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Research Papers

Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy

[+] Author Affiliations
Steffen Rüttinger, Rainer Macdonald

Physikalisch-Technische Bundesanstalt, Berlin, Germany

Benedikt Krämer, Felix Koberling

PicoQuant GmbH, Berlin, Germany

Martin Roos, Eberhardt Hildt

Robert Koch Institut, Berlin, Germany

J. Biomed. Opt. 11(2), 024012 (March 27, 2006). doi:10.1117/1.2187425
History: Received July 27, 2005; Revised November 24, 2005; Accepted December 05, 2005; Published March 27, 2006
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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.

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© 2006 Society of Photo-Optical Instrumentation Engineers

Citation

Steffen Rüttinger ; Benedikt Krämer ; Martin Roos ; Eberhardt Hildt ; Felix Koberling, et al.
"Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy", J. Biomed. Opt. 11(2), 024012 (March 27, 2006). ; http://dx.doi.org/10.1117/1.2187425


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