STED-FCS Nanoscopy of Membrane Dynamics

Abstract

Lipid-lipid and lipid-protein interactions such as the formation of lipid nanodomains (often denoted “rafts”) are considered to play a functional part in a whole range of membrane-associated processes. However, their direct and nonin- vasive observation in living cells is impeded by the resolution limit of >200 nm of a conventional far-field optical microscope. With the superior spatial resolution of STED nanoscopy, it is now possible to directly resolve nanoscale membrane organization. While direct imaging of membrane heterogeneities turns challenging due to their fast dynamics, the combination of STED nanoscopy with tools such as fluorescence correlation spectroscopy (FCS) allows the disclosure of complex nanoscopic dynamical processes. By performing FCS measurements in observation spots tuned to a diameter of down to 30 nm, new details of molecular membrane dynamics have been obtained: Unlike fluorescent phosphoglycerolipids, fluorescent sphingolipids are transiently (~10 ms) trapped on the nanoscale in often cholesterol- and cytoskeleton-assisted molecular complexes. These interactions are distinct for different lipids and may play an important role in cellular functionality. Compari- son of the trapping characteristics to the organization of the different fluorescent lipid analogs in model membranes reveals details of the role of lipid “rafts”. This chapter reviews how STED-FCS may shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity.