Multiphoton fluorescence lifetime imaging microscopy (FLIM) using a streak camera

We report the development, calibration and biomedical application of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (approximately 0.2 μm) and temporal (approximately 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 in the context of quantitative measurement of fluorescence resonance energy transfer (FRET).