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Proceedings Paper

Spectrally resolved fluorescence lifetime and FRET measurements
Author(s): Laimonas Kelbauskas; Sascha Dietrich; Birgit Hoffmann; Thomas Zimmer; Klaus Benndorf; Wolfgang Becker; Axel Bergmann; Nikolaj Klöcker; Christoph Biskup
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Paper Abstract

We present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain in conjunction with a laser scanning microscope and a pulsed excitation source. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting (TCSPC) approach. The complete setup consists of a laser scanning microscope (LSM-510, Zeiss), a polychromator (250is, Chromex), a streak camera (C5680 with M5677 sweep unit, Hamamatsu Photonics) or a 16-channel TCSPC detector head (PML-16, Becker and Hickl) connected to a TCSPC imaging module (SPC-730/SPC-830, Becker and Hickl). With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. The fluorescence emitted by the sample can be recorded in a single measurement. No filters have to be used to separate the contributions of different fluorophores to the overall fluorescence signal. When applied to Forster resonance energy transfer (FRET) measurements, the technique allows to separate the decay components of the donor and acceptor fluorescence. In this way, it is possible to reliably determine FRET efficiencies between acceptor and donor fluorophores in given subcellular structures.

Paper Details

Date Published: 23 February 2006
PDF: 10 pages
Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 608903 (23 February 2006); doi: 10.1117/12.645809
Show Author Affiliations
Laimonas Kelbauskas, Arizona State Univ. (United States)
Sascha Dietrich, Friedrich-Schiller Univ. Jena (Germany)
Birgit Hoffmann, Friedrich-Schiller Univ. Jena (Germany)
Thomas Zimmer, Friedrich-Schiller Univ. Jena (Germany)
Klaus Benndorf, Friedrich-Schiller Univ. Jena (Germany)
Wolfgang Becker, Becker & Hickl GmbH (Germany)
Axel Bergmann, Becker & Hickl GmbH (Germany)
Nikolaj Klöcker, Albert-Ludwigs Univ. Freiburg (Germany)
Christoph Biskup, Friedrich-Schiller Univ. Jena (Germany)

Published in SPIE Proceedings Vol. 6089:
Multiphoton Microscopy in the Biomedical Sciences VI
Ammasi Periasamy; Peter T. C. So, Editor(s)

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