Fig. 1.

Expansion microscopy (ExM) workflow. First, a biological specimen is chemically fixed (Fixation). Next, the specimen is treated with compounds that bind to key biomolecules or labels (Anchoring), so they can be tethered to the hydrogel polymer chains synthesized in the next step. A hydrogel made of closely spaced, densely cross-linked, highly charged monomers is then polymerized evenly throughout the cells or tissue, intercalating between and around the biomolecules or labels (Gelation). Then the embedded specimen goes through a mechanical homogenization step involving denaturation and/or digestion of structural molecules (Mechanical homogenization). The specimen–hydrogel composite is now ready for physical expansion by dialysis in low-salt buffer or water (Expansion). Biomolecules or labels of interest remain bound to the expanded polymer network, which has pulled them apart (as schematized in the dashed box). Images were adapted by permission with Macmillan Publishers Ltd: Nature Biotechnology [4], copyright 2016