3D Probed Lipid Dynamics in Small Unilamellar Vesicles

Single‐molecule fluorescence correlation spectroscopy overcomes the resolution barrier of optical microscopy (10≈–20 nm) and is utilized to look into lipid dynamics in small unilamellar vesicles (SUVs; diameter < 100 nm). The fluorescence trajectories of lipid‐like tracer 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine (DiD) in the membrane bilayers are acquired at a single‐molecule level. The autocorrelation analysis yields the kinetic information on lipid organization, oxygen transport, and lateral diffusion in SUVs' membrane. First, the isomerization feasibility may be restricted by the addition of cholesterols, which form structure conjugation with DiD chromophore. Second, the oxygen transport is prevented from the ultrasmall cluster and cholesterol‐rich regions, whereas it can pass through the membrane region with liquid‐disordered phase (L_d) and defects. Third, by analyzing 2D spectra correlating the lipid diffusion coefficient and triplet‐state lifetime, the heterogeneity in lipid bilayer can be precisely visualized such as lipid domain with different phases, the defects of lipid packing, and DiD‐induced “bouquet” ultrasmall clusters.