  We report the development and detailed calibration of a multiphoton
fluorescence lifetime imaging system (FLIM) using a streak camera. The present
system is versatile with high spatial (0.2 micron) and temporal (50 psec)
resolution and allows rapid data acquisition and reliable and reproducible
lifetime determinations. The system was calibrated with standard fluorescent
dyes and the lifetime values obtained were in very good agreement with values
reported in literature for these dyes. We also demonstrate the applicability of
the system to FLIM studies in cellular specimens including stained pollen
grains and fibroblast cells expressing green fluorescent protein. The lifetime
values obtained matched well with those reported earlier by other groups for
these same specimens. Potential applications of the present system include the
measurement of intracellular physiology and Fluorescence Resonance Energy
Transfer (FRET) imaging which are discussed in the context of live cell
imaging.
