Heterogeneous molecular distribution in supported multicomponent lipid bilayers

Membrane domains contribute important structural and functional attributes to biological membranes. We describe the heterogeneous nanoscale distribution of lipid molecules within microscale membrane domains in multicomponent lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). The lipids were labeled with the fluorescent lipid analogues Bodipy-PC and DiI-C20:0 to identify the distribution of individual membrane components. We used a near-field scanning optical microscope (NSOM) at room temperature to identify the nanoscale structures in the membrane. Simultaneous multicolor NSOM imaging at the emission maxima of the fluorescent analogues revealed a patchy distribution of Bodipy-PC and DiI-C20:0 indicative of phase separations in the bilayer. In a cholesterol-free system (DPPC/DLPC = 1:1), NSOM images proved that the two phosphatidylcholine molecules can coexist in domains at the micrometer level but form nanoscopic patches within the domains; DPPC occurs at the edge of the domains, whereas DLPC is present throughout the domains. In the presence of cholesterol (DPPC/DLPC = 7:3, chol = 18.9%), the two lipid molecules were more miscible but incomplete phase separations also occurred. The average domain sizes were 140-200 nm, well below the resolution capabilities of diffraction-limited light microscopy techniques; the domains were unresolvable by confocal microscopy. Our high-resolution NSOM studies of membrane domain behavior provide a better understanding of complex membrane phase phenomena in multicomponent biological membranes.