Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes

Only a limited number of noninvasive techniques are available to directly measure the dynamic behavior of lipids in model and cell membranes. Here, we explored whether a commercial instrument could be used for fluorescence correlation spectroscopy (FCS) under pulsed stimulated emission depletion (STED). To overcome issues with photobleaching and poor distinction between confocal and STED signals, we implemented resonant line-scan STED with filtered FCS, which has the additional benefit of autocalibrating the dimensions of the point-spread function and obtaining spatially resolved molecular mobility at subdiffraction resolution. With supported lipid bilayers, we achieved a detection spot radius of 40 nm, although at the expense of decreased molecular brightness. We also used this approach to map the dynamics of Atto646N-labeled sphingomyelin and phosphatidylethanolamine in the plasma membrane. Despite the reliability of the method and the demonstration that photobleaching and the photophysical properties of the dye did not influence diffusion measurements, we found great heterogeneities even within one cell. For both lipids, regions of high local density correlated with slow molecular diffusion, indicating trapping of Atto646N-labeled lipids. Future studies with new dyes are needed to reveal the origin of the trapping.