Four-dimensional multiphoton microscopy with time-correlated single-photon counting.

We report on the implementation of fluorescence-lifetime imaging in multiphoton excitation microscopy that uses PC-compatible modules for time-correlated single-photon counting. Four-dimensional data stacks are produced with each pixel featuring fluorescence-decay curves that consist of as many as 4096 bins. Fluorescence lifetime(s) and their amplitude(s) are extracted by statistical methods at each pixel or in arbitrarily defined regions of interest. When employing an avalanche photodiode the width of the temporal response function is 420 ps. Although this response confines the temporal resolution to values greater than several hundreds of picoseconds, the lifetime precision is determined by the signal-to-noise ratio and can be in the range of tens of picosconds. Lifetime changes are visualized in pulsed-laser-deposited fluorescent layers as well as in cyan fluorescent proteins that transfer energy to yellow fluorescent proteins in live mammalian cells.