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 Day, Mike Davidson, Ammasi Periasamy

Research output: Contribution to journalArticle

48 Citations (Scopus)

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.

Original languageEnglish
Article number054009
JournalJournal of Biomedical Optics
Volume14
Issue number5
DOIs
StatePublished - 2009

Fingerprint

resonance fluorescence
Energy transfer
Microscopic examination
energy transfer
microscopy
proteins
Proteins
Lasers
lasers
Wavelength
wavelengths
Argon lasers
excitation
Fluorescence Resonance Energy Transfer
argon lasers
Imaging systems
amino acids
Amino acids
Assays
Fluorescence

Keywords

  • 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

Cite this

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser. / Sun, Yuansheng; Booker, Cynthia F.; Kumari, Sangeeta; Day, Richard; Davidson, Mike; Periasamy, Ammasi.

In: Journal of Biomedical Optics, Vol. 14, No. 5, 054009, 2009.

Research output: Contribution to journalArticle

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