Probing the plasma membrane organization in living cells by spot variation fluorescence correlation spectroscopy.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τ(d) and the squared waist of observation w(2). Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.