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Optical nanoantennas can efficiently confine light into nanoscopic hotspots,enabling single-molecule detection sensitivity at biological relevantconditions. This innovative approach to breach the diffraction limit offers aversatile platform to investigate the dynamics of individual biomolecules inliving cell membranes and their partitioning into cholesterol-dependent lipidnanodomains. Here, we present optical nanoantenna arrays with accessiblesurface hotspots to study the characteristic diffusion dynamics ofphosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane ofliving cells at the nanoscale. Fluorescence burst analysis and fluorescencecorrelation spectroscopy performed on nanoantennas of different gap sizes showthat, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomainsof 10 nm diameter with short characteristic times around 100 {\mu}s. Theremoval of cholesterol led to the free diffusion of SM, consistent with thedispersion of nanodomains. Our results are consistent with the existence ofhighly transient and fluctuating nanoscale assemblies enriched by cholesteroland sphingolipids in living cell membranes, also known as lipid rafts.Quantitative data on sphingolipids partitioning into lipid rafts is crucial tounderstand the spatiotemporal heterogeneous organization of transient molecularcomplexes on the membrane of living cells at the nanoscale. The proposedtechnique is fully biocompatible and thus provides various opportunities forbiophysics and live cell research to reveal details that remain hidden inconfocal diffraction-limited measurements.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells