To Hop or Not to Hop: Exceptions in the FCS Diffusion Law

Diffusion obstacles in membranes have not been directly visualized because of fast membrane dynamics and the occurrence of subresolution molecular complexes. To understand the obstacle characteristics, mobility-based methods are often used as an indirect way of assessing the membrane structure. Molecular movement in biological plasma membranes is often characterized by anomalous diffusion, but the exact underlying mechanisms are still elusive. Imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is a well-established mobility-based method that provides spatially resolved diffusion coefficient maps and is combined with FCS diffusion law analysis to examine subresolution membrane organization. In recent years, although FCS diffusion law analysis has been instrumental in providing new insights into the membrane structure below the optical diffraction limit, there are certain exceptions and anomalies that require further clarification. To this end, we correlate the membrane structural features imaged by atomic force microscopy (AFM) with the dynamics measured using ITIR-FCS. We perform ITIR-FCS measurements on supported lipid bilayers (SLBs) of various lipid compositions to characterize the anomalous diffusion of lipid molecules in distinct obstacle configurations, along with the high-resolution imaging of the membrane structures with AFM. Furthermore, we validate our experimental results by performing simulations on image grids with experimentally determined obstacle configurations. This study demonstrates that FCS diffusion law analysis is a powerful tool to determine membrane heterogeneities implied from dynamics measurements. Our results corroborate the commonly accepted interpretations of imaging FCS diffusion law analysis, and we show that exceptions happen when domains reach the percolation threshold in a biphasic membrane and a network of domains behaves rather like a meshwork, resulting in hop diffusion.