Single-lipid tracking reveals heterogeneities in the nanoscale dynamics of colloid-supported lipid bilayers

In this work, we utilize single-particle tracking photoactivated localization microscopy (sptPALM) to explore lipid dynamics in colloid-supported lipid bilayers (CSLBs) with liquid-like (DOPC), gel-like (DPPC), and phase-separated (DOPC:DPPC:cholesterol) membranes. Using total internal reflection fluorescence illumination, we tracked photoactivatable fluorescent dyes conjugated to lipids within these membranes. Analysis of tracked lipids revealed that bilayers across all compositions have heterogeneous dynamics, with lipid mobility varying over three orders of magnitude. We leveraged the temperature-dependent phase behavior of DPPC to transform gel-like membranes at room temperature into liquid-like membranes above 41 °C, which resulted in increased diffusivity and a surprising decrease in heterogeneity. Finally, we perform single lipid tracking in fluid-rich phases within gel-phase regions to demonstrate their dynamics with reduced lipid mobility because of soft confinement within phase-separated microdomains. Our findings have implications for colloidal assembly strategies that exploit ligand mobility to create controlled and reproducible colloidal superstructures.