Atomic force microscopy of phase separation on ruptured, giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) are synthetic model systems widely used in biophysical studies of lipid membranes. Phase separation behaviors of lipid species in these two model systems differ due to the lipid-substrate interactions that are present only for SLBs. Therefore, GUVs are believed to resemble natural cell membranes more closely, and a very large body of literature focuses on applying nano-characterization techniques to quantify phase separation on GUVs. However, one important technique, atomic force microscopy (AFM), has not yet been used successfully to study phase separation on GUVs. In the present study, we report that in binary systems, certain phase domains on GUVs retain their original shapes and patterns after the GUVs rupture on glass surfaces. This enabled AFM experiments on phase domains from binary GUVs containing 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). These DLPC/DSPC and DLPC/DPPC GUVs both presented two different gel phases, one of which (bright phase) included a relatively high concentration of DiI-C20 but excluded Bodipy-HPC, and the other of which (dark phase) excluded both probes. The bright phases are of interest because they seem to stabilize dark phases against coalescence. Results suggested that the gel phases labeled by DiI-C20 in the DLPC/DSPC membrane, which surround the dark gel phase, is an extra layer of membrane, indicating a highly curved structure that might stabilize the interior dark domains. This phenomenon was not found in the DLPC/DPPC membrane. These results show the utility of AFM on collapsed GUVs, and suggest a possible mechanism for stabilization of lipid domains.