Eggeling Christian, Kask Peet, Winkler Dirk, Jäger Stefan
Max-Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, 37077 Goettingen, Germany.
Biophys J. 2005 Jul;89(1):605-18. doi: 10.1529/biophysj.104.052753. Epub 2005 Apr 22.
In this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Förster resonance energy transfer (FRET) as a very powerful combination for monitoring biochemical reactions on the basis of single molecule events. 2CG-FCS, which is a new variation emerging from the family of fluorescence correlation spectroscopy, globally analyzes the simultaneously recorded auto- and cross-correlation data from two photon detectors monitoring the fluorescence emission of different colors. Overcoming the limitations inherent in mere auto- and cross-correlation analysis, 2CG-FCS is sensitive in resolving and quantifying fluorescent species that differ in their diffusion characteristics and/or their molecular brightness either in one or both detection channels. It is able to account for effects that have often been considered as sources of severe artifacts in two-color and FRET measurements, the most prominent artifacts comprising photobleaching, cross talk, or concentration variations in sample preparation. Because of its very high statistical accuracy, the combination of FRET and 2CG-FCS is suited for high-throughput applications such as drug screening. Employing beam scanning during data acquisition even further enhances this capability and allows measurement times of <2 s. The improved performance in monitoring a FRET sample was verified by following the protease cleavage reaction of a FRET-active peptide. The FRET-inactive subpopulation of uncleaved substrate could be correctly assigned, revealing a substantial portion of inactive or missing acceptor label. The results were compared to those obtained by two-dimensional fluorescence intensity distribution analysis.
在本研究中，我们介绍了双色全荧光相关光谱法（2CG-FCS）与Förster共振能量转移（FRET）相结合的方法，这是一种基于单分子事件监测生化反应的强大组合。2CG-FCS是荧光相关光谱家族中出现的一种新变体，它对来自两个光子探测器同时记录的自相关和互相关数据进行全局分析，这两个探测器监测不同颜色的荧光发射。2CG-FCS克服了单纯自相关和互相关分析固有的局限性，能够灵敏地分辨和量化在一个或两个检测通道中扩散特性和/或分子亮度不同的荧光物质。它能够解释在双色和FRET测量中常常被视为严重伪像来源的效应，最突出的伪像包括光漂白、串扰或样品制备中的浓度变化。由于其极高的统计精度，FRET与2CG-FCS的组合适用于药物筛选等高通量应用。在数据采集过程中采用光束扫描甚至进一步增强了这种能力，并允许测量时间小于2秒。通过跟踪FRET活性肽的蛋白酶切割反应，验证了在监测FRET样品方面的改进性能。未切割底物的FRET非活性亚群能够被正确识别，揭示出相当一部分无活性或缺失的受体标签。将结果与通过二维荧光强度分布分析获得的结果进行了比较。
