Single-lipid dynamics in phase-separated supported lipid bilayers

Phase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Single-molecule diffusion measures a key characteristic of membrane behavior and relates to the effective membrane viscosity. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. We demonstrate agreement between FCS and SPT analyses with both the single-step length distribution and the mean squared displacement of lipids with significant immobile diffusers. Traditionally, SPT is sensitive to diffuser aggregation, whereas FCS largely excludes aggregates from the reported data. Protocols are reported for identifying and culling the aggregates prior to calculating diffusion rates via SPT. With aggregate culling, all diffusion measurement methods provide consistent results. With varying membrane composition and temperature, we demonstrate the importance of the tie-line length that separates the coexisting lipid phases in predicting the differences in diffusion between the Ld and Lo phases. HIGHLIGHTSO_LILipid diffusion varies with the lipid phases, temperature, and aggregation C_LIO_LIAggregate culling yields consistent measurements from single-particle tracking and fluorescence correlation spectroscopy C_LIO_LIMembrane with higher cholesterol content or at low temperature have more aggregates C_LIO_LIA more variation in the diffusion rates occurred between the coexisting lipid phases at low temperatures and low cholesterol content C_LI