areas with high time and spatial resolution

    NASA Astrophysics Data System (ADS)

    Bernardo, César; Belsley, Michael; de Matos Gomes, Etelvina; Gonçalves, Hugo; Isakov, Dmitry; Liebold, Falk; Pereira, Eduardo; Pires, Vladimiro; Samantilleke, Anura; Vasilevskiy, Mikhail; Schellenberg, Peter

    2014-08-01

    We present a flexible fluorescence lifetime imaging device which can be employed to scan large sample areas with a spatial resolution adjustable from many micrometers down to sub-micrometers and a temporal resolution of 20 picoseconds. Several different applications of the system will be presented including protein microarrays analysis, the scanning of historical samples, evaluation of solar cell surfaces and nanocrystalline organic crystals embedded in electrospun polymeric nanofibers. Energy transfer processes within semiconductor quantum dot superstructures as well as between dye probes and graphene layers were also investigated.

  19. Automated High-Throughput Fluorescence Lifetime Imaging Microscopy to Detect Protein-Protein Interactions.

    PubMed

    Guzmán, Camilo; Oetken-Lindholm, Christina; Abankwa, Daniel

    2016-04-01

    Fluorescence resonance energy transfer (FRET) is widely used to study conformational changes of macromolecules and protein-protein, protein-nucleic acid, and protein-small molecule interactions. FRET biosensors can serve as valuable secondary assays in drug discovery and for target validation in mammalian cells. Fluorescence lifetime imaging microscopy (FLIM) allows precise quantification of the FRET efficiency in intact cells, as FLIM is independent of fluorophore concentration, detection efficiency, and fluorescence intensity. We have developed an automated FLIM system using a commercial frequency domain FLIM attachment (Lambert Instruments) for wide-field imaging. Our automated FLIM system is capable of imaging and analyzing up to 50 different positions of a slide in less than 4 min, or the inner 60 wells of a 96-well plate in less than 20 min. Automation is achieved using a motorized stage and controller (Prior Scientific) coupled with a Zeiss Axio Observer body and full integration into the Lambert Instruments FLIM acquisition software. As an application example, we analyze the interaction of the oncoprotein Ras and its effector Raf after drug treatment. In conclusion, our automated FLIM imaging system requires only commercial components and may therefore allow for a broader use of this technique in chemogenomics projects. PMID:26384400

  20. Nanoscale fluorescence lifetime imaging of an optical antenna with a single diamond NV center.

    PubMed

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

    2013-08-14

    Solid-state quantum emitters, such as artificially engineered quantum dots or naturally occurring defects in solids, are being investigated for applications ranging from quantum information science and optoelectronics to biomedical imaging. Recently, these same systems have also been studied from the perspective of nanoscale metrology. In this letter, we study the near-field optical properties of a diamond nanocrystal hosting a single nitrogen vacancy center. We find that the nitrogen vacancy center is a sensitive probe of the surrounding electromagnetic mode structure. We exploit this sensitivity to demonstrate nanoscale fluorescence lifetime imaging microscopy (FLIM) with a single nitrogen vacancy center by imaging the local density of states of an optical antenna. PMID:23815462

  1. 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

  2. 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.

  3. A UV-Visible-NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

    NASA Astrophysics Data System (ADS)

    Urayama, P.; Zhong, W.; Beamish, J. A.; Minn, F. K.; Sloboda, R. D.; Dragnev, K. H.; Dmitrovsky, E.; Mycek, M.-A.

    This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV-visible-NIR excitation (337.1-960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime 1.4 ns) and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime 400 ns).

  4. 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.

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

    PubMed Central

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

    2015-01-01

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

  6. 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

  7. Three-dimensional fluorescence lifetime tomography

    SciTech Connect

    Godavarty, Anuradha; Sevick-Muraca, Eva M.; Eppstein, Margaret J.

    2005-04-01

    Near-infrared fluorescence tomography using molecularly targeted lifetime-sensitive, fluorescent contrast agents have applications for early-stage cancer diagnostics. Yet, although the measurement of fluorescent lifetime imaging microscopy (FLIM) is extensively used in microscopy and spectroscopy applications, demonstration of fluorescence lifetime tomography for medical imaging is limited to two-dimensional studies. Herein, the feasibility of three-dimensional fluorescence-lifetime tomography on clinically relevant phantom volumes is established, using (i) a gain-modulated intensified charge coupled device (CCD) and modulated laser diode imaging system, (ii) two fluorescent contrast agents, e.g., Indocyanine green and 3-3'-Diethylthiatricarbocyanine iodide differing in their fluorescence lifetime by 0.62 ns, and (iii) a two stage approximate extended Kalman filter reconstruction algorithm. Fluorescence measurements of phase and amplitude were acquired on the phantom surface under different target to background fluorescence absorption (70:1, 100:1) and fluorescence lifetime (1:1, 2.1:1) contrasts at target depths of 1.4-2 cm. The Bayesian tomography algorithm was employed to obtain three-dimensional images of lifetime and absorption owing to the fluorophores.

  8. Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells

    NASA Astrophysics Data System (ADS)

    Takahashi, Noriko; Sawada, Wakako; Noguchi, Jun; Watanabe, Satoshi; Ucar, Hasan; Hayashi-Takagi, Akiko; Yagishita, Sho; Ohno, Mitsuyo; Tokumaru, Hiroshi; Kasai, Haruo

    2015-10-01

    It remains unclear how readiness for Ca2+-dependent exocytosis depends on varying degrees of SNARE complex assembly. Here we directly investigate the SNARE assembly using two-photon fluorescence lifetime imaging (FLIM) of Förster resonance energy transfer (FRET) between three pairs of neuronal SNAREs in presynaptic boutons and pancreatic β cells in the islets of Langerhans. These FRET probes functionally rescue their endogenous counterparts, supporting ultrafast exocytosis. We show that trans-SNARE complexes accumulated in the active zone, and estimate the number of complexes associated with each docked vesicle. In contrast, SNAREs were unassembled in resting state, and assembled only shortly prior to insulin exocytosis, which proceeds slowly. We thus demonstrate that distinct states of fusion readiness are associated with SNARE complex formation. Our FRET/FLIM approaches enable optical imaging of fusion readiness in both live and chemically fixed tissues.

  9. Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells

    PubMed Central

    Takahashi, Noriko; Sawada, Wakako; Noguchi, Jun; Watanabe, Satoshi; Ucar, Hasan; Hayashi-Takagi, Akiko; Yagishita, Sho; Ohno, Mitsuyo; Tokumaru, Hiroshi; Kasai, Haruo

    2015-01-01

    It remains unclear how readiness for Ca2+-dependent exocytosis depends on varying degrees of SNARE complex assembly. Here we directly investigate the SNARE assembly using two-photon fluorescence lifetime imaging (FLIM) of Förster resonance energy transfer (FRET) between three pairs of neuronal SNAREs in presynaptic boutons and pancreatic β cells in the islets of Langerhans. These FRET probes functionally rescue their endogenous counterparts, supporting ultrafast exocytosis. We show that trans-SNARE complexes accumulated in the active zone, and estimate the number of complexes associated with each docked vesicle. In contrast, SNAREs were unassembled in resting state, and assembled only shortly prior to insulin exocytosis, which proceeds slowly. We thus demonstrate that distinct states of fusion readiness are associated with SNARE complex formation. Our FRET/FLIM approaches enable optical imaging of fusion readiness in both live and chemically fixed tissues. PMID:26439845

  10. 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

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

    PubMed

    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

  12. Fluorescence lifetime imaging and intravascular ultrasound: co-registration study using ex vivo human coronaries.

    PubMed

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

    2015-01-01

    Fluorescence lifetime imaging (FLIM) has demonstrated potential for robust assessment of atherosclerotic plaques biochemical composition and for complementing conventional intravascular ultrasound (IVUS), which provides information on plaque morphology. The success of such a bi-modal imaging modality depends on accurate segmentation of the IVUS images and proper angular registration between these two modalities. This paper reports a novel IVUS segmentation methodology addressing this issue. The image preprocessing consisted of denoising, using the Wiener filter, followed by image smoothing, implemented through the application of the alternating sequential filter on the edge separability metric images. Extraction of the lumen/intima and media/adventitia boundaries was achieved by tracing the gray-scale peaks over the A-lines of the IVUS preprocessed images. Cubic spline interpolation, in both cross-sectional and longitudinal directions, ensured boundary smoothness and continuity. The detection of the guide-wire artifact in both modalities is used for angular registration. Intraluminal studies were conducted in 13 ex vivo segments of human coronaries. The IVUS segmentation accuracy was assessed against independent manual tracings, providing 91.82% sensitivity and 97.55% specificity. The proposed methodology makes the bi-modal FLIM and IVUS approach feasible for comprehensive intravascular diagnosis by providing co-registered biochemical and morphological information of atherosclerotic plaques. PMID:25163056

  13. Fluorescence Lifetime Imaging and Intravascular Ultrasound: Co-Registration Study Using Ex Vivo Human Coronaries

    PubMed Central

    Gorpas, Dimitris; Fatakdawala, Hussain; Bec, Julien; Ma, Dinglong; Yankelevich, Diego R.; Qi, Jinyi

    2015-01-01

    Fluorescence lifetime imaging (FLIM) has demonstrated potential for robust assessment of atherosclerotic plaques biochemical composition and for complementing conventional intravascular ultrasound (IVUS), which provides information on plaque morphology. The success of such a bi-modal imaging modality depends on accurate segmentation of the IVUS images and proper angular registration between these two modalities. This paper reports a novel IVUS segmentation methodology addressing this issue. The image preprocessing consisted of denoising, using the Wiener filter, followed by image smoothing, implemented through the application of the alternating sequential filter on the edge separability metric images. Extraction of the lumen/intima and media/adventitia boundaries was achieved by tracing the gray-scale peaks over the A-lines of the IVUS preprocessed images. Cubic spline interpolation, in both cross-sectional and longitudinal directions, ensured boundary smoothness and continuity. The detection of the guide-wire artifact in both modalities is used for angular registration. Intraluminal studies were conducted in 13 ex vivo segments of human coronaries. The IVUS segmentation accuracy was assessed against independent manual tracings, providing 91.82% sensitivity and 97.55% specificity. The proposed methodology makes the bi-modal FLIM and IVUS approach feasible for comprehensive intravascular diagnosis by providing co-registered biochemical and morphological information of atherosclerotic plaques. PMID:25163056

  14. In vivo wound healing diagnosis with second harmonic and fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Deka, Gitanjal; Wu, Wei-Wen; Kao, Fu-Jen

    2013-06-01

    Skin wounds heal when a series of cell lineages are triggered, followed by collagen deposition, to reconstruct damaged tissues. This study evaluates the regeneration of collagen and change in cellular metabolic rate in vivo during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy respectively. The metabolic rate of cells is reflected through the lifetime of the autofluorescence from the co-enzyme protein, reduced nicotinamide adenine dinucleotide, due to its change in the relative concentration of bound and free forms. A higher than normal cellular metabolic rate is observed during the first week of healing, which decreases gradually after eight days of wound formation. SHG signal intensity change indicates the net degradation of collagen during the inflammatory phase, and net regeneration begins on day five. Eventually, the quantity of collagen increases gradually to form a scar tissue as the final product. Importantly, this work demonstrates the feasibility of an in vivo imaging approach for a normal wound on rat skin, which has the potential to supplement the noninvasive clinical diagnosis of wounds.

