Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser
Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser
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DOI：
10.1117/1.3227036
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发表时间：
2009-09-01
影响因子：
3.5
通讯作者：
Periasamy, Ammasi
中科院分区：
文献类型：
--
作者：
Sun, Yuansheng;Booker, Cynthia F.;Periasamy, Ammasi
橙色荧光蛋白（FPs）是福斯特共振能量转移（FRET）合作伙伴的有吸引力的候选者，弥补了绿色和红色/远红色FPs之间的差距，但它们对使用常见的固定激光波长构成了重大挑战。我们研究了单体Kusabira橙2 (mKO2) FP作为FRET受体，单体蓝绿色FP （mTFP）作为FRET供体，在FRET标准构建体上使用固定距离的氨基酸连接体，在活细胞中表达。我们在配备白光激光器的徕卡TCS SP5 X成像系统上使用增感光谱FRET显微镜，量化了该结构的表观FRET效率（E%），该系统允许选择从470到670 nm的任何激发波长，以1 nm的增量。在敏化光谱FRET显微镜下获得的E%然后用荧光寿命测量证实。我们的结果表明，在适当的成像设置下，mKO2和mTFP是很好的FRET合作伙伴。氩气458激光线对mTFP进行了最佳激发，选择了白光激光对mKO2的激发波长为540 nm。白光激光器通常扩展使用橙色和红色/远红色FPs在敏化FRET显微镜分析通过裁剪激发和发射精确到FRET对的需要。(C) 2009光学仪器工程师学会。(DOI: 10.1117/1.3227036)
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]