Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy
Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy
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DOI：
10.1016/s0006-3495(98)77976-7
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发表时间：
1998-05-01
影响因子：
3.4
通讯作者：
Herman, B
中科院分区：
文献类型：
--
作者：
Gordon, GW;Berry, G;Herman, B
荧光共振能量转移(FRET)是一种用于量化连接到不同荧光团的两个分子之间的距离的技术。通过结合光学显微镜和FRET，有可能获得关于蛋白质、脂类、酶、DNA和RNA在体内的结合和相互作用的定量的时间和空间信息。随着各种突变绿色荧光蛋白(MtGFP)的最近发展，FRET显微镜提供了在完整的活细胞中测量细胞内分子物种相互作用的可能性，其中供体和受体荧光团实际上是分子本身的一部分。然而，稳态FRET显微镜测量可能会受到几个需要纠正的失真来源的影响，这些来源包括在施主激发波长处对受体的直接激发以及FRET对受体浓度的依赖。我们提出了一种分析荧光显微镜中用标准滤光片装置获得的FRET数据的简单方法。该方法针对串扰(任何利用受体发射滤光片检测施主荧光以及利用施主发射滤光片检测任何受体荧光)以及FRET对供体和受体的浓度的依赖性进行了校正。对蛋白质Bcl2和Beclin(最近发现的一种位于染色体17q21上的Bcl2相互作用蛋白)的相互作用的测量表明，这种方法在校正供体和受体浓度以及不同过滤单元之间的串扰方面是准确的。
Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected, These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.