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Journal of Biomedical Optics

Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy
Author(s): Steffen Rüttinger; Rainer Macdonald; Benedikt Krämer; Felix Koberling; Martin Roos; Eberhardt Hildt
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Paper Abstract

Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Förster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.

Paper Details

Date Published: 1 March 2006
PDF: 9 pages
J. Biomed. Opt. 11(2) 024012 doi: 10.1117/1.2187425
Published in: Journal of Biomedical Optics Volume 11, Issue 2
Show Author Affiliations
Steffen Rüttinger, Physikalisch-Technische Bundesanstalt (Germany)
Rainer Macdonald, Physikalisch-Technische Bundesanstalt (Germany)
Benedikt Krämer, PicoQuant GmbH (Germany)
Felix Koberling, PicoQuant GmbH (Germany)
Martin Roos, Robert Koch-Institut (Germany)
Eberhardt Hildt, Robert Koch-Institut (Germany)

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