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Optimization of Pairings and Detection Conditions for Measurement of FRET between Cyan and Yellow Fluorescent Proteins

Published online by Cambridge University Press:  16 May 2006

Mark A. Rizzo
University of Maryland School of Medicine, Baltimore, MD 21201, USA
Gerald Springer
Department of Molecular Physiology and Biophysics, 735 Light Hall, Vanderbilt University, Nashville, TN 37232, USA
Katsuhisa Segawa
3-204 Murayamadainijutaku, 1-2-46 Fujimicho Higashimurayama-shi, Tokyo 189-0024, Japan
Warren R. Zipfel
Applied & Engineering Physics, 212 Clark Hall, Cornell University, Ithaca, NY 14853, USA
David W. Piston
Department of Molecular Physiology and Biophysics, 735 Light Hall, Vanderbilt University, Nashville, TN 37232, USA
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Detection of Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater Förster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.Note: M. Rizzo and K. Segawa performed this research at Vanderbilt University (same address as Piston). W. Zipfel performed research at Cornell University.

© 2006 Microscopy Society of America

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