Skakun Victor V, Digris Anatoli V, Apanasovich Vladimir V
Department of Systems Analysis and Computer Simulation, Belarusian State University, Minsk, Belarus.
Methods Mol Biol. 2014;1076:719-41. doi: 10.1007/978-1-62703-649-8_33.
In fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis, the same experimental fluorescence intensity fluctuations are used, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding molecular diffusion coefficients and triplet-state parameters such as fraction and decay time. The amplitude distribution of these fluctuations is calculated by PCH analysis yielding the molecular brightness. Both FCS and PCH give information about the molecular concentration. Here we describe a global analysis protocol that simultaneously recovers relevant and common parameters in model functions of FCS and PCH from a single fluorescence fluctuation trace. Application of a global analysis approach allows increasing the information content available from a single measurement that results in more accurate values of molecular diffusion coefficients and triplet-state parameters and also in robust, time-independent estimates of molecular brightness and number of molecules.
在荧光相关光谱法（FCS）和光子计数直方图（PCH）分析中，使用的是相同的实验荧光强度波动，但每种分析方法关注的是信号的不同特性。在FCS中测量荧光波动相关性的时间依赖性衰减，从而得到分子扩散系数和三重态参数，如分数和衰减时间。通过PCH分析计算这些波动的幅度分布，从而得到分子亮度。FCS和PCH都能提供有关分子浓度的信息。在这里，我们描述了一种全局分析协议，该协议可从单个荧光波动轨迹中同时恢复FCS和PCH模型函数中的相关和共同参数。全局分析方法的应用能够增加单次测量可获得的信息量，从而得到更准确的分子扩散系数和三重态参数值，以及更可靠、与时间无关的分子亮度和分子数量估计值。