  15. In vivo wound healing diagnosis with second harmonic and fluorescence lifetime imaging.

    PubMed

    Deka, Gitanjal; Wu, Wei-Wen; Kao, Fu-Jen

    2013-06-01

    Skin wounds heal when a series of cell lineages are triggered, followed by collagen deposition, to reconstruct damaged tissues. This study evaluates the regeneration of collagen and change in cellular metabolic rate in vivo during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy respectively. The metabolic rate of cells is reflected through the lifetime of the autofluorescence from the co-enzyme protein, reduced nicotinamide adenine dinucleotide, due to its change in the relative concentration of bound and free forms. A higher than normal cellular metabolic rate is observed during the first week of healing, which decreases gradually after eight days of wound formation. SHG signal intensity change indicates the net degradation of collagen during the inflammatory phase, and net regeneration begins on day five. Eventually, the quantity of collagen increases gradually to form a scar tissue as the final product. Importantly, this work demonstrates the feasibility of an in vivo imaging approach for a normal wound on rat skin, which has the potential to supplement the noninvasive clinical diagnosis of wounds. PMID:23264966

  16. In vivo wound healing diagnosis with second harmonic and fluorescence lifetime imaging.

    PubMed

    Deka, Gitanjal; Wu, Wei-Wen; Kao, Fu-Jen

    2013-06-01

    Skin wounds heal when a series of cell lineages are triggered, followed by collagen deposition, to reconstruct damaged tissues. This study evaluates the regeneration of collagen and change in cellular metabolic rate in vivo during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy respectively. The metabolic rate of cells is reflected through the lifetime of the autofluorescence from the co-enzyme protein, reduced nicotinamide adenine dinucleotide, due to its change in the relative concentration of bound and free forms. A higher than normal cellular metabolic rate is observed during the first week of healing, which decreases gradually after eight days of wound formation. SHG signal intensity change indicates the net degradation of collagen during the inflammatory phase, and net regeneration begins on day five. Eventually, the quantity of collagen increases gradually to form a scar tissue as the final product. Importantly, this work demonstrates the feasibility of an in vivo imaging approach for a normal wound on rat skin, which has the potential to supplement the noninvasive clinical diagnosis of wounds. PMID:23748703

  17. 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

  18. 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. PMID:27239747

  19. Investigation of signal-to-noise ratio in frequency-domain multiphoton fluorescence lifetime imaging microscopy.

    PubMed

    Zhang, Yide; Khan, Aamir A; Vigil, Genevieve D; Howard, Scott S

    2016-07-01

    Multiphoton microscopy (MPM) combined with fluorescence lifetime imaging microscopy (FLIM) has enabled three-dimensional quantitative molecular microscopy in vivo. The signal-to-noise ratio (SNR), and thus the imaging rate of MPM-FLIM, which is fundamentally limited by the shot noise and fluorescence saturation, has not been quantitatively studied yet. In this paper, we investigate the SNR performance of the frequency-domain (FD) MPM-FLIM with two figures of merit: the photon economy in the limit of shot noise, and the normalized SNR in the limit of saturation. The theoretical results and Monte Carlo simulations find that two-photon FD-FLIM requires 50% fewer photons to achieve the same SNR as conventional one-photon FLIM. We also analytically show that the MPM-FD-FLIM can exploit the DC and higher harmonic components generated by nonlinear optical mixing of the excitation light to improve SNR, reducing the required number of photons by an additional 50%. Finally, the effect of fluorophore saturation on the experimental SNR performance is discussed. PMID:27409702

  20. Real-time analysis of metabolic activity within Lactobacillus acidophilus by phasor fluorescence lifetime imaging microscopy of NADH.

    PubMed

    Torno, Keenan; Wright, Belinda K; Jones, Mark R; Digman, Michelle A; Gratton, Enrico; Phillips, Michael

    2013-04-01

    Nicotinamide adenine dinucleotide (NADH) is an endogenous fluorescent molecule commonly used as a metabolic biomarker. Fluorescence lifetime imaging microscopy (FLIM) is a method in which the fluorescence decay is measured at each pixel of an image. While the fluorescence spectrum of free and protein-bound NADH is very similar, free and protein-bound NADH display very different decay profiles. Therefore, FLIM can provide a way to distinguish free/bound NADH at the level of single bacteria within biological samples. The phasor technique is a graphical method to analyse the entire image and to produce a histogram of pixels with different decay profile. In this study, NADH fluorescence decay profiles within Lactobacillus acidophilus samples treated using different protocols indicated discernible variations. Clear distinctions between fluorescence decay profiles of NADH in samples of artificially heightened metabolic activity in comparison to those of samples lacking an accessible carbon source were obtained. PMID:23233088

  1. 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

  2. Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy.

    PubMed

    Day, Richard N

    2014-03-15

    The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster resonance energy transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the 'FRET standard' fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein-protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1α). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBPα. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. PMID:23806643

  3. Persistent luminescence nanoprobe for biosensing and lifetime imaging of cell apoptosis via time-resolved fluorescence resonance energy transfer.

    PubMed

    Zhang, Lei; Lei, Jianping; Liu, Jintong; Ma, Fengjiao; Ju, Huangxian

    2015-10-01

    Time-resolved fluorescence technique can reduce the short-lived background luminescence and auto-fluorescence interference from cells and tissues by exerting the delay time between pulsed excitation light and signal acquisition. Here, we prepared persistent luminescence nanoparticles (PLNPs) to design a universal time-resolved fluorescence resonance energy transfer (TR-FRET) platform for biosensing, lifetime imaging of cell apoptosis and in situ lifetime quantification of intracellular caspase-3. Three kinds of PLNPs-based nanoprobes are assembled by covalently binding dye-labeled peptides or DNA to carboxyl-functionalized PLNPs for the efficient detection of caspase-3, microRNA and protein. The peptides-functionalized nanoprobe is also employed for fluorescence lifetime imaging to monitor cell apoptosis, which shows a dependence of cellular fluorescence lifetime on caspase-3 activity and thus leads to an in situ quantification method. This work provides a proof-of-concept for PLNPs-based TR-FRET analysis and demonstrates its potential in exploring dynamical information of life process. PMID:26232881

  4. 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

  5. Fluorescence Characteristics and Lifetime Images of Photosensitizers of Talaporfin Sodium and Sodium Pheophorbide a in Normal and Cancer Cells

    PubMed Central

    Awasthi, Kamlesh; Yamamoto, Kazuhito; Furuya, Kazunari; Nakabayashi, Takakazu; Li, Liming; Ohta, Nobuhiro

    2015-01-01

    Fluorescence spectra and fluorescence lifetime images of talaporfin sodium and sodium-pheophorbide a, which can be regarded as photosensitizers for photodynamic therapy, were measured in normal and cancer cells. The reduction of the fluorescence intensity by photoirradiation was observed for both photosensitizers in both cells, but the quenching rate was much faster in cancer cells than in normal cells. These results are explained in terms of the excessive generation of reactive oxygen species via photoexcitation of these photosensitizers in cancer cells. The fluorescence lifetimes of both photosensitizers in cancer cells are different from those in normal cells, which originates from the different intracellular environments around the photosensitizers between normal and cancer cells. PMID:25993516

  6. Detection of enzyme activity in orthotopic murine breast cancer by fluorescence lifetime imaging using a fluorescence resonance energy transfer-based molecular probe

    NASA Astrophysics Data System (ADS)

    Solomon, Metasebya; Guo, Kevin; Sudlow, Gail P.; Berezin, Mikhail Y.; Edwards, W. Barry; Achilefu, Samuel; Akers, Walter J.

    2011-06-01

    Cancer-related enzyme activity can be detected noninvasively using activatable fluorescent molecular probes. In contrast to ``always-on'' fluorescent molecular probes, activatable probes are relatively nonfluorescent at the time of administration due to intramolecular fluorescence resonance energy transfer (FRET). Enzyme-mediated hydrolysis of peptide linkers results in reduced FRET and increase of fluorescence yield. Separation of signal from active and inactive probe can be difficult with conventional intensity-based fluorescence imaging. Fluorescence lifetime (FLT) measurement is an alternative method to detect changes in FRET. Thus, we investigate FLT imaging for in vivo detection of FRET-based molecular probe activation in an orthotopic breast cancer model. Indeed, the measured FLT of the enzyme-activatable molecular probe increases from 0.62 ns just after injection to 0.78 ns in tumor tissue after 4 h. A significant increase in FLT is not observed for an always-on targeted molecular probe with the same fluorescent reporter. These results show that FLT contrast is a powerful addition to preclinical imaging because it can report molecular activity in vivo due to changes in FRET. Fluorescence lifetime imaging exploits unique characteristics of fluorescent molecular probes that can be further translated into clinical applications, including noninvasive detection of cancer-related enzyme activity.

  7. Support vector machine based classification and mapping of atherosclerotic plaques using fluorescence lifetime imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Fatakdawala, Hussain; Gorpas, Dimitris S.; Bec, Julien; Ma, Dinglong M.; Yankelevich, Diego R.; Bishop, John W.; Marcu, Laura

    2016-02-01

    The progression of atherosclerosis in coronary vessels involves distinct pathological changes in the vessel wall. These changes manifest in the formation of a variety of plaque sub-types. The ability to detect and distinguish these plaques, especially thin-cap fibroatheromas (TCFA) may be relevant for guiding percutaneous coronary intervention as well as investigating new therapeutics. In this work we demonstrate the ability of fluorescence lifetime imaging (FLIm) derived parameters (lifetime values from sub-bands 390/40 nm, 452/45 nm and 542/50 nm respectively) for generating classification maps for identifying eight different atherosclerotic plaque sub-types in ex vivo human coronary vessels. The classification was performed using a support vector machine based classifier that was built from data gathered from sixteen coronary vessels in a previous study. This classifier was validated in the current study using an independent set of FLIm data acquired from four additional coronary vessels with a new rotational FLIm system. Classification maps were compared to co-registered histological data. Results show that the classification maps allow identification of the eight different plaque sub-types despite the fact that new data was gathered with a different FLIm system. Regions with diffuse intimal thickening (n=10), fibrotic tissue (n=2) and thick-cap fibroatheroma (n=1) were correctly identified on the classification map. The ability to identify different plaque types using FLIm data alone may serve as a powerful clinical and research tool for studying atherosclerosis in animal models as well as in humans.

  8. Flavin fluorescence lifetime imaging of living peripheral blood mononuclear cells on micro and nano-structured surfaces

    NASA Astrophysics Data System (ADS)

    Teplicky, T.; Horilova, J.; Bruncko, J.; Gladine, C.; Lajdova, I.; Mateasik, A.; Chorvat, D.; Marcek Chorvatova, A.

    2015-03-01

    Fabricated micro- and nano-structured surfaces were evaluated for use with living cells. Metabolic state was tested by means of endogenous flavin fluorescence of living peripheral blood mononuclear cells (PBMC) positioned on a coverslip, non-covered, or covered with micro- or nano-structured surfaces (OrmoComp polymer structures produced by 2-photon photopolymerisation, or Zinc Oxide (ZnO) layer fabricated by pulsed laser deposition). Confocal microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) were employed to gather flavin fluorescence lifetime images of living PBMC on structured surfaces. Gathered data are the first step towards monitoring of the live cell interaction with different micro/nano-structured surfaces and thus evaluate their potential applicability in the biomedical field.

  9. Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols.

    PubMed

    Mueller-Harvey, Irene; Feucht, Walter; Polster, Juergen; Trnková, Lucie; Burgos, Pierre; Parker, Anthony W; Botchway, Stanley W

    2012-03-16

    Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ~1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime (τ(2)=1.9-3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there is significant nuclear absorption of flavanols. This advanced imaging using two-photon excitation and biophysical techniques described here will prove valuable for probing the intracellular trafficking and functions of flavanols, such as EGCG, which is the major flavanol of green tea. PMID:22340533

  10. Nuclear uptake of ultrasmall gold-doxorubicin conjugates imaged by fluorescence lifetime imaging microscopy (FLIM) and electron microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Xuan; Shastry, Sathvik; Bradforth, Stephen E.; Nadeau, Jay L.

