Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy
Transcripción
External stimulators activate cells and induce three-dimensional circular membrane protrusion, or membrane ruffle formation, essential for various cellular activities.
To visualize membrane ruffle formation, begin with a multi-well plate containing a coverslip at the base. The coverslip's top has cultured macrophages.
Treat these macrophages with stimulator molecules that activate the cells, facilitating cytoskeleton reorganization and membrane protrusion formation. Overlay the cells with a fixative solution, cross-linking the proteins, and preserving the cellular structure.
Treat the fixed macrophages with increasing alcohol concentrations for complete dehydration. Desiccate these macrophages using a critical point dryer, eliminating any traces of water for better imaging.
Mount the coverslip onto a scanning electron microscopy, or SEM, specimen holder using conductive tape. Sputter coat the macrophage-containing coverslip with a thin metal layer, ensuring good electronic conductivity during imaging.
Place the coverslip in the SEM chamber. Focus the electron beam on the coverslip. The electron beam hits the metal-coated macrophage's membrane, causing low-energy secondary electron generation from the membrane surface.
The three-dimensional protrusions scatter electrons differently than the rest of the membrane, highlighting these features on the cell surface. The detector collects the scattered electrons from various cell surface regions, generating a contrast image.
In the SEM image, macrophages appear darker with bright circular protrusions on the surface, confirming ruffle formation.
