Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope w ith the capability to perform optical sectioning is described. The excitati on source is a mode-locked Ti:Sapphire laser that is regeneratively amplifi ed and frequency doubled to 415 nm. Time-gated fluorescence intensity image s at increasing delays after excitation are acquired using a gated microcha nnel plate image intensifier combined with an intensified CCD camera. By fi tting 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 componen t of the fluorescence lifetime. This FLIM instrument was demonstrated to ex hibit a temporal discrimination of better than 10 ps. It has been applied t o chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorop hore environment. Initially, standard fluorescent dyes were studied and the n this FLIM microscope was applied to the imaging of biological tissue, suc cessfully contrasting different tissues and different states of tissue usin g autofluorescence. To demonstrate the potential for real-world application s, the FLIM microscope has been configured using potentially compact, porta ble and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structure d illumination technique.