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Abstract：
Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.
摘要：
Förster共振能量转移（FRET）光谱法是一种用于从FRET效率的分布确定蛋白质寡聚体的四级结构的方法，该FRET效率的分布是从表达目标蛋白质的细胞的荧光图像的像素中提取的。FRET光谱法方案目前依赖于从基于强度的实验获得光谱分辨的荧光数据。另一种成像方法，荧光寿命成像显微镜（FLIM），是从由FRET引起的供体的荧光寿命的减少来计算图像中的每个像素的FRET效率的广泛使用的替代方案。在具有不同比例的供体和受体的寡聚体的FLIM研究中，供体寿命可以通过将时间分辨的荧光衰减数据与预定数量的指数衰减曲线进行拟合来获得。然而,这需要了解样品中荧光蛋白的数量和相对排列，这正是FRET光谱法的目标，因此产生了一个难题，该难题阻止了FLIM仪器的用户执行FRET光谱法。这里,我们描述了通过使用基于积分的方法从荧光衰减曲线计算FRET效率，在时间分辨荧光显微镜上实现FRET光谱法的尝试。这种方法,我们称之为时间集成FRET(或tiFRET)，在活细胞的细胞质中表达的寡聚荧光蛋白构建体进行测试。目前的结果表明，tiFRET是实现FRET光谱法的一种有前途的方法，并建议对仪器进行潜在的调整，以提高此类研究的准确性和分辨率。
