Chemical Imaging of Lipid Segregation: Determining Different Length Scales of Heterogeneity with Compressive-Sensing Sum Frequency Generation Microscopy and Brewster Angle Microscopy.

Membranes of various phospholipids may separate into different domains at micrometer length scales at the air-water interface. A significant challenge is to visualize the molecular organization and obtain chemical information on this surface. Langmuir-Blodgett monolayers of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) on fused silica were investigated with compressive sensing sum frequency generation (SFG) microscopy. Before and after mixing, SFG spectra for DSPC and DLPC revealed significant differences, indicating a structural change. Brewster angle microscopy images show phase separations that directly correlate the morphology of phospholipid mixtures with SFG images. Exploiting vibrational contrast in SFG images, exchange between the two phases was discovered, and quantitative thermodynamic analysis of lipid compositions in liquid-condensed and liquid-expanded (LE) phases was provided. Local SFG spectra reveal significant differences from one another, indicating the heterogeneity resulting from domain areas with distinct molecular orientation and conformation. Further heterogeneity across the domain boundary was presented on a finer scale, revealing an effect on DLPC due to the condensed phase DSPC, and the terminal methyl of DLPC perturbs the ordering of DSPC in the LE phase. This work demonstrates the heterogeneity of a two-dimensional binary lipid system mainly due to the aliphatic chain length and transition temperature difference.