Accurate Single-Particle Tracking and Diffusion Measurement in Freestanding Lipid Bilayers and Model Membranes

Single-particle tracking (SPT) in lipid bilayers offers a powerful means to investigate the dynamic interaction of individual molecules in a variety of model membranes. However, accurate diffusion characterization in freestanding bilayers remains challenging due to membrane fragility, the potential for curvature, limited optical signal-to-background ratio, and the practical need for label concentrations that do not approximate infinite dilution. Here, we report a dual-mode fluorescence microscope configuration for bilayers formed on a microelectrode cavity array (MECAopto-inv chip) that combines widefield SPT with confocal fluorescence correlation spectroscopy (FCS). By using FCS to calibrate the SPT algorithm, we demonstrate improved measurement accuracy for diffusion constants. Importantly, we show that proper calibration resists measurement bias at high fluorescent particle concentrations. Supporting simulations confirm the robustness of this approach. When calibrated with FCS and corrected for blur, our measurements help resolve the variability in diffusion constants reported in prior literature. Furthermore, the strategy is broadly applicable to any planar bilayer system. The method provides a rigorous, quantitative benchmark for future studies involving nanopores, membrane protein assemblies, and ion-channel transport in lipid bilayers, especially when both optical and electrical information is desired.