Van Munster E B, Kremers G J, Adjobo-Hermans M J W, Gadella T W J
Centre for Advanced Microscopy, Section Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, the Netherlands.
J Microsc. 2005 Jun;218(Pt 3):253-62. doi: 10.1111/j.1365-2818.2005.01483.x.
Fluorescence resonance energy transfer (FRET) is an extremely effective tool to detect molecular interaction at suboptical resolutions. One of the techniques for measuring FRET is acceptor photobleaching: the increase in donor fluorescence after complete acceptor photobleaching is a measure of the FRET efficiency. However, in wide-field microscopy, complete acceptor photobleaching is difficult due to the low excitation intensities. In addition, the method is sensitive to inadvertent donor bleaching, autofluorescence and bleed-through of excitation light. In the method introduced in this paper, donor and acceptor intensities are monitored continuously during acceptor photobleaching. Subsequently, curve fitting is used to determine the FRET efficiency. The method was demonstrated on cameleon (YC2.1), a FRET-based Ca(2+) indicator, and on a CFP-YFP fusion protein expressed in HeLa cells. FRET efficiency of cameleon in the presence of 1 mm Ca(2+) was 31 +/- 3%. In the absence of Ca(2+) a FRET efficiency of 15 +/- 2% was found. A FRET efficiency of 28% was found for the CFP-YFP fusion protein in HeLa cells. Advantages of the method are that it does not require complete acceptor photobleaching, it includes correction for spectral cross-talk, donor photobleaching and autofluorescence, and is relatively simple to use on a normal wide-field microscope.
荧光共振能量转移（FRET）是一种在亚光学分辨率下检测分子相互作用的极其有效的工具。测量FRET的技术之一是受体光漂白：完全受体光漂白后供体荧光的增加是FRET效率的一种度量。然而，在宽场显微镜中，由于激发强度低，完全受体光漂白很困难。此外，该方法对无意的供体漂白、自发荧光和激发光的串扰很敏感。在本文介绍的方法中，在受体光漂白过程中连续监测供体和受体强度。随后，使用曲线拟合来确定FRET效率。该方法在基于FRET的Ca(2+)指示剂cameleon（YC2.1）以及在HeLa细胞中表达的CFP-YFP融合蛋白上得到了验证。在存在1 mM Ca(2+)的情况下，cameleon的FRET效率为31±3%。在不存在Ca(2+)的情况下，发现FRET效率为15±2%。在HeLa细胞中，CFP-YFP融合蛋白的FRET效率为28%。该方法的优点是不需要完全受体光漂白，它包括对光谱串扰、供体漂白和自发荧光的校正，并且在普通宽场显微镜上使用相对简单。
