Ferrand Patrick, Wenger Jérôme, Rigneault Hervé
Mosaic Group, Institut Fresnel, CNRS, Aix-Marseille Université, Ecole Central Marseille, Marseille, France.
Methods Mol Biol. 2011;783:181-95. doi: 10.1007/978-1-61779-282-3_10.
Fluorescence correlation spectroscopy (FCS), implemented in microscopy, relies on performing an autocorrelation of the time fluctuating intensity arising from individual molecules diffusing through a confocal volume. It allows us to investigate a large variety of dynamic processes and to quantify photophysical, photochemical, interaction, diffusion, and transport properties of molecules. This tutorial chapter is intended to give an "hands on" view of FCS. After a brief introduction on the principle of FCS, the major theoretical assumptions are emphasized, and the main analytical expression are given. Then the key parameters that have to be considered when building a FCS system are discussed. The complete method of operation is described, including calibration, measurement, and data treatment. The major difficulties that are encountered when performing for the first time FCS are illustrated by examples of measurements, and possible solutions are proposed.
荧光相关光谱法（FCS）应用于显微镜中，它通过对单个分子扩散通过共聚焦体积时产生的随时间波动的强度进行自相关分析来实现。它使我们能够研究各种动态过程，并量化分子的光物理、光化学、相互作用、扩散和传输特性。本章教程旨在提供对FCS的“实际操作”视角。在简要介绍FCS原理之后，强调了主要的理论假设，并给出了主要的分析表达式。然后讨论了构建FCS系统时必须考虑的关键参数。描述了完整的操作方法，包括校准、测量和数据处理。首次进行FCS测量时遇到的主要困难通过测量示例进行说明，并提出了可能的解决方案。
