Skakun Victor V, Engel Ruchira, Digris Anatoli V, Borst Jan Willem, Visser Antonie J W G
Department of Systems Analysis, Belarusian State University, Minsk, Belarus.
Front Biosci (Elite Ed). 2011 Jan 1;3(2):489-505. doi: 10.2741/e264.
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, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH 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. The global analysis approach is described and tested with experimental fluorescence fluctuation data of enhanced green-fluorescent protein (eGFP) and dimeric eGFP (two eGFP molecules connected by a six amino acid long linker) in aqueous buffer. Brightness values and diffusion constants are recovered with good precision elucidating novel excited-state and motional properties of both proteins.
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，例如可得出分子扩散系数。通过PCH计算这些波动的幅度分布，可得出分子亮度。FCS和PCH都能提供有关分子浓度的信息。在此，我们描述了一种全局分析方案，该方案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。我们用增强型绿色荧光蛋白（eGFP）和二聚体eGFP（由六个氨基酸长的接头连接的两个eGFP分子）在水性缓冲液中的实验荧光波动数据描述并测试了全局分析方法。亮度值和扩散常数能够以良好的精度恢复，阐明了这两种蛋白质新的激发态和运动特性。