    2014-11-01

    Fluorescence lifetime imaging microscopy (FLIM) has been used to image free and encapsulated doxorubicin (Dox) uptake into cells, since interaction of Dox with DNA leads to a characteristic lifetime change. However, none of the reported Dox conjugates were able to enter cell nuclei. In this work, we use FLIM to show nuclear uptake of 2.7 nm mean diameter Au nanoparticles conjugated to Dox. The pattern of labelling differed substantially from what was seen with free Dox, with slower nuclear entry and stronger cytoplasmic labelling at all time points. As the cells died, the pattern of labelling changed further as intracellular structures disintegrated, consistent with association of Au-Dox to membranes. The patterns of Au distribution and intracellular structure changes were confirmed using electron microscopy, and indicate different mechanisms of cytotoxicity with stable Au-Dox conjugates compared to Dox alone. Such conjugates are promising tools for overcoming resistance in Dox-resistant cancers.Fluorescence lifetime imaging microscopy (FLIM) has been used to image free and encapsulated doxorubicin (Dox) uptake into cells, since interaction of Dox with DNA leads to a characteristic lifetime change. However, none of the reported Dox conjugates were able to enter cell nuclei. In this work, we use FLIM to show nuclear uptake of 2.7 nm mean diameter Au nanoparticles conjugated to Dox. The pattern of labelling differed substantially from what was seen with free Dox, with slower nuclear entry and stronger cytoplasmic labelling at all time points. As the cells died, the pattern of labelling changed further as intracellular structures disintegrated, consistent with association of Au-Dox to membranes. The patterns of Au distribution and intracellular structure changes were confirmed using electron microscopy, and indicate different mechanisms of cytotoxicity with stable Au-Dox conjugates compared to Dox alone. Such conjugates are promising tools for overcoming resistance in

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    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

  13. 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

  14. 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

  15. Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm.

    PubMed

    Li, David D-U; Arlt, Jochen; Tyndall, David; Walker, Richard; Richardson, Justin; Stoppa, David; Charbon, Edoardo; Henderson, Robert K

    2011-09-01

    A high-speed and hardware-only algorithm using a center of mass method has been proposed for single-detector fluorescence lifetime sensing applications. This algorithm is now implemented on a field programmable gate array to provide fast lifetime estimates from a 32 × 32 low dark count 0.13 μm complementary metal-oxide-semiconductor single-photon avalanche diode (SPAD) plus time-to-digital converter array. A simple look-up table is included to enhance the lifetime resolvability range and photon economics, making it comparable to the commonly used least-square method and maximum-likelihood estimation based software. To demonstrate its performance, a widefield microscope was adapted to accommodate the SPAD array and image different test samples. Fluorescence lifetime imaging microscopy on fluorescent beads in Rhodamine 6G at a frame rate of 50 fps is also shown. PMID:21950926

  16. 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.

  17. 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.

  18. phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy.

    PubMed

    Van Munster, E B; Gadella, T W J

    2004-01-01

    In conventional wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity-modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded at various phase differences between excitation and intensifier gain modulation, the phase and modulation depth of the emitted light is obtained. The fluorescence lifetime is determined from the delay and the decrease in modulation depth of the emission relative to the excitation. A minimum of three images is required, but in this case measurements become susceptible to aliasing caused by the presence of higher harmonics. Taking more images to avoid this is not always possible owing to phototoxicity or movement. A method is introduced, phiFLIM, requiring only three recordings that is not susceptible to aliasing. The phase difference between the excitation and the intensifier is scanned over the entire 360 degrees range following a predefined phase profile, during which the image produced by the intensifier is integrated onto the CCD camera, yielding a single image. Three different images are produced following this procedure, each with a different phase profile. Measurements were performed with a conventional wide-field frequency-domain FLIM system based on an acousto-optic modulator for modulation of the excitation and a microchannel-plate image intensifier coupled to a CCD camera for the detection. By analysis of the harmonic content of measured signals it was found that the third harmonic was effectively the highest present. Using the conventional method with three recordings, phase errors due to aliasing of up to +/- 29 degrees and modulation depth errors of up to 30% were found. Errors in lifetimes of YFP-transfected HeLa cells were as high as 100%. With phiFLIM, using the same specimen and settings, systematic errors due to aliasing did not occur. PMID:14678510

  19. 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). PMID:25755202

  20. 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

  1. 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

  2. Quantification of the Metabolic State in Cell-Model of Parkinson’s Disease by Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Chakraborty, Sandeep; Nian, Fang-Shin; Tsai, Jin-Wu; Karmenyan, Artashes; Chiou, Arthur

    2016-01-01

    Intracellular endogenous fluorescent co-enzymes, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), play a pivotal role in cellular metabolism; quantitative assessment of their presence in living cells can be exploited to monitor cellular energetics in Parkinson’s disease (PD), a neurodegenerative disorder. Here, we applied two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to noninvasively measure the fluorescence lifetime components of NADH and FAD, and their relative contributions in MPP+ (1-methyl-4-phenylpyridinium) treated neuronal cells, derived from PC12 cells treated with nerve growth factor (NGF), to mimic PD conditions. A systematic FLIM data analysis showed a statistically significant (p < 0.001) decrease in the fluorescence lifetime of both free and protein-bound NADH, as well as free and protein-bound FAD in MPP+ treated cells. On the relative contributions of the free and protein-bound NADH and FAD to the life time, however, both the free NADH contribution and the corresponding protein-bound FAD contribution increase significantly (p < 0.001) in MPP+ treated cells, compared to control cells. These results, which indicate a shift in energy production in the MPP+ treated cells from oxidative phosphorylation towards anaerobic glycolysis, can potentially be used as cellular metabolic metrics to assess the condition of PD at the cellular level. PMID:26758390

  3. Mapping of intracellular concentrations of macromolecules by two-photon excited fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Liu, Lixin; Pliss, Artem; Peng, Xiao; Kuzmin, Andrey; Qu, Junle; Prasad, Paras N.

    2016-03-01

    Measurements and monitoring of concentrations of macromolecules in live cells and sub-cellular structures is of tremendous interest in cell biology and translational medicine. In this report we demonstrate a breakthrough potential of FLIM for real-time quantitative mapping of macromolecular distribution in the cell. In our approach we exploit a correlation existing between the fluorescence lifetime of fluorophores, refractive index and local concentrations of cellular macromolecules in the of fluorophore's microenvironment. We show a value of our approach for fundamental cell science and cellular diagnostic assays.

  4. Spectral variation of fluorescence lifetime near single metal nanoparticles

    NASA Astrophysics Data System (ADS)

    Li, Jia; Krasavin, Alexey V.; Webster, Linden; Segovia, Paulina; Zayats, Anatoly V.; Richards, David

    2016-02-01

    We explore the spectral dependence of fluorescence enhancement and the associated lifetime modification of fluorescent molecules coupled to single metal nanoparticles. Fluorescence lifetime imaging microscopy and single-particle dark-field spectroscopy are combined to correlate the dependence of fluorescence lifetime reduction on the spectral overlap between the fluorescence emission and the localised surface plasmon (LSP) spectra of individual gold nanoparticles. A maximum lifetime reduction is observed when the fluorescence and LSP resonances coincide, with good agreement provided by numerical simulations. The explicit comparison between experiment and simulation, that we obtain, offers an insight into the spectral engineering of LSP mediated fluorescence and may lead to optimized application in sensing and biomedicine.

  5. 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.

  6. Fluorescence lifetime imaging using a compact, low-cost, diode-based all-solid-state regenerative amplifier

    NASA Astrophysics Data System (ADS)

    Mendez, E.; Elson, D. S.; Koeberg, M.; Dunsby, C.; Bradley, D. D. C.; French, P. M. W.

    2004-05-01

    A fluorescence lifetime imaging (FLIM) system is described that utilizes a new compact and low-cost ultrafast laser source based on a gain-switched laser diode-seeded all-solid-state Cr:LiSAF regenerative amplifier that has been designed for this application. The pulse parameters of this source (0.5 μJ, 827 nm, 100 ps, 5 kHz) are shown to be appropriate to time-domain FLIM using a gated optical intensifier and the application to functional imaging of biological tissue is demonstrated, as well as the first evaluation of organic light emitting diodes using FLIM.

  7. Combined nonlinear laser imaging (two-photon excitation fluorescence, second and third-harmonic generation, and fluorescence lifetime imaging microscopies) in ovarian tumors

    NASA Astrophysics Data System (ADS)

    Adur, J.; Pelegati, V. B.; de Thomaz, A. A.; Bottcher-Luiz, F.; Andrade, L. A. L. A.; Almeida, D. B.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue.

  8. 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

  9. Fluorescence lifetime distributions in proteins.

    PubMed Central

    Alcala, J. R.; Gratton, E.; Prendergast, F. G.

    1987-01-01

    The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most proteins can be satisfactorily described only using several exponential components. Here it is proposed that continuous lifetime distributions can better represent the observed decay. An approach based on protein dynamics is presented, which provides fluorescence lifetime distribution functions for single tryptophan residue proteins. First, lifetime distributions for proteins interconverting between two conformations, each characterized by a different lifetime value, are derived. The evolution of the lifetime values as a function of the interconversion rate is studied. In this case lifetime distributions can be obtained from a distribution of rates of interconversion between the two conformations. Second, the existence of a continuum of energy substates within a given conformation was considered. The occupation of a particular energy substate at a given temperature is proportional to the Boltzmann factor. The density of energy states of the potential well depends upon the width of the well, which determines the degree of freedom the residue can move in the conformational space. Lifetime distributions can be obtained by association of each energy substate with a different lifetime value and assuming that the average conformation can change as the energy of the substate is increased. Finally, lifetime distributions for proteins interconverting between two conformations, each characterized by a quasi-continuum of energy substates, are presented. The origin of negative components

  10. Pinhole shifting lifetime imaging microscopy.

    PubMed

    Ramshesh, Venkat K; Lemasters, John J

    2008-01-01

    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 (Eu(3+)), 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 Eu(3+) 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 micros was estimated for the Eu(3+) microspheres, consistent with independent measurements. This simple adaptation is the basis for quantitative 3-D lifetime imaging microscopy. PMID:19123648

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

    PubMed Central

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

    2012-01-01

    Abstract. 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. PMID:23224011

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Spectroscopy and Fluorescence Lifetime Imaging Microscopy To Probe the Interaction of Bovine Serum Albumin with Graphene Oxide.

    PubMed

    Kuchlyan, Jagannath; Kundu, Niloy; Banik, Debasis; Roy, Arpita; Sarkar, Nilmoni

    2015-12-29

    The interaction of graphene oxide (GO) with bovine serum albumin (BSA) in aqueous buffer solution has been investigated with various spectroscopic and imaging techniques. At single molecular resolution this interaction has been performed using fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) techniques. The conformational dynamics of BSA on GO's influence have been explored by FCS and circular dichroism (CD) spectroscopy. For the FCS studies BSA was labeled covalently by a fluorophore, Alexa Fluor 488. On the addition of GO in phosphate buffer of 10 mM at pH 7.4 the diffusion time (τD) and the hydrodynamic radius (Rh) of BSA increase due to adsorption of BSA. Conformational relaxation time components of native BSA drastically vary with the addition of GO, signifying the change of conformational dynamics of BSA after addition of GO. The adsorption isotherm also indicates significant adsorption of BSA on the GO surface. Adsorption of BSA on the GO surface has shown in direct images of atomic force microscopy (AFM) and FLIM. Fluorescence quenching study of BSA with addition of GO also indicates that there is strong interaction between BSA and GO. PMID:26646418

  14. Time-domain fluorescence lifetime imaging microscopy: a quantitative method to follow transient protein-protein interactions in living cells.

