Special Section on Visible Fluorescent Proteins

Characterization of an improved donor fluorescent protein for Förster resonance energy transfer microscopy

[+] Author Affiliations
Richard N. Day, Cynthia F. Booker

University of Virginia Health System, Departments of Medicine and Cell Biology, P. O. Box 800578, Charlottesville, Virginia 22908-0578

Ammasi Periasamy

University of Virginia, W.M. Keck Center for Cellular Imaging, Departments of Biology and Biomedical Engineering, Gilmer Hall, Charlottesville, Virginia 22904-4328

J. Biomed. Opt. 13(3), 031203 (June 10, 2008). doi:10.1117/1.2939094
History: Received August 09, 2007; Revised January 08, 2008; Accepted January 09, 2008; Published June 10, 2008
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The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Typically, the Cerulean and Venus variants of the cyan and yellow FPs are used for FRET studies, but there are limitations to their use. Here, Cerulean and the newly developed monomeric Teal FP (mTFP) are compared as FRET donors for Venus using spectral and fluorescence lifetime measurements from living cells. The results demonstrate that when compared to Cerulean, mTFP has increased brightness, optimal excitation using the standard 458-nm laser line, increased photostability, and improved spectral overlap with Venus. In addition, the two-photon excitation and fluorescence lifetime characteristics are determined for mTFP. Together, these measurements indicate that mTFP is an excellent donor fluorophore for FRET studies, and that its use may improve the detection of interactions involving proteins that are difficult to express, or that need to be produced at low levels in cells.

Figures in this Article
© 2008 Society of Photo-Optical Instrumentation Engineers

Citation

Richard N. Day ; Cynthia F. Booker and Ammasi Periasamy
"Characterization of an improved donor fluorescent protein for Förster resonance energy transfer microscopy", J. Biomed. Opt. 13(3), 031203 (June 10, 2008). ; http://dx.doi.org/10.1117/1.2939094


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