Measuring Hindered Diffusion Dynamics in Live Cell Plasma Membranes with Confocal and Super-Resolution Imaging

Abstract

The cellular plasma membrane is a highly heterogeneous structure organised on nano-scales and displays a crucial interaction platform for proteins, lipids and soluble ligands. Investigating the molecular membrane organisation by measuring diffusion dynamics offers a better understanding of its biological function. Fluorescence correlation spectroscopy (FCS) is one of the prominent tools to elucidate these dynamics in living cells but can only report on the dynamics at one given spatial position at a time. Using scanning fluorescence correlation spectroscopy (sFCS), we obtain a multitude of FCS measurements at different spatial locations. Here, we present a statistical analysis pipeline for sFCS data which allows for the accurate determination of the diffusion dynamics and the differentiation of free (Brownian) from hindered (non-Brownian) diffusion modes. We show free diffusion for phospholipids in model membranes and cells but reveal hindered diffusion of sphingolipids and GPI-anchored proteins in cells. Notably, these measurements can be performed using standard fluorescent dyes or proteins on a conventional confocal laser scanning microscope. To further investigate the dynamics on single cell level, we combine sFCS with stimulated emission depletion (STED) microscopy and by alternating conventional and super-resolved excitation we introduce line interleaved excitation scanning STED-FCS (LIESS-FCS). With LIESS-FCS the diffusion modes can be directly determined at multiple spots within the cellular plasma membrane providing detailed insights into organisation and function. Overall we are presenting a novel toolkit to investigate nano-scale molecular diffusion dynamics for shedding a new light on membrane organisation and heterogeneity.