Four-dimensional multiphoton microscopy with time-correlated single-photoncounting

We report on the implementation of fluorescence-lifetime imaging in multiph oton excitation microscopy that uses PC-compatible modules for time-correla ted single-photon counting. Four-dimensional data stacks are produced with each pixel featuring fluorescence-decay curves that consist of as many as 4 096 bins. Fluorescence lifetime(s) and their amplitude(s) are extracted by statistical methods at each pixel or in arbitrarily defined regions of inte rest. When employing an avalanche photodiode the width of the temporal resp onse function is 420 ps. Although this response confines the temporal resol ution to values greater than several hundreds of picoseconds, the lifetime precision is determined by the signal-to-noise ratio and can be in the rang e of tens of picosconds. Lifetime changes are visualized in pulsed-laser-de posited fluorescent layers as well as in cyan fluorescent proteins that tra nsfer energy to yellow fluorescent proteins in live mammalian cells. (C) 20 00 Optical Society of America. OCIS codes: 110.0110, 120.4640, 170.6320, 18 0.2520.