    PubMed

    Padilla-Parra, Sergi; Audugé, Nicolas; Tramier, Marc; Coppey-Moisan, Maïté

    2015-06-01

    Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein-protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value (E) and the fraction of the donor-tagged protein that undergoes FRET (fD). To simultaneously obtain fD and E values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET (mfD), obtained from the mathematical minimization of fD. Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that mfD constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells. PMID:26034312

  15. Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM).

    PubMed

    Margineanu, Anca; Chan, Jia Jia; Kelly, Douglas J; Warren, Sean C; Flatters, Delphine; Kumar, Sunil; Katan, Matilda; Dunsby, Christopher W; French, Paul M W

    2016-01-01

    We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements. PMID:27339025

  16. Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

    PubMed Central

    Margineanu, Anca; Chan, Jia Jia; Kelly, Douglas J.; Warren, Sean C.; Flatters, Delphine; Kumar, Sunil; Katan, Matilda; Dunsby, Christopher W.; French, Paul M. W.

    2016-01-01

    We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements. PMID:27339025

  17. Asante Calcium Green and Asante Calcium Red—Novel Calcium Indicators for Two-Photon Fluorescence Lifetime Imaging

    PubMed Central

    Jahn, Karolina; Hille, Carsten

    2014-01-01

    For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720–900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca2+-free and Ca2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca2+-dependent way, unraveling in vitro dissociation constants KD of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca2+-free and Ca2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ KD of 180 nM was determined. Thus, quantitative [Ca2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems. PMID:25140519

  18. Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

    PubMed

    Jahn, Karolina; Hille, Carsten

    2014-01-01

    For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+)-free and Ca(2+)-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+)-dependent way, unraveling in vitro dissociation constants K(D) of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca(2+)-free and Ca(2+)-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D) of 180 nM was determined. Thus, quantitative [Ca(2+)]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+)]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+) indicator dye for 2P-FLIM recordings applicable in diverse biological systems. PMID:25140519

  19. 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. PMID:26500051

  20. 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.

  1. 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

  2. A dual-modality optical coherence tomography and fluorescence lifetime imaging microscopy system for simultaneous morphological and biochemical tissue characterization

    PubMed Central

    Park, Jesung; Jo, Javier A.; Shrestha, Sebina; Pande, Paritosh; Wan, Qiujie; Applegate, Brian E.

    2010-01-01

    Most pathological conditions elicit changes in the tissue optical response that may be interrogated by one or more optical imaging modalities. Any single modality typically only furnishes an incomplete picture of the tissue optical response, hence an approach that integrates complementary optical imaging modalities is needed for a more comprehensive non-destructive and minimally-invasive tissue characterization. We have developed a dual-modality system, incorporating optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM), that is capable of simultaneously characterizing the 3-D tissue morphology and its biochemical composition. The Fourier domain OCT subsystem, at an 830 nm center wavelength, provided high-resolution morphological volumetric tissue images with an axial and lateral resolution of 7.3 and 13.4 µm, respectively. The multispectral FLIM subsystem, based on a direct pulse-recording approach (upon 355 nm laser excitation), provided two-dimensional superficial maps of the tissue autofluorescence intensity and lifetime at three customizable emission bands with 100 µm lateral resolution. Both subsystems share the same excitation/illumination optical path and are simultaneously raster scanned on the sample to generate coregistered OCT volumes and FLIM images. The developed OCT/FLIM system was capable of a maximum A-line rate of 59 KHz for OCT and a pixel rate of up to 30 KHz for FLIM. The dual-modality system was validated with standard fluorophore solutions and subsequently applied to the characterization of two biological tissue types: postmortem human coronary atherosclerotic plaques, and in vivo normal and cancerous hamster cheek pouch epithelial tissue. PMID:21258457

  3. Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

    The epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by the heterochromatin protein 1 (HP1). In earlier studies, we showed that the CCAAT/enhancer-binding protein alpha (C/EBPα), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1α. Here, deletion and mutagenesis are combined with live-cell imaging approaches to characterize these protein interactions. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1α and the BZip protein, and co-expression studies indicated that the mobile fractions of these nuclear proteins diffuse independently of one another. The steady-state interactions of these proteins in regions of heterochromatin were monitored using Förster resonance energy transfer (FRET). A point mutation in HP1α, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1α W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1α or HP1αW174A. The functional significance of these interactions is discussed.

  4. Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis

    PubMed Central

    Barber, P.R.; Ameer-Beg, S.M.; Gilbey, J.; Carlin, L.M.; Keppler, M.; Ng, T.C.; Vojnovic, B.

    2008-01-01

    Förster resonance energy transfer (FRET) detected via fluorescence lifetime imaging microscopy (FLIM) and global analysis provide a way in which protein–protein interactions may be spatially localized and quantified within biological cells. The FRET efficiency and proportion of interacting molecules have been determined using bi-exponential fitting to time-domain FLIM data from a multiphoton time-correlated single-photon counting microscope system. The analysis has been made more robust to noise and significantly faster using global fitting, allowing higher spatial resolutions and/or lower acquisition times. Data have been simulated, as well as acquired from cell experiments, and the accuracy of a modified Levenberg–Marquardt fitting technique has been explored. Multi-image global analysis has been used to follow the epidermal growth factor-induced activation of Cdc42 in a short-image-interval time-lapse FLIM/FRET experiment. Our implementation offers practical analysis and time-resolved-image manipulation, which have been targeted towards providing fast execution, robustness to low photon counts, quantitative results and amenability to automation and batch processing.

  5. Microtubule Affinity Regulating Kinase Activity in Living Neurons Was Examined by a Genetically Encoded Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging-based Biosensor

    PubMed Central

    Timm, Thomas; von Kries, Jens Peter; Li, Xiaoyu; Zempel, Hans; Mandelkow, Eckhard; Mandelkow, Eva-Maria

    2011-01-01

    Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ∼18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons. PMID:21984823

  6. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging.

    PubMed

    Pliss, Artem; Peng, Xiao; Liu, Lixin; Kuzmin, Andrey; Wang, Yan; Qu, Junle; Li, Yuee; Prasad, Paras N

    2015-01-01

    Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine. PMID:26155309

  7. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging

    PubMed Central

    Pliss, Artem; Peng, Xiao; Liu, Lixin; Kuzmin, Andrey; Wang, Yan; Qu, Junle; Li, Yuee; Prasad, Paras N

    2015-01-01

    Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine. PMID:26155309

  8. 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.

  9. Spectral variation of fluorescence lifetime near single metal nanoparticles

    PubMed Central

    Li, Jia; Krasavin, Alexey V.; Webster, Linden; Segovia, Paulina; Zayats, Anatoly V.; Richards, David

    2016-01-01

    We explore the spectral dependence of fluorescence enhancement and the associated lifetime modification of fluorescent molecules coupled to single metal nanoparticles. Fluorescence lifetime imaging microscopy and single-particle dark-field spectroscopy are combined to correlate the dependence of fluorescence lifetime reduction on the spectral overlap between the fluorescence emission and the localised surface plasmon (LSP) spectra of individual gold nanoparticles. A maximum lifetime reduction is observed when the fluorescence and LSP resonances coincide, with good agreement provided by numerical simulations. The explicit comparison between experiment and simulation, that we obtain, offers an insight into the spectral engineering of LSP mediated fluorescence and may lead to optimized application in sensing and biomedicine. PMID:26876780

  10. Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy

    NASA Astrophysics Data System (ADS)

    Schweitzer, Dietrich; Deutsch, Lydia; Klemm, Matthias; Jentsch, Susanne; Hammer, Martin; Peters, Sven; Haueisen, Jens; Müller, Ulrich A.; Dawczynski, Jens

    2015-06-01

    The time-resolved autofluorescence of the eye is used for the detection of metabolic alteration in diabetic patients who have no signs of diabetic retinopathy. One eye from 37 phakic and 11 pseudophakic patients with type 2 diabetes, and one eye from 25 phakic and 23 pseudophakic healthy subjects were included in the study. After a three-exponential fit of the decay of autofluorescence, histograms of lifetimes τi, amplitudes αi, and relative contributions Qi were statistically compared between corresponding groups in two spectral channels (490lifetimes. Median and mean of the histograms of τ2, τ3, and α3 in ch1 show the greatest differences between phakic diabetic patients and age-matched controls (p<0.000004). The lack of pixels with a τ2 of ˜360 ps, the increased number of pixels with τ2>450 ps, and the shift of τ3 from ˜3000 to 3700 ps in ch1 of diabetic patients when compared with healthy subjects indicate an increased production of free flavin adenine dinucleotide, accumulation of advanced glycation end products (AGE), and, probably, a change from free to protein-bound reduced nicotinamide adenine dinucleotide at the fundus. AGE also accumulated in the crystalline lens.

  11. Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy.

    PubMed

    Schweitzer, Dietrich; Deutsch, Lydia; Klemm, Matthias; Jentsch, Susanne; Hammer, Martin; Peters, Sven; Haueisen, Jens; Müller, Ulrich A; Dawczynski, Jens

    2015-06-01

    The time-resolved autofluorescence of the eye is used for the detection of metabolic alteration in diabetic patients who have no signs of diabetic retinopathy. One eye from 37 phakic and 11 pseudophakic patients with type 2 diabetes, and one eye from 25 phakic and 23 pseudophakic healthy subjects were included n the study. After a three-exponential fit of the decay of autofluorescence, histograms of lifetimes τ(i), amplitudes α(i), and relative contributions Q(i) were statistically compared between corresponding groups in two spectral channels (490 < ch1 < 560 nm, 560 < ch2 < 700 nm). The change in single fluorophores was estimated by applying the Holm–Bonferroni method and by calculating differences in the sum histograms of lifetimes. Median and mean of the histograms of τ(2), τ(3), and α(3) in ch1 show the greatest differences between phakic diabetic patients and age-matched controls (p < 0.000004). The lack of pixels with a τ(2) of ∼360 ps, the increased number of pixels with τ(2) > 450 ps, and the shift of τ(3) from ∼3000 to 3700 ps in ch1 of diabetic patients when compared with healthy subjects indicate an increased production of free flavin adenine dinucleotide, accumulation of advanced glycation end products (AGE), and, probably, a change from free to protein-bound reduced nicotinamide adenine inucleotide at the fundus. AGE also accumulated in the crystalline lens. PMID:25769278

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

    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.

  13. Nanodimentional Aggregates In Organic Monolayers Studied With Atomic Force Microscopy (AFM) And Fluorescence Lifetime Imaging Microscopy (FLIM)

    NASA Astrophysics Data System (ADS)

    Ivanov, George R.; Burov, Julian

    2007-04-01

    Organic monolayers from a fluorescently labeled phospholipid (DPPE-NBD) were deposited on solid supports under special conditions that form stable nanometer wide bilayers cylinders that protrude from the monolayer. This molecule was frequently used in sensor applications due to its sensitivity to environment changes. The proposed configuration should provide both fast response times (ultra thin film) and increased sensitivity (greatly increased surface area). AFM can clearly distinguish between the different phases. The height difference between the solid-expanded and the liquid-expanded phase was measured to be 1.4 nm while the bilayer thickness was 5.6 nm. The solid domains show a 20 % decrease in fluorescence lifetime in comparison to the monolayer as measured by FLIM. This difference in lifetimes is explained in the model of fluorescence self quenching in the solid phase due to the molecules being closer to each other.

