Understanding the effect of a solid support on the dynamical properties of a supported lipid bilayer (SLB) is a prerequisite for the applications of SLB as a model biomembrane. Here, we applied two-dimensional fluorescence lifetime correlation spectroscopy to examine the effect of solution pH on the diffusion of lipids in the proximal/distal leaflets of a zwitterionic SLB. Leaflet-specific diffusion analyses at various pH revealed that the diffusion of lipids in the proximal leaflet facing a glass surface becomes slower by decreasing pH with the transition pH of ∼7.4. We attributed it to the reduction of the surface charge density of a glass support. Furthermore, the data clearly showed that the lipid diffusion in the distal leaflet facing a bulk solution is insensitive to the change in the diffusion property of the proximal leaflet. This reflects a weak interleaflet coupling between the proximal and distal leaflets of the SLB.

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