Fluorescence lifetime imaging by time‐correlated single‐photon counting

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.