Journal
MICROSCOPY RESEARCH AND TECHNIQUE
Volume 63, Issue 1, Pages 58-66Publisher
WILEY-BLACKWELL
DOI: 10.1002/jemt.10421
Keywords
fluorescence lifetime imaging; FLIM; time-correlated single photon counting; TCSPC; multiphoton microscopy; confocal microscopy; multi-wavelength; fluorescence resonance energy transfer; FRET
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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.
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