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SpRET: Highly Sensitive and Reliable Spectral Measurement of Absolute FRET Efficiency

Published online by Cambridge University Press:  21 February 2011

Shiri Levy ,
Christian D. Wilms ,
Eliaz Brumer ,
Joy Kahn ,
Lilach Pnueli ,
Yoav Arava ,
Jens Eilers  and
Daniel Gitler
Shiri Levy
Affiliation:
Department of Physiology and Neurobiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
Christian D. Wilms
Affiliation:
Carl-Ludwig-Institut für Physiologie, Liebigstr. 27, 04103 Leipzig, Germany
Eliaz Brumer
Affiliation:
Department of Physiology and Neurobiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
Joy Kahn
Affiliation:
Department of Physiology and Neurobiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
Lilach Pnueli
Affiliation:
Department of Biology, Technion—Israel Institute of Technology, Haifa 32000, Israel
Yoav Arava
Affiliation:
Department of Biology, Technion—Israel Institute of Technology, Haifa 32000, Israel
Jens Eilers
Affiliation:
Carl-Ludwig-Institut für Physiologie, Liebigstr. 27, 04103 Leipzig, Germany
Daniel Gitler*
Affiliation:
Department of Physiology and Neurobiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
*
Corresponding author. E-mail: [email protected]
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Abstract

Contemporary research aims to understand biological processes not only by identifying participating proteins, but also by characterizing the dynamics of their interactions. Because Förster's Resonance Energy Transfer (FRET) is invaluable for the latter undertaking, its usage is steadily increasing. However, FRET measurements are notoriously error-prone, especially when its inherent efficiency is low, a not uncommon situation. Furthermore, many FRET methods are either difficult to implement, are not appropriate for observation of cellular dynamics, or report instrument-specific indices that hamper communication of results within the scientific community. We present here a novel comprehensive spectral methodology, SpRET, which substantially increases both the reliability and sensitivity of FRET microscopy, even under unfavorable conditions such as weak fluorescence or the presence of noise. While SpRET overcomes common pitfalls such as interchannel crosstalk and direct excitation of the acceptor, it also excels in removal of autofluorescence or background contaminations and in correcting chromatic aberrations, often overlooked factors that severely undermine FRET experiments. Finally, SpRET quantitatively reports absolute rather than relative FRET efficiency values, as well as the acceptor-to-donor molar ratio, which is critical for full and proper interpretation of FRET experiments. Thus, SpRET serves as an advanced, improved, and powerful tool in the cell biologist's toolbox.

Keywords

FRETfluorescent proteinsmicroscopyfluorescencespectral unmixingmolecular interactions
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 17 , Issue 2 , April 2011 , pp. 176 - 190
Copyright
Copyright © Microscopy Society of America 2011

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Footnotes

Current address: Wolfson Institute for Biomedical Research, University College London, Gower Street, London NW6 2NE, United Kingdom

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