A detailed protocol for expansion microscopy of <i>Paracentrotus lividus</i> embryos and larvae: Incorporating decalcification for improved imaging

Expansion microscopy (ExM) enables superresolution imaging by embedding biological specimens in a swellable hydrogel, followed by optical clearing and physical expansion. Here we present a detailed protocol for applying ExM to embryonic stages of Paracentrotus lividus, a widely used model in developmental biology. Embryos at cleavage and gastrula stages, as well as pluteus larvae, were successfully expanded fourfold after proteinase digestion. Preexpansion Airyscan imaging provided only limited subcellular information due to autofluorescence, whereas post-expansion samples displayed markedly reduced background and resolved fine structures. To address distortions caused by the calcified skeleton in pluteus larvae, we incorporated a decalcification step with EDTA, which preserved morphology and enabled isotropic expansion. Distinct NHS ester dyes further allowed differential labelling of the fertilisation envelope and blastomeres, illustrating the versatility of this approach. Together, these adaptations establish a reproducible workflow for ExM in marine invertebrates, offering a valuable methodological resource and a foundation for future applications in developmental and environmental research.