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Proceedings Paper

The characterization of optimized fluorescent proteins for Förster resonance energy transfer microscopy
Author(s): Richard N. Day; Yuansheng Sun; Cynthia F. Booker; Sangeeta Kumari; Ammasi Periasamy; Mike Davidson
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Paper Abstract

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. Currently, the FPs most commonly used for live-cell FRET studies are the Cerulean and Venus variants of the cyan and yellow FPs. However, there are problems associated with this donor-acceptor pair, and these might be overcome by exploiting the characteristics of some of the newer FPs. For example, earlier we showed that the monomeric teal FP (mTFP) has advantages over Cerulean as a FRET donor for Venus. Here, using mTFP as the common donor fluorophore, we characterize a variety of different yellow, orange and red FPs as potential acceptors of FRET. We employed a "FRET standard" genetic construct to minimize variability in the separation distance and positioning of the fused donor and acceptor FPs. Using spectral FRET imaging and fluorescence lifetime measurements from living cells expressing the fused proteins, we characterized both sensitized acceptor emission and the shortening of the donor lifetime resulting from quenching for each of the fused FP pairs. Surprisingly, we found disagreements between the spectral FRET and lifetime measurements for some of the different FP pairs. Our results appear to indicate that some of the orange and red FPs can quench the mTFP donor while yielding little sensitized emission. We are characterizing the basis for this observation.

Paper Details

Date Published: 25 February 2009
PDF: 9 pages
Proc. SPIE 7183, Multiphoton Microscopy in the Biomedical Sciences IX, 718306 (25 February 2009); doi: 10.1117/12.814918
Show Author Affiliations
Richard N. Day, Univ. of Virginia Health System (United States)
Yuansheng Sun, Univ. of Virginia (United States)
Cynthia F. Booker, Univ. of Virginia Health System (United States)
Sangeeta Kumari, Univ. of Virginia Health System (United States)
Ammasi Periasamy, Univ. of Virginia (United States)
Mike Davidson, Florida State Univ. (United States)

Published in SPIE Proceedings Vol. 7183:
Multiphoton Microscopy in the Biomedical Sciences IX
Ammasi Periasamy; Peter T. C. So, Editor(s)

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