Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale

A frequency domain fluorescence lifetime imaging microscope (FLIM) has been developed. A continuous wave laser excitation source of an epi-illumination fluorescence microscope is modulated at a high frequency fA. The lifetime of the modulated fluorescence emission is determined from the phase delay and modulation depth of the fluorescence signal relative to that of the excitation light. Phase detection is accomplished simultaneously at every location in the image by modulating the high voltage amplification stage of a microchannel plate image intensifier at a frequency near (heterodyne method) or at (homodyne method) fA. The heterodyne or homodyne image output of the intensifier is focused onto a cooled high resolution charge-coupled-device camera for digital recording and subsequent analysis of phase and modulation. The technique has the sensitivity of normal steady state microscopy, and is relatively simple to employ. We present several examples illustrating the applications of FLIM for determining prompt fluorescence lifetimes in picoliter homogeneous solutions, for lifetime imaging of single cells, and for phase suppressing particular lifetime components in fluorescence images. Several unique aspects of lifetime resolved image processing are featured and discussed, including the analysis, statistical evaluation, and display of the data. Coupling of the spatial and temporal aspects of fluorescence images extends considerably the possibilities for quantitative fluorescence microscopy.