Spectrally resolved fluorescence lifetime and FRET measurements

Laimonas Kelbauskas; Sascha Dietrich; Birgit Hoffmann; Thomas Zimmer; Klaus Benndorf; Wolfgang Becker; Axel Bergmann; Nikolaj Klöcker; Christoph Biskup

doi:10.1117/12.645809

23 February 2006 Spectrally resolved fluorescence lifetime and FRET measurements

Laimonas Kelbauskas, Sascha Dietrich, Birgit Hoffmann, Thomas Zimmer, Klaus Benndorf, Wolfgang Becker, Axel Bergmann, Nikolaj Klöcker, Christoph Biskup

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Proceedings Volume 6089, Multiphoton Microscopy in the Biomedical Sciences VI; 608903 (2006) https://doi.org/10.1117/12.645809
Event: SPIE BiOS, 2006, San Jose, California, United States

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.

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Laimonas Kelbauskas, Sascha Dietrich, Birgit Hoffmann, Thomas Zimmer, Klaus Benndorf, Wolfgang Becker, Axel Bergmann, Nikolaj Klöcker, and Christoph Biskup "Spectrally resolved fluorescence lifetime and FRET measurements", Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 608903 (23 February 2006); https://doi.org/10.1117/12.645809

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KEYWORDS

Luminescence

Fluorescence resonance energy transfer

Microscopes

Molecules

Sensors

Proteins

Photons

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