Abstract
Fluorescence correlation spectroscopy (FCS) is a single-molecule fluorescence technique used to monitor molecular dynamics. It can be applied to living cells expressing fluorescently labeled proteins and lipids to determine the diffusion timescales and the total number of diffusing fluorescent molecules in the cell. A practical step-by-step approach to performing FCS with a commercial spectroscopy/microscopy system, the Zeiss Confocor 3, how to set up live-cell FCS experiments, acquire reliable data, and finally how to analyze the data acquired, are all described in this unit.