title: Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser.
abstract: Orange fluorescent proteins (FPs) are attractive candidates as Forster 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.
authors.affiliation: University of Virginia, Department of Biology, W. M. Keck Center for Cellular Imaging, McCormick Road, Charlottesville, Virginia 22904, USA.
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Sun
authors.forename: Cynthia F
authors.initials: CF
authors.lastname: Booker
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Kumari
authors.forename: Richard N
authors.initials: RN
authors.lastname: Day
authors.forename: Mike
authors.initials: M
authors.lastname: Davidson
authors.forename: Ammasi
authors.initials: A
authors.lastname: Periasamy
mesh_terms: D000818:Animals; D002460:Cell Line; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D007834:Lasers; D008164:Luminescent Proteins / Q000032:analysis*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D010902:Pituitary Gland / Q000166:cytology* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein