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10 July 2003 High resolution TCSPC lifetime imaging
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Abstract
Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging in laser scanning microscopes can be combined with a multi-detector technique that allows to record time-resolved images in several wavelength channels simultaneously. The technique is based on a multi-dimensional histogramming process that records the photon density versus the time within the fluorescence decay function, the x-y coordinates of the scanning area and the detector channel number. It avoids any time gating or wavelength switching and therefore yields a near-ideal counting efficiency. We show an instrument that records dual wavelength lifetime images with up to 512 x 512 pixels, and single wavelength lifetime images with up to 1024 x 1024 pixels. It resolves the components of double-exponential decay functions down to 30 ps, and works at the full scanning speed of a two-photon laser scanning microscope. The performance of the instrument is demonstrated for simultaneous lifetime imaging of the donor and acceptor fluorescence in CFP/YFP FRET systems and for tissue samples stained with several fluorophores.
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Wolfgang Becker, Axel Bergmann, Christoph Biskup, Laimonas Kelbauskas, Thomas Zimmer, Nikolaj Klocker, and Klaus Benndorf "High resolution TCSPC lifetime imaging", Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003); doi: 10.1117/12.472866; https://doi.org/10.1117/12.472866
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