Phase separation in supported phospholipid bilayers visualized by near-field scanning optical microscopy in aqueous solution

An experimental approach for near-field scanning optical microscopy (NSOM) of biological samples in an aqueous environment with a resolution of < 100 nm has been developed. This was accomplished by using high-throughput bent optical fiber probes prepared by a two-step chemical etching method followed by focused ion beam milling to fabricate a reproducible aperture. The utility of the method for high-resolution fluorescence imaging of biological samples in liquid was demonstrated using phase separated, supported phospholipid bilayers as models for natural membranes. Bilayers with a phase separated dipalmitoyl-phosphatidylcholine/dilauroyl-phosphatidylcholine (DPPC/DLPC) mixture in one or both leaflets were imaged by both atomic force microscopy and NSOM. The addition of dihexadecanoyl-sn-glycero-3-phosphoethanolamine-Texas Red (DHPE-TR) was used to visualize fluid and gel phases for the NSOM fluorescence measurements. Hybrid bilayers with 7:3 DLPC/DPPC in the bottom leaflet and DPPC on top showed phase separation to give striped DPPC domains similar to those observed for the corresponding monolayer. By contrast, the DLPC/DPPC mixture in the upper leaflet gave primarily large condensed DPPC domains. A bilayer with 7:3 DLPC/DPPC in both leaflets showed clear evidence for superposition of DPPC domains in the two layers. The resolution and image quality obtained for bilayers to aqueous solution clearly demonstrate the potential of NSOM for applications to biological imaging under physiological conditions.