The imaging FCS diffusion law in the presence of multiple diffusive modes

•FCS diffusion law determines the diffusive modes of proteins.•The method provides information about membrane organization below the diffraction limit.•Multiple diffusive modes of one particle can co-exist and can be distinguished by relative changes in the FCS diffusion law.•Particles with different diffusive modes can be distinguished even within a single FCS measurement. The cellular plasma membrane is the barrier over which cells exchange materials and communicate with their surroundings, and thus plays the central role in cellular sensing and metabolism. Therefore, the investigation of plasma membrane organization and dynamics is required for understanding of cellular functions. The plasma membrane is a heterogeneous matrix. The presence of structures such as lipid and protein domains and the cytoskeleton meshwork poses a hindrance to the free diffusion of membrane associated biomolecules. However, these domains and the cytoskeleton meshwork barriers are below the optical diffraction limit with potentially short lifetimes and are not easily detected even in super-resolution microscopy. Therefore, dynamic measurements are often used to indirectly prove the existence of domains and barriers by analyzing the mode of diffusion of probe molecules. One of these tools is the Fluorescence Correlation Spectroscopy (FCS) diffusion law. The FCS diffusion law is a plot of diffusion time (τd) versus observation area. For at least three different diffusive modes – free, domain confined, and meshwork hindered hop diffusion – the expected plots have been characterized, typically by its y-intercept (τ0) when fit with a linear model, and have been verified in many cases. However, a description of τ0 has only been given for pure diffusive modes. But in many experimental cases it is not evident that a protein will undergo only one kind of diffusion, and thus the interpretation of the τ0 value is problematic. Here, we therefore address the question about the absolute value of τ0 in the case of complex diffusive modes, i.e. when either one molecule is domain confined and cytoskeleton hindered or when two molecules exhibit different diffusive behavior at the same position in a sample. In addition, we investigate how τ0 changes when the diffusive mode of a probe alters upon disruption of domains or the cytoskeleton by drug treatments. By a combination of experimental studies and simulations, we show that τ0 is not influenced equally by the different diffusive modes as typica