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Fluorescence Lifetime Imaging Microscopy (fluorescence + lifetime_imaging_microscopy)
Selected AbstractsCharacterization of yeast strains by fluorescence lifetime imaging microscopyFEMS YEAST RESEARCH, Issue 1 2008Hemant Bhatta Abstract The results of fluorescence lifetime imaging microscopy of selected yeast strains were presented and the fact that the lifetime distributions can be successfully used for strain characterization and differentiation was demonstrated. Four strains of industrially relevant yeast Saccharomyces were excited at 405 nm and the autofluorescence observed within 440,540 nm. Using statistical tools such as empirical cumulative distribution functions with Kolmogorov,Smirnov testing, the four studied strains were categorized into three different groups for normal sample size of 70 cells slide,1 at a significance level of 5%. The differentiation of all of the examined strains from one another was shown to be possible by increasing the sample size to 420 cells, which is achievable by taking the lifetime data at six different positions in the slide. [source] Fluorescent proteins for single-molecule fluorescence applicationsJOURNAL OF BIOPHOTONICS, Issue 1 2008Britta Seefeldt Abstract We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurementsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2009Aymeric Leray Abstract Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system. Microsc. Res. Tech., 2009. © 2008 Wiley-Liss, Inc. [source] Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitationMICROSCOPY RESEARCH AND TECHNIQUE, Issue 4 2008Stanley Walter Botchway Abstract The real-time uptake of serotonin, a neurotransmitter, by rat leukemia mast cell line RBL-2H3 and 5-hydroxytryptophan by Chinese hamster V79 cells has been studied by fluorescence lifetime imaging microscopy (FLIM), monitoring ultraviolet (340 nm) fluorescence induced by two-photon subpicosecond 630 nm excitation. Comparison with two-photon excitation with 590 nm photons or by three-photon excitation at 740 nm shows that the use of 630 nm excitation provides optimal signal intensity and lowered background from auto-fluorescence of other cellular components. In intact cells, we observe using FLIM three distinct fluorescence lifetimes of serotonin and 5-hydroxytryptophan according to location. The normal fluorescence lifetimes of both serotonin (3.8 ns) and 5-hydroxytryptophan (3.5 ns) in solution are reduced to ,2.5 ns immediately on uptake into the cell cytosol. The lifetime of internalized serotonin in RBL-2H3 cells is further reduced to ,2.0 ns when stored within secretory vesicles. Microsc. Res. Tech., 2008. © 2007 Wiley-Liss, Inc. [source] Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cellsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 4-5 2004H.E. Grecco Abstract In this work, we characterized streptavidin-conjugated quantum dots (QDs) manufactured by Quantum Dot Corporation. We present data on: (1) two-photon excitation; (2) fluorescence lifetimes; (3) ensemble and single QD emission anisotropy; (4) QDs as donors for Förster resonance energy transfer (FRET); and (5) spectral conversion of QDs exposed to high-intensity illumination. We also demonstrate the utility of QDs for (1) imaging the binding and uptake of biotinylated transferrin on living cells, and (2) resolving by fluorescence lifetime imaging microscopy (FLIM) signals originating from QDs from those of spatially and spectrally overlapping visible fluorescent proteins (VFPs). Microsc. Res. Tech. 65:169,179, 2004. © 2005 Wiley-Liss, Inc. [source] Quantitative FRET Analysis With the E0GFP-mCherry Fluorescent Protein PairPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2009Lorenzo Albertazzi Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein,protein interaction and even protein modifications in living cells. Here, we analyze the E0GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro. The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R0 = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E0GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo. Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo) reveals that this new pair can be used for very effective quantitative FRET imaging. [source] | |||||||||||||