Fluorescence Correlation Spectroscopy for the Study of Membrane Dynamics and Organization in Giant U

Fluorescence correlation spectroscopy (FCS) is a powerful technique to study the lateral organization of membranes. It measures fluorescence intensity fluctuations in the single molecule regime and allows the determination of diffusion coefficients. When applied to lipid membranes, their fluidity and lipid phase can be estimated from the diffusion rates of fluorescent particles partitioned to the membrane. Here, we describe the theoretical basis of FCS and discuss the z-scan approach for measurements on lipid membranes. We also list the materials necessary for a FCS experiment on giant unilamellar vesicles (GUVs). Finally, we present simple protocols for the preparation of GUVs and the acquisition and analysis of FCS data on the vesicles, so that diffusion coefficients of fluorescent probes within lipid membranes can be estimated.