Resolving Nanoscale Curvature on Lipid Bilayers with Polarized Localization Microscopy

Abstract

The lateral sorting of membrane lipids and proteins in conjunction with membrane curvature, are postulated to provide a physical basis to initiate and regulate many complex cellular processes such as endocytosis/exocytosis. However, many hypotheses concerning these processes are unanswered because of the diffraction-limited resolution of most optical techniques (∼200 nm). In addition, present imaging techniques are incapable of observing dynamic nanoscale membrane bending and the reorganization of membrane bound molecules around the curvature site. To overcome these experimental limitations, we aim to detect and resolve biological processes involving nanoscale cell membrane curvature by Polarized Localization Microscopy (PLM). Selective excitation of fluorophores embedded in the lipid membrane by p- or s-polarized light reveal distinct vertical versus lateral membrane regions with 18 nm localization uncertainty. To exploit PLM experimental capabilities and improved resolution over current imaging techniques, PLM was performed on systems with induced curvature. For instance, small unilaminar lipid vesicles (50 -/+ 20 nm diameter) provide regions of curvature bound to a supported lipid bilayer (SLBs) with a lipid composition of POPC and 0.3% DiI. Additional samples include SLBs on polymerized large unilaminar vesicles (100 nm -/+ 20 diameter) that induces membrane deformation. The novel technique of PLM detects the presence of the vesicles and resolves their sizes and lateral positions in a super-resolution image. Further, we aim to utilize PLM to investigate the interplay between membrane orientations and nanoscale membrane lipid phase dynamics. Moreover, we anticipate to demonstrate the effect of various membrane orientations on the redistribution of trans-membrane molecules and proteins sorting. These studies will provide fundamental insights of curvature sensitive biological mechanisms that have been previously intractable, including neuronal communication, immunological signaling, and viral infections.