| Popis: |
The plasma membrane of mammalian cells links transmembrane receptors, various structural components, and membrane-binding proteins to subcellular processes, allowing inter- and intracellular communication. Therefore, 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) reveal detailed information about the cellular membrane topology, including the structure and curvature. However, electron microscopy cannot identify proteins associated with 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 with 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 and 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 scanning electron microscopy (SEM) imaging. The combination of STED microscopy and platinum replica SEM or TEM provides the 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. |
