Revisiting staining of biological samples for electron microscopy: perspectives for recent research

Abstract

This review revisits staining protocols for electron microscopy focussing on the visualization of active sites, i.e. enzymes, metabolites or proteins, in cells and tissues, which were never established as standard protocols in electron microscopy.

How Membrane Contact Sites Shape the Phagophore

“…While fluorescence microscopy is well-suited for characterizing the interactions and dynamics of specific proteins even in live cells, it is limited in resolution and leaves all unlabeled structures in the dark. In contrast, electron microscopy methods reveal the entire local cellular environment at a higher resolution, making use of the inherent contrast of the biological structures, sometimes enhanced with nonspecific stainings (Kuchenbrod et al, 2021). However, given the small mean free path of electrons, imaging needs to be done in vacuum, necessitating sample fixation by chemical methods or vitrification.…”

“…The growing membrane is further modified by conjugation of the ubiquitin-like protein Atg8 (or its homologs LC3/GABARAP in higher eukaryotes) to the lipid phosphatidylethanolamine (PE). Among other functions, Atg8 interacts with selective autophagy receptors to mediate the enwrapping of specific cargo by the growing phagophore ( Johansen and Lamark, 2020 ). Phagophore closure is a membrane scission process and was suggested to depend on the ESCRT (endosomal sorting complex required for transport) machinery ( Takahashi et al, 2018 ; Zhou et al, 2019).…”