Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM
Begemann, I., Keller, U., Nüsse, H., Klingauf, J., & Galic, M.
scanning electron microscopy, SE/BSE, CLEM, immunogold labelling, curvature-sensitive protein, BAR domain
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.