Fluorescence Correlation Spectroscopy

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.