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Supported lipid bilayers have been studied intensively over the past twodecades. In this work, we study the diffusion of single gold nanoparticles(GNPs) with diameter of 20 nm attached to GM1 ganglioside or DOPE lipids atdifferent concentrations in supported DOPC bilayers. The indefinitephotostability of GNPs combined with the high sensitivity of interferometricscattering microscopy (iSCAT) allows us to achieve 1.9 nm spatial precision at1 ms temporal resolution, while maintaining long recording times. Ourtrajectories visualize strong transient confinements within domains as small as20 nm, and the statistical analysis of the data reveals multiple mobilities anddeviations from normal diffusion. We present a detailed analysis of ourfindings and provide interpretations regarding the effect of the supportingsubstrate and GM1 clustering. We also comment on the use of high-speed iSCATfor investigating diffusion of lipids, proteins or viruses in lipid membraneswith unprecedented spatial and temporal resolution.

Tracking single particles on supported lipid membranes: multi-mobility diffusion and nanoscopic confinement