Two-colour Imaging Fluorescence Correlation Spectroscopy (ImFCS) probes the influence of the juxta-membrane actin-cortex

Here we extend Imaging Fluorescence Correlation Spectroscopy (ImFCS), together with bright, photostable fluorophores, to quantify how the juxta-membranous actin cortex influences plasma membrane dynamics. This problem is technically challenging because the membrane is only a few nanometres thick, whereas the axial resolution of live-cell fluorescence imaging is on the order of 100 nm. We address this by engineering a membrane-anchored actin reporter that combines a weakly interacting actin-binding domain with bright, photostable fluorophores optimized for two-colour ImFCS. This design enables the generation of simultaneous, co-registered diffusion maps where the mobility of the reporter serves as a dynamic sensor for the local density of the cortical actin network. The actin-binding reporter displays diffusion coefficients spanning more than two orders of magnitude, in stark contrast to an otherwise similar reporter that cannot bind actin, which diffuses much faster and within a much narrower range. Thus, the mobility of the reporter becomes a sensitive spatial readout of actin-mediated constraints. By comparing, on a pixel-by-pixel basis, the diffusion of the actin-binding reporter with that of the non-binding mutant, we construct an actin-occupancy map that quantifies the fraction of the membrane functionally influenced by cortical actin. Using this approach, we find that the juxta-membrane actin cortex modulates transport over more than 80% of the cell surface. Our two-channel ImFCS strategy establishes diffusion-based comparison as a general methodology to map the functional footprint of the cortical cytoskeleton on live cell membranes.