期刊
JOURNAL OF BIOMEDICAL OPTICS
卷 14, 期 5, 页码 -出版社
SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS
DOI: 10.1117/1.3227036
关键词
Forster resonance energy transfer (FRET); spectral FRET (sFRET); fluorescence lifetime imaging FRET (FLIM-FRET); white-light laser; monomeric Kusabira orange 2 (mKO2); monomeric teal fluorescent protein (mTFP); instrument response function (IRF); protein-protein interactions
资金
- National Center for Research Resources
- National Institutes of Health [RR025616]
- NATIONAL CENTER FOR RESEARCH RESOURCES [S10RR025616] Funding Source: NIH RePORTER
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. (C) 2009 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3227036]
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