Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.

SummaryA whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a modelocked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diodepumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.

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This article describes a wide-field time-domain fluorescence lifetime imaging (FLIM) microscope with optical sectioning. The FLIM system utilizes a wide-field time-gated optical image intensifier, with a minimum gate width of 85 ps, to achieve high temporal resolution of fluorescence decays induced by ultrashort laser pulses. Different configurations, using excitation pulses of picojoule energy at 80 MHz repetition rate and of nanojoule energy at 10 kHz, are compared. The instrument has a temporal dynamic range spanning from 100 ps to tens of μs and is shown to have a temporal discrimination better than 10 ps. When applied to laser dye samples, it has produced FLIM maps demonstrating sensitivity to variations in both chemical species and local environment, e.g., viscosity. Wide-field optical sectioning is achieved using the technique of structured illumination, which is applied to remove out-of-focus light that can result in lifetime artifacts. The sectioning strength, which may be adjusted by choosing an appropriate spatial modulation frequency, is characterized and shown to be comparable to that of a confocal microscope. Practical considerations concerned with improving the quality of sectioned fluorescence lifetime maps, including using a large bit depth camera, are discussed.