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

Yuansheng Sun, Cynthia F. Booker, Sangeeta Kumari, Richard N. Day, Mike Davidson, Ammasi Periasamy

Research output: Contribution to journalArticle

49 Scopus citations


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.

Original languageEnglish (US)
Article number054009
JournalJournal of Biomedical Optics
Issue number5
StatePublished - Dec 1 2009



  • Fluorescence lifetime imaging FRET (FLIM-FRET)
  • Forster resonance energy transfer (FRET)
  • Instrument response function (IRF)
  • Monomeric Kusabira orange 2 (mKO2)
  • Monomeric teal fluorescent protein (mTFP)
  • Protein-protein interactions
  • Spectral FRET (sFRET)
  • White-light laser

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biomaterials
  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics

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