Polarized Localization Microscopy Detects Membrane Curvature and Reveals the Interplay between Membrane Orientation and Protein Dynamics

Abstract

Nanoscale membrane curvature is a necessary component of countless cellular processes. However, the understanding of these processes is limited by the experimental techniques and its inability to detect and resolve nanoscale membrane curvature. Here we present Polarized Localization Microscopy (PLM), a novel optical microscopy technique that detects and resolves membrane curvature with 15 nm resolution. PLM combines the advantages of polarized total internal reflection fluorescence microscopy (TIRF) and fluorescence localization microscopy to reveal single-fluorophore locations and orientations without sacrificing the localization precision of the point spread function. We demonstrate PLM capability of resolving membrane orientations by draping a supported lipid bilayer over polystyrene nanoparticles of varying sizes on a glass coverslip, thus creating a model membrane with coexisting flat and curved regions and membrane radii of curvature as small as 20 nm. Further, we introduce theoretical analysis that enables mapping out the membrane topology from the density of detected localizations in fluorescence excitation polarization. Using this approach, the interplay between membrane topology and cholera toxin subunit B (CTxB) dynamics was addressed. CTxB partitioned to curved membrane over the nanoparticle and further drove formation of lipid tubules. PLM detected the binding of an exocytosis protein complex to nanoscale membrane curvature. PLM was utilized to perform high throughput single particle tracking to reveal the correlated effects of membrane curvature, dynamics, and protein distribution. By providing super-resolution of membrane topology, PLM is able to reveal the critical components in various cellular processes such as endocytosis/exocytosis, viral infections, and neurotransmission.