Z-STED Imaging and Spectroscopy to Investigate Nanoscale Membrane Structure and Dynamics

Aurélien Barbotin,Iztok Urbančič,Silvia Galiani,Christian Eggeling,Martin Booth,Erdinc Sezgin

doi:10.1016/j.bpj.2020.04.006

Abstract

Super-resolution stimulated emission depletion (STED) microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy) or axially (z). Two-dimensional STED has been extensively used to elucidate the nanoscale membrane structure and dynamics via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of ∼100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles, or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively.

Highlights

Cellular membranes are hubs for cellular signaling [1]
The phase mask was created by an spatial light modulators (SLMs), and as such, it could be swapped to any other phase mask to change the stimulated emission depletion (STED) confinement mode without changing the optical layout
Certain STED microscopes use a combination of the 2D and z-STED depletion patterns to increase the resolution along all dimensions

Summary

Introduction

Cellular membranes are hubs for cellular signaling [1]. They are heterogeneous structures accommodating clusters, domains, and nanoassemblies [2,3], and this heterogeneity is crucial for cellular signaling [4]. There has been extensive effort to resolve the mystery of nanoscale structure and dynamics of cellular membranes. Super-resolution imaging technologies have been extremely useful for shedding light on the nanoscale architecture and the supramolecular organization of the cells [5,6,7,8,9,10,11]. Super-resolution can be used to increase the lateral and the axial resolution of microscopes.

Methods

Results

Conclusion