High resolution TCSPC lifetime imaging

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Abstract
Time-correlated single photon counting (TCSPC) allows ps resolved multi-wavelength imaging in laser scanning microscopes. The technique is based on a four-dimensional histogramming process that records the photon density over the time within the fluorescence decay function, the x-y coordinates of the scanning area and the wavelength channel number. It avoids any time gating or wavelength scanning and therefore yields a near-perfect counting efficiency. It does, however, require a very large data memory and therefore was limited to image sizes smaller than the scanning resolution of the microscopes and a time bin width that does not fully exploit the time resolution of the best detectors. We show an improved version of TCSPC imaging that allows for recording of lifetime images with 1024 x 1024 pixels and a time bin width of 1 ps at the full scanning speed of a Zeiss LSM-510. The performance is demonstrated for resolving fast lifetime components in CFP/YFP FRET systems.

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