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

J. Microsc., Vol. 203, Pt. 3, Sep. 2001, 246-257. M.J. Cole, J. Siegel, S.E.D. Webb, et al.

A whole-field time-domain fluorescence lifetime imaging(FLIM) microscope with the capability to perform opticalsectioning is described. The excitation source is a modelockedTi:Sapphire laser that is regeneratively amplifiedand frequency doubled to 415 nm. Time-gated fluorescenceintensity images at increasing delays afterexcitation are acquired using a gated microchannel plateimage intensifier combined with an intensified CCDcamera. By fitting a single or multiple exponential decayto each pixel in the field of view of the time-gatedimages, 2-D FLIM maps are obtained for each componentof the fluorescence lifetime. This FLIM instrument wasdemonstrated to exhibit a temporal discrimination ofbetter than 10 ps. It has been applied to chemicallyspecific imaging, quantitative imaging of concentrationratios of mixed fluorophores and quantitative imaging ofperturbations to fluorophore environment. Initially, standardfluorescent dyes were studied and then this FLIMmicroscope was applied to the imaging of biologicaltissue, successfully contrasting different tissues anddifferent states of tissue using autofluorescence. Todemonstrate the potential for real-world applications,the FLIM microscope has been configured using potentiallycompact, portable and low cost all-solid-state diodepumpedlaser technology. Whole-field FLIM with opticalsectioning (3D FLIM) has been realized using astructured illumination technique.

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