  14. Fluorescence lifetimes of some Rauwolfia alkaloids

    NASA Astrophysics Data System (ADS)

    Hidalgo, J.; Arjona, D. Gonzalez; Roldan, E.; Sanchez, M.

    1986-03-01

    The natural fluorescence lifetimes of the following Rauwolfia alkaloids, Reserpine, Rescinnamine, Corynanthine, Yohimbine, --- Ajmalicine, Serpentine and Ajmaline, have been calculated from a modified form of the Strickler-Berg equation. The actual lifetimes were derived from the quantum yields and the calculated natural lifetimes.

  15. Bi-modal imaging of atherosclerotic plaques: Automated method for co-registration between fluorescence lifetime imaging and intravascular ultrasound data

    NASA Astrophysics Data System (ADS)

    Gorpas, Dimitris; Fatakdawala, Hussain; Bec, Julien; Ma, Dinglong; Yankelevich, Diego R.; Bishop, John W.; Qi, Jinyi; Marcu, Laura

    2014-03-01

    The risk of atherosclerosis plaque rupture cannot be assessed by the current imaging systems and thus new multi-modal technologies are under investigation. This includes combining a new fluorescence lifetime imaging (FLIm) technique, which is sensitive to plaque biochemical features, with conventional intravascular ultrasound (IVUS), which provides information on plaque morphology. In this study we present an automated method allowing for the co-registration of imaging data acquired based on these two techniques. Intraluminal studies were conducted in ex-vivo segments of human coronaries with a multimodal catheter integrating a commercial IVUS (40 MHz) and a rotational side-viewing fiber based multispectral FLIm system (355 nm excitation, 390+/-20, 452+/-22 and 542+/-25 nm acquisition wavelengths). The proposed method relies on the lumen/intima boundary extraction from the IVUS polar images. Image restoration is applied for the noise reduction and edge enhancement, while gray-scale peak tracing over the A-lines of the IVUS polar images is applied for the lumen boundary extraction. The detection of the guide-wire artifact is used for the angular registration between FLIm and IVUS data, after which the lifetime values can be mapped onto the segmented lumen/intima interface. The segmentation accuracy has been assessed against manual tracings, providing 0.120+/-0.054 mm mean Hausdorff distance. This method makes the bi-modal FLIm and IVUS approach feasible for comprehensive intravascular diagnostic by providing co-registered biochemical and morphological information about atherosclerotic plaques.

  16. 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. PMID:27430158

  17. 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

  18. 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.

  19. Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions inside cells.

    PubMed

    Duncan, R R

    2006-11-01

    Recent developments in cellular imaging spectroscopy now permit the minimally invasive study of protein dynamics inside living cells. These advances are of interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Until recently, all protein interactions had to be determined in vitro using biochemical approaches: this biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with high spatial resolution. These techniques can detect and quantify protein behaviours down to the single-molecule level, all inside living cells. More recent developments in TCSPC (time-correlated single-photon counting) imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. PMID:17052173

  20. 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

  1. 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.

  2. Fluorescence lifetime excitation cytometry by kinetic dithering.

    PubMed

    Li, Wenyan; Vacca, Giacomo; Castillo, Maryann; Houston, Kevin D; Houston, Jessica P

    2014-07-01

    Flow cytometers are powerful high-throughput devices that capture spectroscopic information from individual particles or cells. These instruments provide a means of multi-parametric analyses for various cellular biomarkers or labeled organelles and cellular proteins. However, the spectral overlap of fluorophores limits the number of fluorophores that can be used simultaneously during experimentation. Time-resolved parameters enable the quantification of fluorescence decay kinetics, thus circumventing common issues associated with intensity-based measurements. This contribution introduces fluorescence lifetime excitation cytometry by kinetic dithering (FLECKD) as a method to capture multiple fluorescence lifetimes using a hybrid time-domain approach. The FLECKD approach excites fluorophores by delivering short pulses of light to cells or particles by rapid dithering and facilitates measurement of complex fluorescence decay kinetics by flow cytometry. Our simulations demonstrated a resolvable fluorescence lifetime value as low as 1.8 ns (±0.3 ns) with less than 20% absolute error. Using the FLECKD instrument, we measured the shortest average fluorescence lifetime value of 2.4 ns and found the system measurement error to be ±0.3 ns (SEM), from hundreds of monodisperse and chemically stable fluorescent microspheres. Additionally, we demonstrate the ability to detect two distinct excited state lifetimes from fluorophores in single cells using FLECKD. This approach presents a new ability to resolve multiple fluorescence lifetimes while retaining the fluidic throughput of a cytometry system. The ability to discriminate more than one average fluorescence lifetime expands the current capabilities of high-throughput and intensity-based cytometry assays as the need to tag one single cell with multiple fluorophores is now widespread. PMID:24668857

  3. Fluorescence lifetime excitation cytometry by kinetic dithering

    PubMed Central

    Li, Wenyan; Vacca, Giacomo; Castillo, Maryann; Houston, Kevin D; Houston, Jessica P

    2014-01-01

    Flow cytometers are powerful high-throughput devices that capture spectroscopic information from individual particles or cells. These instruments provide a means of multi-parametric analyses for various cellular biomarkers or labeled organelles and cellular proteins. However, the spectral overlap of fluorophores limits the number of fluorophores that can be used simultaneously during experimentation. Time-resolved parameters enable the quantification of fluorescence decay kinetics, thus circumventing common issues associated with intensity-based measurements. This contribution introduces fluorescence lifetime excitation cytometry by kinetic dithering (FLECKD) as a method to capture multiple fluorescence lifetimes using a hybrid time-domain approach. The FLECKD approach excites fluorophores by delivering short pulses of light to cells or particles by rapid dithering and facilitates measurement of complex fluorescence decay kinetics by flow cytometry. Our simulations demonstrated a resolvable fluorescence lifetime value as low as 1.8 ns (±0.3 ns) with less than 20% absolute error. Using the FLECKD instrument, we measured the shortest average fluorescence lifetime value of 2.4 ns and found the system measurement error to be ±0.3 ns (SEM), from hundreds of monodisperse and chemically stable fluorescent microspheres. Additionally, we demonstrate the ability to detect two distinct excited state lifetimes from fluorophores in single cells using FLECKD. This approach presents a new ability to resolve multiple fluorescence lifetimes while retaining the fluidic throughput of a cytometry system. The ability to discriminate more than one average fluorescence lifetime expands the current capabilities of high-throughput and intensity-based cytometry assays as the need to tag one single cell with multiple fluorophores is now widespread. PMID:24668857

  4. Fluorescence lifetime imaging and FRET-induced intracellular redistribution of Tat-conjugated quantum dot nanoparticles through interaction with a phthalocyanine photosensitiser.

    PubMed

    Yaghini, Elnaz; Giuntini, Francesca; Eggleston, Ian M; Suhling, Klaus; Seifalian, Alexander M; MacRobert, Alexander J

    2014-02-26

    The interaction of Tat-conjugated PEGylated CdSe/ZnS quantum dots (QD) with the amphiphilic disulfonated aluminium phthalocyanine photosensitiser is investigated in aqueous solution and in a human breast cancer cell line. In aqueous solution, the QDs and phthalocyanine form stable nanocomposites. Using steady-state and time-resolved fluorescence measurements combined with singlet oxygen detection, efficient Förster resonance energy transfer (FRET) is observed with the QDs acting as donors, and the phthalocyanine photosensitiser, which mediates production of singlet oxygen, as acceptors. In cells, the Tat-conjugated QDs localise in lysosomes and the QD fluorescence lifetimes are close to values observed in aqueous solution. Strong FRET-induced quenching of the QD lifetime is observed in cells incubated with the nanocomposites using fluorescence lifetime imaging microscopy (FLIM). Using excitation of the QDs at wavelengths where phthalocyanine absorption is negligible, FRET-induced release of QDs from endo/lysosomes is confirmed using confocal imaging and FLIM, which is attributed to photooxidative damage to the endo/lysosomal membranes mediated by the phthalocyanine acceptor. PMID:24031023

  5. Optimal estimator for tomographic fluorescence lifetime multiplexing

    PubMed Central

    Hou, Steven S.; Bacskai, Brian J.; Kumar, Anand T. N.

    2016-01-01

    We use the model resolution matrix to analytically derive an optimal Bayesian estimator for multiparameter inverse problems that simultaneously minimizes inter-parameter cross talk and the total reconstruction error. Application of this estimator to time-domain diffuse fluorescence imaging shows that the optimal estimator for lifetime multiplexing is identical to a previously developed asymptotic time-domain (ATD) approach, except for the inclusion of a diagonal regularization term containing decay amplitude uncertainties. We show that, while the optimal estimator and ATD provide zero cross talk, the optimal estimator provides lower reconstruction error, while ATD results in superior relative quantitation. The framework presented here is generally applicable to other multiplexing problems where the simultaneous and accurate relative quantitation of multiple parameters is of interest. PMID:27192234

  6. 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.

  7. 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.

  8. Combined fiber probe for fluorescence lifetime and Raman spectroscopy

    PubMed Central

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Marple, Eric; Urmey, Kirk; Wachsmann-Hogiu, Sebastian; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-01-01

    In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. PMID:26093843

  9. Fluorescence lifetime spectroscopy of glioblastoma multiforme.

    PubMed

    Marcu, Laura; Jo, Javier A; Butte, Pramod V; Yong, William H; Pikul, Brian K; Black, Keith L; Thompson, Reid C

    2004-01-01

    Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time-resolved, laser-induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time-resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time-resolved spectra were detected in a 360-550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral- (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high-grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectral- and the time domain can enhance the ability of fluorescence-based techniques to diagnose and detect brain tumor margins intraoperatively. PMID:15339216

  10. Measurement of Rydberg positronium fluorescence lifetimes

    NASA Astrophysics Data System (ADS)

    Deller, A.; Alonso, A. M.; Cooper, B. S.; Hogan, S. D.; Cassidy, D. B.

    2016-06-01

    We report measurements of the fluorescence lifetimes of positronium (Ps) atoms with principal quantum numbers n =10 -19 . Ps atoms in Rydberg-Stark states were produced via a two-color two-step 1 3S→2 3P→n 3S/n lifetimes of the Rydberg levels, yielding values ranging from 3 μ s to 26 μ s . Our data are in accord with the expected radiative lifetimes of Rydberg-Stark states of Ps.

  11. Fluorescence lifetime resolution with phase fluorometry

    NASA Astrophysics Data System (ADS)

    Ide, Geert; Engelborghs, Yves; Persoons, Andre

    1983-07-01

    A phase fluorometer for the measurement of fluorescence lifetimes was constructed from commercially available components. The instrument was tested by using optical delays of 4 and 2 cm, showing an accuracy of 10 ps. Lifetimes, as short as 0.3 ns, obtained by the quenching of fluorescein by KI, were analyzed with a standard deviation of 3 ps. The lifetime resolving power was checked using mixtures of acridine and quinine-sulphate and least-squares fitting procedures. Accurate amplitude ratios were obtained with the technique of phase-sensitive detection [J. R. Lakowicz and H. Cherek, J. Biochem. Biophys. Methods 5, 19 (1981)].

  12. 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.

  13. Characterization of thylakoid membrane in a heterocystous cyanobacterium and green alga with dual-detector fluorescence lifetime imaging microscopy with a systematic change of incident laser power.

