Phase Separation in Supported Phospholipid Bilayers Visualized by Near-Field Scanning Optical Microscopy in Aqueous Solution

Abstract

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 sepa...