Electrostatic force microscopy analysis of lipid miscibility in two-component monolayers

Electrostatic force microscopy (EFM) was used to assess lipid miscibility and phase behavior in two-component Langmuir-Blodgett (LB) monolayers composed of cationic dioctadecyldimethylammonium bromide (DOMA) and nonionic methyl stearate (SME) lipids. The surface potential measurements were calibrated by applying known bias voltages to the sample during several line scans, thus creating surface potential "scale bars" on the images from which it was determined that circular domains were 50 mV more positive than the surrounding phase. As the spatially averaged surface potential of DOMA was over 400 mV more positive than that of SME, this 50-mV surface potential difference is too low to correspond to lipid phase separation (immiscibility) in the two-component film. Rather, the surface potential contrast was attributed to an increased packing density and a more orthogonal orientation of lipids in the domains resulting in a greater contribution of dipoles to the measured (normal) surface potential. Monolayers prepared by sequentially spreading the two lipids resulted in irregular domains that were 50-450 mV more positive than the surrounding phase, representing varying degrees of lipid mixing, restricted by two-dimensional diffusion at the interface. Fluorescent images of monolayers stained with negatively charged dye supported the EFM miscibility prediction and assignment of surface potential. These results demonstrate a new approach using EFM to quantitatively measure surface potential in order to assess the lateral distribution of components in thin films as well as predict adsorption patterns to heterogeneous interfaces.