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Journal of Biomedical Optics

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser
Author(s): Yuansheng Sun; Cynthia F. Booker; Sangeeta Kumari; Richard N. Day; Michael W. Davidson; Ammasi Periasamy
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Paper Abstract

Orange fluorescent proteins (FPs) are attractive candidates as Förster resonance energy transfer (FRET) partners, bridging the gap between green and red/far-red FPs, but they pose significant challenges using common fixed laser wavelengths. We investigated monomeric Kusabira orange 2 (mKO2) FP as a FRET acceptor for monomeric teal FP (mTFP) as donor on a FRET standard construct using a fixed-distance amino acid linker, expressed in live cells. We quantified the apparent FRET efficiency (E%) of this construct, using sensitized spectral FRET microscopy on the Leica TCS SP5 X imaging system equipped with a white-light laser that allows choosing any excitation wavelength from 470 to 670 nm in 1-nm increments. The E% obtained in sensitized spectral FRET microscopy was then confirmed with fluorescence lifetime measurements. Our results demonstrate that mKO2 and mTFP are good FRET partners given proper imaging setups. mTFP was optimally excited by the Argon 458 laser line, and the 540-nm wavelength excitation for mKO2 was chosen from the white-light laser. The white-light laser generally extends the usage of orange and red/far-red FPs in sensitized FRET microscopy assays by tailoring excitation and emission precisely to the needs of the FRET pair.

Paper Details

Date Published: 1 September 2009
PDF: 11 pages
J. Biomed. Opt. 14(5) 054009 doi: 10.1117/1.3227036
Published in: Journal of Biomedical Optics Volume 14, Issue 5
Show Author Affiliations
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)
Richard N. Day, Indiana Univ. School of Medicine (United States)
Michael W. Davidson, Florida State Univ. (United States)
Ammasi Periasamy, Univ. of Virginia (United States)

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