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Originally published as Biophys J. BioFAST on December 21, 2004.
doi:10.1529/biophysj.104.055442
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Biophysical Journal 88:L14-L16 (2005)
© 2005 The Biophysical Society

High-Contrast Imaging of Fluorescent Protein FRET by Fluorescence Polarization Microscopy

Mark A. Rizzo and David W. Piston

Department of Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee, 37232

Correspondence: Address reprint requests and inquiries to David W. Piston, Tel.: 615-322-7030; Fax:615-322-7236; E-mail: dave.piston{at}vanderbilt.edu.

Detection of Förster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.




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