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Proceedings Paper

High resolution TCSPC lifetime imaging
Author(s): Wolfgang Becker; Axel Bergmann; Christoph Biskup; Laimonas Kelbauskas; Thomas Zimmer; Nikolaj Klocker; Klaus Benndorf
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Paper 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.

Paper Details

Date Published: 10 July 2003
PDF: 10 pages
Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003); doi: 10.1117/12.472866
Show Author Affiliations
Wolfgang Becker, Becker & Hickl GmbH (Germany)
Axel Bergmann, Becker & Hickl GmbH (Germany)
Christoph Biskup, Friedrich-Schiller-Univ. Jena (Germany)
Laimonas Kelbauskas, Friedrich-Schiller-Univ. Jena (Germany)
Thomas Zimmer, Friedrich-Schiller-Univ. Jena (France)
Nikolaj Klocker, Albert-Ludwigs-Univ. Freiburg (Germany)
Klaus Benndorf, Friedrich-Schiller-Univ. Jena (Germany)


Published in SPIE Proceedings Vol. 4963:
Multiphoton Microscopy in the Biomedical Sciences III
Ammasi Periasamy; Peter T. C. So, Editor(s)

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