Expansion microscopy (ExM) physically magnifies specimens, allowing to obtain super-resolution images using a conventional diffraction-limited microscope such as confocal microscopy. By optimizing several steps of this method, we demonstrated that the cell ultrastructure can be preserved after expansion and thus reveals details that were previously only accessible by transmission electron microscopy. As a result, we called this method ultrastructure expansion microscopy (U-ExM). Here we describe the step-by-step U-ExM protocol, as well as pitfalls and how to avoid them. We explain which steps may be modified in order to optimize the expansion and preservation of the structure of interest. Finally, we are demonstrating that U-ExM can be successfully applied to isolated macromolecular structures, unicellular organisms and human cells in culture.
Keywords: Centrioles; Isotropicity; Macromolecular assemblies; Ultrastructure expansion microscopy.
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