Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM

Abstract

Scanning electron microscopy (SEM) takes advantage of distinct detectors to visualise secondary and back-scattering electrons. Here, we report an integrated approach that relies on these two detection methods to simultaneously acquire correlated information on plasma membrane topography and curvature-sensitive cytosolic protein localization in intact cell samples. We further provide detailed preparation and staining protocols, as well as a thorough example-based discussion for imaging optimisation. Collectively, the presented method enables rapid and precise analysis of cytosolic proteins adjacent to cellular membranes with a resolution of ~100 nm, without time-consuming preparations or errors induced by sequential visualisation present in fluorescence-based correlative approaches.

Keywords

• scanning electron microscopy
• SE/BSE
• CLEM
• immunogold labelling
• curvature-sensitive protein
• BAR domain