Millington Michael, Grindlay G Joan, Altenbach Kirsten, Neely Robert K, Kolch Walter, Bencina Mojca, Read Nick D, Jones Anita C, Dryden David T F, Magennis Steven W
Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), The University of Edinburgh, King's Buildings, Edinburgh EH9 3JZ, UK.
Biophys Chem. 2007 May;127(3):155-64. doi: 10.1016/j.bpc.2007.01.008. Epub 2007 Feb 1.
We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.
我们使用宽场光子计数荧光寿命成像技术（FLIM），利用对照荧光共振能量转移（FRET）对来研究固定细胞和活细胞中的FRET。我们研究了表达青色荧光蛋白（CFP）或CFP与黄色荧光蛋白（YFP）融合蛋白的固定哺乳动物细胞，以及表达天蓝色荧光蛋白（Cerulean）或天蓝色荧光蛋白与维纳斯荧光蛋白（Venus）融合蛋白的活真菌细胞。我们发现哺乳动物细胞和真菌细胞中的荧光行为基本相同。重要的是，高精度的FLIM数据能够可重复地解析多个荧光衰减，从而揭示有关发生FRET的蛋白质群体比例的新信息，并减少供体-受体距离测量中的误差。我们对这个简单对照系统的研究结果表明，对更复杂的蛋白质-蛋白质相互作用进行体内FLIM-FRET研究将从这种定量测量中受益匪浅。
