Becker, W.W.BeckerBergmann, A.A.BergmannHink, M.A.M.A.HinkKönig, K.K.KönigBenndorf, K.K.BenndorfBiskup, C.C.Biskup2022-03-032022-03-032004https://publica.fraunhofer.de/handle/publica/20564710.1002/jemt.10421We 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.en610620502journal article