Bacia Kirsten, Haustein Elke, Schwille Petra
Cold Spring Harb Protoc. 2014 Jul 1;2014(7):709-25. doi: 10.1101/pdb.top081802.
Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed.
荧光相关光谱法（FCS）用于研究极低浓度下生物分子的运动和相互作用，能产生具有良好空间和时间分辨率的结果。通过一系列技术发展，FCS已成为一种通用技术，可用于研究多种样品类型，并能与其他方法有效结合。与其他基于荧光的技术不同，FCS数据分析并非基于荧光发射的平均强度，而是检测热平衡时因与均值的自发偏差而引起的微小强度波动。这些波动可能源于分子移动导致的局部浓度变化，或源于低浓度下荧光标记生物分子的特征性分子间或分子内反应。在此，我们对FCS进行基本介绍，包括其技术发展和理论基础、FCS系统的实验设置、设置调整、数据采集以及FCS测量分析。最后，讨论了FCS在脂质双层膜和活细胞研究中的应用。
