Picosecond fluorescence lifetime microscopy by TCSPC imaging

Wolfgang Becker; Axel Bergmann; Karsten Koenig; Uday Tirlapur

doi:10.1117/12.424584

24 April 2001 Picosecond fluorescence lifetime microscopy by TCSPC imaging

Wolfgang Becker, Axel Bergmann, Karsten Koenig, Uday Tirlapur

Proceedings Volume 4262, Multiphoton Microscopy in the Biomedical Sciences; (2001) https://doi.org/10.1117/12.424584
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

A new Time-Correlated Single Photon Counting (TCSPC) imaging technique delivers combined intensity-lifetime images in a two-photon laser scanning microscope. The sample is excited by laser pulses of 150 fs duration and 80 MHz repetition rate. The microscope scans the sample with a pixel dwell time in the +s range. The fluorescence is detected with a fast PMT at the non-descanned port of the laser scanning microscope. The single photon pulses from the PMT and the scan control signals from the scanning head are used to build up a three-dimensional histogram of the photon density over the time within the decay function and the image coordinates x and y. Analysis of the recorded data delivers images containing the intensity as brightness and the lifetime as colour, images within selected time windows or decay curves in selected pixels. The performance of the system is shown for typical applications such as FRET measurements, Ca imaging and discrimination of endogenous fluorophores or different dyes in living cells and tissues.

Citation Download Citation

Wolfgang Becker, Axel Bergmann, Karsten Koenig, and Uday Tirlapur "Picosecond fluorescence lifetime microscopy by TCSPC imaging", Proc. SPIE 4262, Multiphoton Microscopy in the Biomedical Sciences, (24 April 2001); https://doi.org/10.1117/12.424584

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