    PubMed

    Nozue, Shuho; Mukuno, Akira; Tsuda, Yumi; Shiina, Takashi; Terazima, Masahide; Kumazaki, Shigeichi

    2016-01-01

    Fluorescence Lifetime Imaging Microscopy (FLIM) has been applied to plants, algae and cyanobacteria, in which excitation laser conditions affect the chlorophyll fluorescence lifetime due to several mechanisms. However, the dependence of FLIM data on input laser power has not been quantitatively explained by absolute excitation probabilities under actual imaging conditions. In an effort to distinguish between photosystem I and photosystem II (PSI and PSII) in microscopic images, we have obtained dependence of FLIM data on input laser power from a filamentous cyanobacterium Anabaena variabilis and single cellular green alga Parachlorella kessleri. Nitrogen-fixing cells in A. variabilis, heterocysts, are mostly visualized as cells in which short-lived fluorescence (≤0.1 ns) characteristic of PSI is predominant. The other cells in A. variabilis (vegetative cells) and P. kessleri cells show a transition in the status of PSII from an open state with the maximal charge separation rate at a weak excitation limit to a closed state in which charge separation is temporarily prohibited by previous excitation(s) at a relatively high laser power. This transition is successfully reproduced by a computer simulation with a high fidelity to the actual imaging conditions. More details in the fluorescence from heterocysts were examined to assess possible functions of PSII in the anaerobic environment inside the heterocysts for the nitrogen-fixing enzyme, nitrogenase. Photochemically active PSII:PSI ratio in heterocysts is tentatively estimated to be typically below our detection limit or at most about 5% in limited heterocysts in comparison with that in vegetative cells. PMID:26474523

  14. 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

  15. Influence of the refractive index on EGFP fluorescence lifetimes in mixtures of water and glycerol

    NASA Astrophysics Data System (ADS)

    Suhling, Klaus; Davis, Daniel M.; Petrasek, Zdenek; Siegel, Jan; Phillips, David

    2001-07-01

    As a precursor to applying fluorescence lifetime imaging (FLIM) to studies of intercellular communication in molecular immunology, we have investigated the fluorescence lifetime of enhanced green fluorescent protein (EGFP) in mixtures of water and glycerol using time-correlated single photon counting (TCSPC). We find that the EGFP lifetime decreases with increasing glycerol content. This is accounted for quantitatively by the refractive index dependence of the fluorescence lifetime as predicted by the Strickler Berg formula which relates the fluorescence lifetime to the absorption spectrum. The solvent viscosity has no influence on the fluorescence lifetime. We also discuss the refractive index dependence of the GFP fluorescence lifetime in more complex systems. The findings are particularly relevant for the interpretation of FLIM of GFP expressed in environments such as bacteria and cells.

  16. 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.

  17. Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy.

    PubMed

    Boens, Noël; Qin, Wenwu; Basarić, Nikola; Hofkens, Johan; Ameloot, Marcel; Pouget, Jacques; Lefèvre, Jean-Pierre; Valeur, Bernard; Gratton, Enrico; vandeVen, Martin; Silva, Norberto D; Engelborghs, Yves; Willaert, Katrien; Sillen, Alain; Rumbles, Garry; Phillips, David; Visser, Antonie J W G; van Hoek, Arie; Lakowicz, Joseph R; Malak, Henryk; Gryczynski, Ignacy; Szabo, Arthur G; Krajcarski, Don T; Tamai, Naoto; Miura, Atsushi

    2007-03-01

    A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-l-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 degrees C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and the solvent, the excitation wavelengths used range from 284 to 575 nm, the emission from 330 to 630 nm. These lifetime standards may be used to either calibrate or test the resolution of time- and frequency-domain instrumentation or as reference compounds to eliminate the color effect in photomultiplier tubes. Statistical analyses by means of two-sample charts indicate that there is no laboratory bias in the lifetime determinations. Moreover, statistical tests show that there is an excellent correlation between the lifetimes estimated by the time-domain and frequency-domain fluorometries. Comprehensive tables compiling the results for 20 (fluorescence lifetime standard/solvent) combinations are given. PMID:17269654

  18. Automated analysis of multimodal fluorescence lifetime imaging and optical coherence tomography data for the diagnosis of oral cancer in the hamster cheek pouch model

    PubMed Central

    Pande, Paritosh; Shrestha, Sebina; Park, Jesung; Gimenez-Conti, Irma; Brandon, Jimi; Applegate, Brian E.; Jo, Javier A.

    2016-01-01

    It is known that the progression of oral cancer is accompanied by changes in both tissue biochemistry and morphology. A multimodal imaging approach combining functional and structural imaging modalities could therefore provide a more comprehensive prognosis of oral cancer. This idea forms the central theme of the current study, wherein this premise is examined in the context of a multimodal imaging system that combines fluorescence lifetime imaging (FLIM) and optical coherence tomography (OCT). Towards this end, in the first part of the present study, the diagnostic advantage obtained by using both fluorescence intensity and lifetime information is assessed. In the second part of the study, the diagnostic potential of FLIM-derived biochemical features is compared with that of OCT-derived morphological features. For an objective assessment, several quantitative biochemical and morphological features from FLIM and OCT data, respectively, were obtained using signal and image processing techniques. These features were subsequently used in a statistical classification framework to quantify the diagnostic potential of different features. The classification accuracy for combined FLIM and OCT features was estimated to be 87.4%, which was statistically higher than accuracy based on only FLIM (83.2%) or OCT (81.0%) features. Moreover, the complimentary information provided by FLIM and OCT features, resulted in highest sensitivity and specificity for the combined FLIM and OCT features for discriminating benign (88.2% sens., 92.0% spec.), pre-cancerous (81.5% sens., 96.0% spec.), and cancerous (90.1% sens., 92.0% spec.) classes. PMID:27231638

  19. Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Sanchez, Washington Y.; Prow, Tarl W.; Sanchez, Washington H.; Grice, Jeffrey E.; Roberts, Michael S.

    2010-07-01

    Ex vivo human skin has been used extensively for cosmeceutical and drug delivery studies, transplantable skin allografts, or skin flaps. However, it has a half-life of a few days due to ischemic necrosis. Traditional methods of assessing viability can be time-consuming and provide limited metabolic information. Using multiphoton tomography and fluorescence lifetime imaging (MPT-FLIM) we assess ischemic necrosis of ex vivo skin by NAD(P)H autofluorescence intensity and fluorescence lifetime. Ex vivo skin is stored in the presence and absence of nutrient media (Dulbecco Modified Eagle Medium) at -20, 4, and 37 °C and room temperature over a 7-day time course to establish different rates of metabolic deterioration. At higher temperatures we observe a decrease in NAD(P)H autofluorescence, higher image noise, and a significant increase in the average fluorescence lifetime (τm) from ~1000 to 2000 ps. Additionally, significant distortions in NAD(P)H fluorescence lifetime histograms correspond to the reduction in autofluorescence. Skin kept at 4 °C, with or without media, showed the least change. Our findings suggest that MPT-FLIM enables useful noninvasive optical biopsies to monitor the metabolic state and deterioration of human skin for research and clinical purposes.

  20. 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.

  1. Compact imaging system with single-photon sensitivity and picosecond time resolution for fluorescence-guided surgery with lifetime imaging capability

    NASA Astrophysics Data System (ADS)

    Powolny, F.; Bruschini, C.; Dubikovskaya, E.; Grigoriev, E.; Michielin, O.; Muehlethaler, K.; Prior, J. O.; Rimoldi, D.; Sinisi, R.; Charbon, E.

    2013-06-01

    We present a single-photon camera for fluorescence imaging capable of providing both intensity and images, with an accuracy better than 100ps; the camera was fabricated in standard CMOS technology. As a first step towards the study of biologically relevant samples, it was used to characterize in-vitro cultured melanoma cells labeled with indocyanine green (ICG) and ICG conjugated with cyclic pentapeptide (RGDfK). The application field would be fluorescence-guided surgical oncology.

  2. 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.

  3. Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements.

    PubMed

    George Abraham, Bobin; Sarkisyan, Karen S; Mishin, Alexander S; Santala, Ville; Tkachenko, Nikolai V; Karp, Matti

    2015-01-01

    Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM). PMID:26237400

  4. Triplet lifetime and delayed fluorescence of azulene

    NASA Astrophysics Data System (ADS)

    Kray, Hans-Joachim; Nickel, Bernhard

    1980-11-01

    With solutions of azulene (Az) and fluoranthene (Fl) in isopentane a delayed fluorescence (DF) S 2(Az) → S 0(Az), resulting from hetero-triplet—triplet annihilation T 1(Az) + T 1(Fl) → S 2(Az) + S 0(Fl), can be observed. From the time-dependence of this DF after laser flash excitation the triplet lifetime of azulene can be calculated. The triplet lifetime has been determined in the temperature range from 131 K to 201 K. The temperature-dependence of the triplet lifetime is explained by thermally activated intersystem crossing (ISC) T 1 ⇝ S 1, followed by internal conversion S 1 ⇝ S 0; the corresponding activation energy approximately equals the difference of the excitation energies of S 1 and T 1. The extrapolated low-temperature value of the triplet lifetime (48 ± 2) μs. The quantum efficiency of the ISC S 1 ⇝ T 1 is estimated to be of the order of magnitude of 4 × 10 -6, and for the quantum efficiency of the ISC S 2 ⇝ T 1 an upper bound of 0.04 is obtained. The experimental conditions for the observation of the phosphorescence T 1 ⇝ S 0 and the E-type DF S 1 → S 0 are discussed.

  5. Fluorescence lifetime imaging of DAPI-stained nuclei as a novel diagnostic tool for the detection and classification of B-cell chronic lymphocytic leukemia.

    PubMed

    Yahav, Gilad; Hirshberg, Abraham; Salomon, Ophira; Amariglio, Ninette; Trakhtenbrot, Luba; Fixler, Dror

    2016-07-01

    B-cell chronic lymphocytic leukaemia (B-CLL) and B-cell precursor acute lymphoblastic leukaemia (B-ALL) are the most common type of leukaemia in adults and children, respectively. Today, fluorescence in situ hybridization (FISH) is the standard for detecting chromosomal aberrations that reflect adverse and favorable outcome. This study revealed a new, simple, and fast diagnostic tool to detect pathological cells by measuring and imaging the fluorescence lifetime (FLT) using FLT imaging microscopy (FLIM) of the peripheral blood (PB) cells of B-CLL samples that were labeled with the DNA binder, DAPI. The FLT of DAPI in healthy individuals was found to be 2.66 ± 0.12 ns. In contrast, PB cells of B-CLL and BM cells of B-ALL patients were characterized by a specific group distribution of the FLT values. The FLT of DAPI was divided into four subgroups, relative to 2.66 ns: short+, normal, prolonged, and prolonged+. These alterations could be related to different chromatin arrangements of B-CLL and B-ALL interphase nuclei. Notably, extremely long FLT of nuclear DAPI correlate with the presence of extra chromosome 12, while moderate increases compared to normal characterize the deletion of p53. Such correlations potentially enable a FLT-based rapid automatic diagnosis and classification of B-CLL even when the frequency of genetic and chromosomal abnormalities is low. © 2016 International Society for Advancement of Cytometry. PMID:27315046

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

    PubMed Central

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

    2015-01-01

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

  7. Non-Invasive In Vivo Imaging of Near Infrared-labeled Transferrin in Breast Cancer Cells and Tumors Using Fluorescence Lifetime FRET

