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 homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.

The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a puked light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. We have carried out time-resolved emission anisotropy studies of the tryptophan fluorescence of a series of proteins to determine the angular range and kinetics of internal rotational motions of this chromophore. Nanosecond emission anisotropy studies have previously provided information concerning the segmental flexibility of domains of immunoglobulins (7, 8) and myosin (9). These studies had a time resolution of several nanoseconds and used extrinsic fluorescent probes. By using synchrotron radiation, we are now able to monitor directly the rotational motions of tryptophan residues in proteins, obviating any perturbations that may be caused by the insertion of an extrinsic probe. The distinctive properties of synchrotron radiation for these studies are its subnanosecond pulse width, high repetition rate and reproducibility, and high intensity in the ultraviolet region (10, 11). Proteins with a single tryptophan residue were studied because the interpretation of their emission anisotropy kinetics is more definitive than for proteins with multiple tryptophans. The molecules investigated were human serum albumin (69,000 daltons) (12), Staphylococcus aureus nuclease B (20,000 daltons) (13), human basic Al myelin protein (18,000 daltons) (14), and Pseudomonas aeruginosa holoazurin and apoazurin (14,000 daltons) (15). THEORY AND ANALYSISIn a time-resolved emission anisotropy experiment, an isotropic sample is excited by a pulse of y-polarized (vertically polarized) light, which produces an ensemble of preferentially aligned excited molecules. The orientations of the excited molecules then become randomized by rotational Brownian motion. For a fluorescent chromophore in a macromolecule, the rate of randomization depends both on the degree of flexibility of this group with respect to the macromolecule and on the size, shape, and internal motions of the macromolecule. These rotational motions can be monitored by measuring y(t) and x(t), the intensities of the y-polarized and x-polarized (horizontally polarized) components of the fluorescence emission as a function of time (for reviews, see refs. 16 and 17). The total fluorescence intensity F(t) and the emission anisotropy A(t) are defined by F(t) = y(t) + 2x(t)The simplest case is a chromophore with a single excited-state lifetime rotating in common with a rigid sphere. F(t) and A(t) are then given by[41 in which Fo is the initial fluorescence intensity and r is the excited state lifetime. For a rigid sphere, the rotational correlation time . is given by...

Fluorescence lifetime imaging (FLIM) is a functional imaging methodology that can provide information, not only concerning the localisation of specific fluorophores, but also about the local fluorophore environment. It may be implemented in scanning confocal or multi-photon microscopes, or in wide-field microscopes and endoscopes. When applied to tissue autofluorescence, it reveals intrinsic excellent contrast between different types and states of tissue. This article aims to review our recent progress in developing time-domain FLIM technology for microscopy and endoscopy and applying it to biological tissue.

We therefore believe that FLIM has a potential future clinical role in imaging BCCs for rapid and noninvasive tumour delineation and as an aid to determine adequate excision margins with best preservation of normal tissue.

We apply fluorescence lifetime imaging to the membrane phase-sensing dye di-4-ANEPPDHQ in model membranes and live cells. We show that the 1700 ps lifetime shift between liquid-disordered and liquid-ordered phases offers greater contrast than the 60 nm spectral shift previously reported. Detection of cholesterol-rich membrane microdomains is confirmed by observation of the temperature dependence of membrane order and by cholesterol depletion using methyl-beta-cyclodextrin.

Abstract. The measurement of the strength and velocity of atmospheric optical turbulence using a generalised SCIDAR technique is outlined and demonstrated. This method allows the full turbulent profile to be characterised from the telescope pupil up to any desired altitude. A number of example profiles from various astronomical observing sites are presented.

Measurements have been made of the concentration dependence of the rise and decay time characteristics of the monomer and excimer fluorescence of deoxygenated solutions of pyrene in cyclohexane at temperatures from 293 to 340 °K. Two independent methods were employed, one using a pulsed light source and a pulse-sampling oscilloscope, and the other a modulated light source and a phase and modulation fluorometer. In conjunction with observations of the monomer and excimer fluorescence quantum efficiencies, the results have been analyzed to determine the six rate parameters which describe the behaviour of the system. Values of 6.8 x 10 -7 and 0.9 x 10 -7 s are obtained for the radiative lifetimes of the monomer and excimer, respectively. Excimer formation is shown to be a diffusion-controlled collision process, in which every collision between excited and unexcited molecules is effective. From the difference in the activation energies for excimer dissociation and formation, the excimer binding energy is found to be 0.34eV.

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.