AUTHOR=Puchkov Dmytro , Müller Paul Markus , Lehmann Martin , Matthaeus Claudia 

TITLE=Analyzing the cellular plasma membrane by fast and efficient correlative STED and platinum replica EM

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=Volume 11 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2023.1305680

DOI=10.3389/fcell.2023.1305680

ISSN=2296-634X

ABSTRACT=The plasma membrane of mammalian cells links transmembrane receptors, various structural components and membrane binding proteins to subcellular processes allowing inter- and intracellular communication. Thereby, membrane binding proteins together with structural components such as actin filaments modulate the cell membrane in their flexibility, stiffness, and curvature. Investigating membrane components and curvature in cells remains challenging due to the diffraction limit in light microscopy. Preparation of 5-15 nm thin plasma membrane sheets and subsequent inspection by metal replica transmission electron microscopy (TEM) reveals detailed information about the cellular membrane topology including structure and curvature. However, electron microscopy cannot identify proteins associated to specific plasma membrane domains. Here, we describe a novel adaptation of correlative super-resolution light microscopy and platinum replica TEM (CLEM-PREM) allowing the analysis of plasma membrane sheets in respect to their structural details, curvature and associated protein composition. We suggest a number of shortcuts and troubleshooting solutions to contemporary PREM protocols. Thus, implementation of super-resolution stimulated emission depletion (STED) microscopy offers significant reduction in sample preparation time as well as reduced technical challenges for imaging and analysis. Additionally, highly technical challenges associated with replica preparation and transfer on a TEM grid can be overcome by the use of scanning electron microscopy (SEM) imaging. The combination of STED microscopy and platinum replica SEM or TEM provides highest spatial resolution of plasma membrane proteins and their underlying membrane and is therefore a suitable method to study cellular events like endocytosis, membrane trafficking or membrane tension adaptations.