    PubMed Central

    Periasamy, Ammasi; Intes, Xavier; Barroso, Margarida

    2013-01-01

    The conjugation of anti-cancer drugs to endogenous ligands has proven to be an effective strategy to enhance their pharmacological selectivity and delivery towards neoplasic tissues. Since cell proliferation has a strong requirement for iron, cancer cells express high levels of transferrin receptors (TfnR), making its ligand, transferrin (Tfn), of great interest as a delivery agent for therapeutics. However, a critical gap exists in the ability to non-invasively determine whether drugs conjugated to Tfn are internalized into target cells in vivo. Due to the enhanced permeability and retention (EPR) effect, it remains unknown whether these Tfn-conjugated drugs are specifically internalized into cancer cells or are localized non-specifically as a result of a generalized accumulation of macromolecules near tumors. By exploiting the dimeric nature of the TfnR that binds two molecules of Tfn in close proximity, we utilized a Förster Resonance Energy Transfer (FRET) based technique that can discriminate bound and internalized Tfn from free, soluble Tfn. In order to non-invasively visualize intracellular amounts of Tfn in tumors through live animal tissues, we developed a novel near infrared (NIR) fluorescence lifetime FRET imaging technique that uses an active wide-field time gated illumination platform. In summary, we report that the NIR fluorescence lifetime FRET technique is capable of non-invasively detecting bound and internalized forms of Tfn in cancer cells and tumors within a live small animal model, and that our results are quantitatively consistent when compared to well-established intensity-based FRET microscopy methods used in in vitro experiments. PMID:24278268

  8. A Macrocyclic Fluorophore Dimer with Flexible Linkers: Bright Excimer Emission with a Long Fluorescence Lifetime.

    PubMed

    Osaki, Hiroshi; Chou, Chih-Ming; Taki, Masayasu; Welke, Kai; Yokogawa, Daisuke; Irle, Stephan; Sato, Yoshikatsu; Higashiyama, Tetsuya; Saito, Shohei; Fukazawa, Aiko; Yamaguchi, Shigehiro

    2016-06-13

    Bright fluorescent molecules with long fluorescence lifetimes are important for the development of lifetime-based fluorescence imaging techniques. Herein, a molecular design is described for simultaneously attaining long fluorescence lifetime (τ) and high brightness (ΦF ×ɛ) in a system that features macrocyclic dimerization of fluorescent π-conjugated skeletons with flexible linkers. An alkylene-linked macrocyclic dimer of bis(thienylethynyl)anthracene was found to show excimer emission with a long fluorescence lifetime (τ≈19 ns) in solution, while maintaining high brightness. A comparison with various relevant derivatives revealed that the macrocyclic structure and the length of the alkylene chains play crucial roles in attaining these properties. In vitro time-gated imaging experiments were conducted as a proof-of-principle for the superiority of this macrocyclic fluorophore relative to the commercial fluorescent dye Alexa Fluor 488. PMID:27121201

  9. 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

  10. eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM).

    PubMed

    Schmitt, Franz-Josef; Thaa, Bastian; Junghans, Cornelia; Vitali, Marco; Veit, Michael; Friedrich, Thomas

    2014-09-01

    The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO

  11. 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.

  12. Angiotensin II-induced angiotensin II type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

    Upon activation, the angiotensin (Ang) II type 1 receptor (AT1Rs) rapidly undergoes endocytosis. After a series of intracellular processes, the internalized AT1Rs recycle back to the plasma membrane or are trafficked to proteasomes or lysosomes for degradation. We recently reported that AT1Rs degrades in proteasomes upon stimulation of the D5 dopamine receptor (D5R) in human renal proximal tubule and HEK-293 cells. This is in contrast to the degradation of AT1R in lysosomes upon binding Ang II. However, the dynamic regulation of the AT1Rs in lysosomes is not well understood. Here we investigated the AT1Rs lysosomal degradation using FRET-FLIM in HEK 293 cells heterologously expressing the human AT1R tagged with EGFP as the donor fluorophore. Compared to its basal state, the lifetime of AT1Rs decreased after a 5-minute treatment with Ang II treatment and colocalized with Rab5 but not Rab7 and LAMP1. With longer Ang II treatment (30 min), the AT1Rs lifetime decreased and co-localized with Rab5, as well as Rab7 and LAMP1. The FLIM data are corroborated with morphological and biochemical co-immunoprecipitation studies. These data demonstrate that Ang II induces the internalization of AT1Rs into early sorting endosomes prior to trafficking to late endosomes and subsequent degradation in lysosomes.

  13. 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. PMID:26753623

  14. Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

    PubMed

    Hickey, James L; James, Janine L; Henderson, Clare A; Price, Katherine A; Mot, Alexandra I; Buncic, Gojko; Crouch, Peter J; White, Jonathan M; White, Anthony R; Smith, Trevor A; Donnelly, Paul S

    2015-10-01

    The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease. PMID:26397162

  15. 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. PMID:25394476

  16. Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    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.

  17. Estimation of Fluorescence Lifetimes Via Rotational Invariance Techniques.

    PubMed

    Yu, Hongqi; Saleeb, Rebecca; Dalgarno, Paul; Day-Uei Li, David

    2016-06-01

    Estimation of signal parameters via rotational invariance techniques is a classical algorithm widely used in array signal processing for direction-of-arrival estimation of emitters. Inspired by this method, a new signal model and new fluorescence lifetime estimation via rotational invariance techniques (FLERIT) were developed for multiexponential fluorescence lifetime imaging (FLIM) experiments. The FLERIT only requires a few time bins of a histogram generated by a time-correlated single-photon counting FLIM system, greatly reducing the data throughput from the imager to the signal processing units. As a noniterative method, the FLERIT does not require initial conditions, prior information nor model selection that are usually required by widely used traditional fitting methods, including nonlinear least square methods or maximum-likelihood methods. Moreover, its simplicity means it is suitable for implementations in embedded systems for real-time applications. FLERIT was tested on synthesized and experimental fluorescent cell data showing the potentials to be widely applied in FLIM data analysis. PMID:26571506

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

    NASA Astrophysics Data System (ADS)

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

    2006-10-01

    We have recently developed a wide-field photon-counting detector (the H33D detector) having high-temporal and highspatial 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.

  19. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    SciTech Connect

    Crissman, Harry A.; Cui, H. H.; Steinkamp, J. A.

    2002-01-01

    Most flow cytometry (FCM) applications monitor fluorescence intensity to quantitate the various cellular parameters; however, the fluorescence emission also contains information relative to the fluorescence lifetime. Recent developments in FCM (Pinsky et al., 1993; Steinkamp & Crissman, 1993; Steinkamp et al., 1993), provide for the measurement of fluorescence lifetime which is also commonly referred to as fluorescence decay, or the time interval in which a fluorochrome remains in the excited state. Many unbound fluorochromes have characteristic lifetime values that are determined by their molecular structure; however, when the probe becomes bound, the lifetime value is influenced by a number of factors that affect the probe interaction with a target molecule. Monitoring the changes in the lifetime of the probe yields information relating to the molecular conformation, the functional state or activity of the molecular target. In addition, the lifetime values can be used as signatures to resolve the emissions of multiple fluorochrome labels with overlapping emission spectra that cannot be resolved by conventional FCM methodology. Such strategies can increase the number of fluorochrome combinations used in a flow cytometer with a single excitation source. Our studies demonstrate various applications of lifetime measurements for the analysis of the binding of different fluorochromes to DNA in single cells. Data presented in this session will show the utility of lifetime measurements for monitoring changes in chromatin structure associated with cell cycle progression, cellular differentiation, or DNA damage, such as induced during apoptosis. Several studies show that dyes with specificity for nucleic acids display different lifetime values when bound to DNA or to dsRNA. The Phase Sensitive Flow Cytometer is a multiparameter instrument, capable of performing lifetime measurements in conjunction with all the conventional FCM measurements. Future modifications of this

  20. Revisit laser scanning fluorescence microscopy performance under fluorescence-lifetime-limited regime

    NASA Astrophysics Data System (ADS)

    Chan, Antony C.; Wong, Terence T. W.; Wong, Kenneth K. Y.; Lam, Edmund Y.; Tsia, Kevin K.

    2014-03-01

    Continuing desire for higher-speed laser scanning fluorescence microscopy (LSFM) and progressive advancement in ultrafast and sensitive photodetectors might imply that our conventional understanding of LSFM is not adequate when approaching to the intrinsic speed limit — fluorescence lifetime. In this regard, we here revisit the theoretical framework of LSFM and evaluate its general performance in lifetime-limited and noise-limited regimes. Our model suggests that there still exists an order-of-magnitude gap between the current LSFM speed and the intrinsic limit. An imaging frame rate of > 100 kHz could be viable with the emerging laser-scanning techniques using ultrafast wavelength-swept sources, or optical time-stretch.

  1. Interpretation of fluorescence decays in proteins using continuous lifetime distributions.

    PubMed Central

    Alcala, J R; Gratton, E; Prendergast, F G

    1987-01-01

    The decay of the tryptophanyl emission in proteins is often complex due to the sensitivity of the tryptophan excited state to its surroundings. The traditional analysis of the decay curve using exponential components is based on the identification of each component with a particular protein conformation. An alternative approach assumes that proteins can exhibit a large number of conformations and that, at room temperature, the interconversion rate between conformations can be of the same order of magnitude as the excited-state decay rate. Following this assumption, the analysis of the protein emission was performed using continuous distributions of lifetime values. The number of average protein conformations, the range of mobility around each conformation, and the rate of interconversion between conformations determines the characteristics of the lifetime distribution. The fluorescence decay from some single tryptophan proteins was measured using multifrequency phase fluorometry and analyzed using a sum of exponentials, unimodal and bimodal probability-density functions, and the analytical form for lifetime distribution obtained for a model in which the tryptophan residue can move in a single potential well. For ribonuclease T1 and neurotoxin variant 3, the sum of two exponentials and bimodal probability-density functions gave comparable results, whereas for phospholipase A2, the description of the decay required three exponentials or bimodal probability-density functions. Also the temperature dependence of the fluorescence decay was investigated. It was found that the lifetime distribution was broader and shifted toward longer lifetime values at lower temperature. The analysis of the decay of tryptophan in buffer and of some tryptophan derivatives gave single-exponential decays. The single-potential well lifetime distribution, which has only three adjustable parameters, gave good fits for all cases investigated, but in the case of phopholipase A2, the temperature

  2. 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

  3. Fluorescence Lifetimes of Normal and Carcinomatous Human Nasopharyngeal Tissues

    NASA Astrophysics Data System (ADS)

    Chen, M.; Li, H.; Li, B.; Chen, R.; Zheng, G.; Song, C.

    2016-03-01

    Time-resolved fluorescence spectra of normal and carcinomatous in vitro human nasopharyngeal tissues are compared. By fitting the time-resolved emission with exponential decays, mean lifetimes were obtained. There were marked differences between the lifetimes of the carcinomatous and the normal tissues. Thus, early diagnosis of nasopharyngeal carcinoma is possible. In general, comprehensive information from human tissue autofluorescence can be acquired via both time-resolved and steady-state fluorescence spectra.

  4. 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

  5. Anomalous temperature dependence of the fluorescence lifetime of phycobiliproteins

    NASA Astrophysics Data System (ADS)

    Maksimov, E. G.; Schmitt, F.-J.; Hätti, P.; Klementiev, K. E.; Paschenko, V. Z.; Renger, G.; Rubin, A. B.

