Fluorescence correlation spectroscopy (FCS) is an emerging technique employed in biophysical studies that exploits the temporal autocorrelation of fluorescence intensity fluctuations measured in a tiny volume (in the order of fL). The autocorrelation curve derived from the fluctuations can then be fitted with diffusion models to obtain parameters such as diffusion time and number of particles in the diffusion volume/area. Application of FCS to membranes allows studying membrane component dynamics, which includes mobility and interactions between the components. However, FCS encounters several difficulties like accurate positioning and stability of the setup when applied to membranes. Here, we describe the theoretical basis of point FCS as well as the scanning FCS (SFCS) approach, which is a practical way to address the challenges of FCS with membranes. We also list materials necessary for FCS experiments on two model membrane systems: (1) supported lipid bilayers and (2) giant unilamellar vesicles. Finally, we present simple protocols for the preparation of these model membrane systems, calibration of the microscope setup for FCS, and acquisition and analysis of point FCS and SFCS data so that diffusion coefficients and concentrations of fluorescent probes within lipid membranes can be calculated.
