Fluorescence lifetime imaging by time-correlated single-photon counting |
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Authors: | Becker W Bergmann A Hink M A König K Benndorf K Biskup C |
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Affiliation: | Becker & Hickl GmbH, D-12277 Berlin, Germany. becker@becker-hickl.com |
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Abstract: | 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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