    2013-05-01

    Using a single photon counting technique we have investigated fluorescence decay spectra of phycobiliproteins with picosecond time resolution. The studies were performed in a wide range of temperatures—from 4 to 300 K. Comparing the fluorescence decay kinetics of samples rapidly frozen in liquid nitrogen with samples that were frozen slowly revealed that the temperature-dependent changes of phycobiliproteins fluorescence lifetime reflect the presence of three different stages, with a phase transition between 273 and 263 K that strongly depends on the rate of freezing. When the temperature decreases from 300 to 273 K, the fluorescence lifetime increases from 1.6 to 1.8 ns. In the region from 273 to 263 K we observed a decrease of the fluorescence lifetime, which strongly depends on the freezing rate: a slight decrease at high freezing rate and a drop down to 200 ps lifetime at slow freezing rate. In the low-temperature regime from 263 to 4 K a linear increase in the fluorescence lifetime was observed for all samples. It was found that the strong temperature dependence of the phycobiliprotein fluorescence, especially in the range between 263 and 273 K, is due to the interaction of the solvent with the chromophore bound to the protein. This feature is explained by a photoisomerization of the phycobiliproteins into a quenching form which is naturally prevented by the protein environment. The formation of ice microcrystals at low freezing rate eliminates this ‘protective’ effect of the protein environment.

  6. Temperature Dependent Fluorescence Lifetime Measurements in a Phosphor

    NASA Astrophysics Data System (ADS)

    Nettles, Charles J.; Smith, R. Seth; Heath, Jonathan J.

    2012-03-01

    This poster will describe an undergraduate senior research project involving fluorescence lifetime measurements in a LaSO4:Eu phosphor compound. Specifically, this project seeks to determine the temperature dependence of the lifetime. The temperature of the phosphor will be varied using a heater block with temperature control. The phosphor will be excited with the 337 nm output of a Nitrogen Laser. An Oriel Monochromator will be used to disperse the fluorescence, and the lifetime for a particular wavelength will be determined from a photomultiplier tube signal. At the time of the presentation, this project will be nearing completion; and I will discuss my progress, successes, and challenges.

  7. 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

  8. Fluorescence lifetime-based glucose sensor using NADH

    NASA Astrophysics Data System (ADS)

    von Ketteler, A.; Siegberg, D.; Herten, D. P.; Horn, C.; Petrich, W.

    2012-03-01

    Fluorescence lifetime-based glucose sensing does not depend on fluctuations of the intensity of the light source, light scattering, or changes in the transmission of optical components. Here we demonstrate the sensing of glucose based on the fluorescence lifetime properties of dihydro nicotinamide adenine dinucleotide (NADH), which is reduced from NAD in the presence of glucose and glucose dehydrogenase. In particular we use the difference in the fluorescence properties of free and protein-bound NADH and calculate an average fluorescence lifetime, which arises from the two short lifetimes τ1=0.28ns and τ2=0.60ns (representing free NADH) and the longer lifetime of τ3=2.9ns (for the protein-bound NADH). While initial results were derived from measurements in aqueous solution, we also demonstrate the suitability of this method for determining the concentration of glucose in blood using test strips. We find that the average fluorescence lifetime changes linearly by a factor of 0.17 per 100mg/dl change in glucose concentration. As an alternative the ratio between free and protein-bound components Rs/l may also be used for quantification. Rs/l increases by a factor of 0.74 per 100mg/dl change in glucose concentration.

  9. Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy.

    PubMed Central

    Barzda, V; de Grauw, C J; Vroom, J; Kleima, F J; van Grondelle, R; van Amerongen, H; Gerritsen, H C

    2001-01-01

    Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood. PMID:11423435

  10. 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.

  11. 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.

  12. Digital analysis and sorting of fluorescence lifetime by flow cytometry.

    PubMed

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

    2010-09-01

    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 (RF)-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 micros, 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 degrees to 1.6 degrees. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 micros 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 RF-modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to approximately 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

  13. Emission Lifetimes of a Fluorescent Dye under Shock Compression.

    PubMed

    Liu, Wei-long; Bassett, Will P; Christensen, James M; Dlott, Dana D

    2015-11-01

    The emission lifetimes of rhodamine 6G (R6G) were measured under shock compression to 9.1 GPa, with the dual intents 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 the 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 to 9 GPa, with a slope of -0.22 ns·GPa(-1). The linear relationship makes it simple to convert lifetimes into pressures. Lifetime measurements in shocked microenvironments may be better than emission intensity measurements, because lifetimes are sensitive to the surrounding environment, but insensitive to intensity variations associated with the motion and optical properties of a dynamically changing structure. PMID:26469397

  14. Measuring and Sorting Cell Populations Expressing Isospectral Fluorescent Proteins with Different Fluorescence Lifetimes

    PubMed Central

    Naivar, Mark; Houston, Jessica P.; Brent, Roger

    2014-01-01

    Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs) fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP) and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼1.5 ns vs ∼3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a “pseudophasor” that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation. PMID:25302964

  15. 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.

  16. Fluorescence lifetime measurements of boronate derivatives to determine glucose concentration

    SciTech Connect

    Gable, J H

    2000-06-01

    A novel investigation into the fluorescence lifetimes of molecules, both established and newly designed, was performed. These molecules are the basis of a continuous, minimally invasive, glucose sensor based on fluorescence lifetime measurements. This sensor, if coupled with an automated insulin delivery device, would effectively create an artificial pancreas allowing for the constant monitoring and control of glucose levels in a person with diabetes. The proposed sensor includes a fluorescent molecule that changes its' fluorescence properties upon binding selectively and reversibly to glucose. One possible sensor molecule is N-methyl-N-(9-methylene anthryl)-2-methylenephenylboronic acid (AB). The fluorescence intensity of AB was shown to change in response to changing glucose concentrations. (James, 1994) James proposed that when glucose binds to AB the fluorescence intensity increases due to an enhancement of the N{yields}B dative bond which prevents photoinduced electron transfer (PET). PET from the amine (N) to the fluorophore (anthracene) quenches the fluorescence. The dative bond between the boron and the amine can prevent PET by involving the lone pair of electrons on the amine in interactions with the boron rather than allowing them to be transferred to the fluorophore. Results of this research show the average fluorescence lifetime of AB also changes with glucose concentration. It is proposed that fluorescence is due to two components: (1) AB with an enhanced N{yields}B interaction, and no PET, and (2) AB with a weak N{yields}B interaction, resulting in fluorescence quenching by PET. Lifetime measurements of AB as a function of both the pH of the solvent and glucose concentration in the solution were made to characterize this two component system and investigate the nature of the N{yields}B bond. Measurements of molecules similar to AB were also performed in order to isolate behavior of specific AB constituents. These molecules are 9-(Methylaminomethyl

  17. Fluorescence lifetimes of molecular dye ensembles near interfaces

    SciTech Connect

    Danz, Norbert; Heber, Joerg; Braeuer, Andreas; Kowarschik, Richard

    2002-12-01

    Fluorescence lifetimes of thin, rhodamine 6G-doped polymer layers in front of a mirror have been determined as a function of the emitter-mirror separation and the conditions of excitation and observation. Lifetime is well known to depend on the spatial emitter-mirror separation. The explanation of experimental data needs to consider direction, polarization, and numerical aperture of the experimental system. As predicted theoretically, experimental results depend on the conditions of illumination and observation, because of the different lifetimes of emitters aligned horizontally or vertically with respect to the plane of interfaces and their selection by the experimental system. This effect is not observable when ions are used as a source of fluorescence, because ensemble averaging depends on the properties of sources.

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

    PubMed

    Ishii, Kunihiko; Tahara, Tahei

    2013-10-01

    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. PMID:23977902

  19. 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.

  20. 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.

  1. The Number of Accumulated Photons and the Quality of Stimulated Emission Depletion Lifetime Images

    SciTech Connect

    Syed, Aleem; Lesoine, Michael D; Bhattacharjee, Ujjal; Petrich, Jacob W; Smith, Emily A

    2014-03-03

    Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion (STED) fluorescence lifetime decay curves to determine how it affects the resulting lifetime image. The fluorescence lifetime of the fluorophore, Alexa Fluor 594 phalloidin, bound to F-actin is probed in cultured S2 cells at a spatial resolution of ~40 nm. This corresponds to a tenfold smaller probe volume compared to confocal imaging, and a reduced number of photons contributing to the signal. Pixel-by-pixel fluorescence lifetime measurements and error analysis show that an average of 40 ± 30 photon counts in the peak channel with a signal-to-noise ratio of 20 is enough to calculate a reliable fluorescence lifetime from a single exponential fluorescence decay. No heterogeneity in the actin cytoskeleton in different regions of the cultured cells was measured in the 40- to 400-nm spatial regime.

  2. 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

  3. Photoacoustic imaging of the excited state lifetime of fluorophores

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

    Photoacoustic (PA) imaging using pump-probe excitation has been shown to allow the detection and visualization of fluorescent contrast agents. The technique relies upon inducing stimulated emission using pump and probe pulses at excitation wavelengths that correspond to the absorption and fluorescence spectra. By changing the time delay between the pulses, the excited state lifetime of the fluorophore is modulated to vary the amount of thermalized energy, and hence PA signal amplitude, to provide fluorophore-specific PA contrast. In this study, this approach was extended to the detection of differences in the excited state lifetime of fluorophores. PA waveforms were measured in solutions of a near-infrared fluorophore using simultaneous and time-delayed pump-probe excitation. The lifetime of the fluorophore solutions was varied by using different solvents and quencher concentrations. By calculating difference signals and by plotting their amplitude as a function of pump-probe time delay, a correlation with the excited state lifetime of the fluorophore was observed. The results agreed with the output of a forward model of the PA signal generation in fluorophores. The application of this method to tomographic PA imaging of differences in the excited state lifetime was demonstrated in tissue phantom experiments.

  4. A fluorescence high-temperature sensor based on fluorescence lifetime

    NASA Astrophysics Data System (ADS)

    Wu, Jinling; Wang, Yutian; Wang, Xinian

    2006-11-01

    A kind of fluorescence optic-fiber temperature sensor is devised based on the alexandrite crystal. In this system, a new optic- fiber probe fabrication techniques is proposed. This system is particularly adapted to the temperature measurement in the range of room temperature to 650°C. During the cause of experimentation, using the PLD-PMTR (termed the Pulse Modulated Phase-locked detection with Two References) signal processing scheme. This temperature measurement method is proved to be effective and useful for its highly resolution and precision. It ensured the detected fluorescence signal to noise ratio was high enough to be measurable when the temperature is raised to 650°C.

  5. 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.

  6. Near-infrared spark source excitation for fluorescence lifetime measurements

    NASA Astrophysics Data System (ADS)

    Birch, D. J. S.; Hungerford, G.; Imhof, R. E.

    1991-10-01

    We have extended the range of excitation wavelengths from spark sources used in single photon timing fluorometry into the near infrared by means of the all-metal coaxial flashlamp filled with an argon-hydrogen gas mixture. At 750 nm this mixture gives ˜15 times the intensity available from pure hydrogen for a comparable pulse duration. Measurements are demonstrated by using the laser dye IR-140 in acetone, for which a fluorescence lifetime of 1.20 ns is recorded.

  7. 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.

  8. Principles and applications of fluorescence lifetime correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Beranová, Lenka; Humpolícková, Jana; Hof, Martin

    2009-05-01

    Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photon counting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new technique with several experimental benefits. In FLCS experiments, pulsed excitation is used and data are stored in a special time-tagged time-resolved mode. Mathematical treatment of TCSPC decay patterns of distinct fluorophores and their mixture enables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties and concentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed and information on interaction of the species can be obtained. This technique is particularly helpful for distinguishing different states of the same fluorophore in different microenvironments. The first application of that concept represents the simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dye is considerably quenched in the layer near the light-absorbing surface. This concept was also used in other applications: a) investigation of a conformational change of a labeled protein, b) detection of small amounts of labeled oligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNA molecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.

  9. 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.

  10. 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.