Research Papers

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser

[+] Author Affiliations
Yuansheng Sun

University of Virginia, Department of Biology, W. M. Keck Center for Cellular Imaging, McCormick Road, Charlottesville, Virginia 22904

Cynthia F. Booker, Sangeeta Kumari

University of Virginia Health System, Department of Medicine and Department of Cell Biology, Charlottesville, Virginia 22908

Richard N. Day

Indiana University, School of Medicine, Department of Cellular and Integrative Physiology, 635 Barnhill Drive, Indianapolis, Indiana 46202

Mike Davidson

The Florida State University, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310

Ammasi Periasamy

University of Virginia, Department of Biology, W. M. Keck Center for Cellular Imaging and Department of Biomedical Engineering, McCormick Road, Charlottesville, Virginia 22904

J. Biomed. Opt. 14(5), 054009 (September 16, 2009). doi:10.1117/1.3227036
History: Received January 15, 2009; Revised July 13, 2009; Accepted July 16, 2009; Published September 16, 2009
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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 470to670nm 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.

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© 2009 Society of Photo-Optical Instrumentation Engineers

Citation

Yuansheng Sun ; Cynthia F. Booker ; Sangeeta Kumari ; Richard N. Day ; Mike Davidson, et al.
"Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser", J. Biomed. Opt. 14(5), 054009 (September 16, 2009). ; http://dx.doi.org/10.1117/1.3227036


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