Rapid analysis of Forster resonance energy transfer by two-color global fluorescence correlation spectroscopy:: Trypsin proteinase reaction
Rapid analysis of Forster resonance energy transfer by two-color global fluorescence correlation spectroscopy:: Trypsin proteinase reaction
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DOI：
10.1529/biophysj.104.052753
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发表时间：
2005-07-01
影响因子：
3.4
通讯作者：
Jäger, S
中科院分区：
文献类型：
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作者：
Eggeling, C;Kask, P;Jäger, S
在这项研究中，我们介绍了双色全局荧光相关光谱 (2CG-FCS) 和福斯特共振能量转移 (FRET) 的组合，作为基于单分子事件监测生化反应的非常强大的组合。 2CG-FCS 是荧光相关光谱家族中出现的一种新变体，可对来自监测不同颜色荧光发射的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。 2CG-FCS 克服了单纯自相关和互相关分析固有的局限性，能够灵敏地解析和定量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。它能够解释双色和 FRET 测量中通常被认为是严重伪影来源的效应，最突出的伪影包括光漂白、串扰或样品制备中的浓度变化。由于其统计准确性非常高，FRET 和 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进一步增强了这种能力，并允许测量时间
In this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Forster 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