A user's guide for characterizing plasma membrane subdomains in living cells by spot variation fluorescence correlation spectroscopy.

Due to the intrinsic molecular Brownian agitation within plasma membrane and the vast diversity of membrane components, it is expected that the plasma membrane organization is highly heterogeneous with the formation of local complex multicomponent assemblies of lipids and proteins on different time scales. Still, deciphering this lateral organization on living cells and on the appropriate length and temporal scales has been challenging but is crucial to advance our knowledge on the biological function of the plasma membrane. Among the methodological developments based on biophotonics, the spot variation FCS (svFCS), a fluorescent correlation spectroscopy (FCS)-based method, has allowed the significant progress in the characterization of cell membrane lateral organization at the suboptical level, including to providing compelling evidence for the in vivo existence of lipid-dependent nanodomains. The aim of this chapter is to serve as a guide for setting and applying the svFCS methodology to study the plasma membrane of both adherent and nonadherent cell types.