Expansion of Toxoplasma gondii cysts in vitro using enzymatically enhanced ultrastructure expansion microscopy

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked super-resolution imaging for a broader scientific circle, lowering the cost and entry skill requirements to the field. One of its branches, ultrastructure ExM (U-ExM), has become popular among research groups studying Apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. The chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localisation. Here, we solve the cyst"s resistance to denaturation required for successful U-ExM of the encapsulated parasites. As the cyst's main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal culture without interference with the epitopes of the cyst-wall associated proteins. Using StcE-enhanced U-ExM, we clarified the shape and location of the GRA2 protein important for establishing a normal cyst. Expanded cysts revealed GRA2 granules spanning across the cyst wall, with a notable presence observed outside on both sides of the CST1-positive layer.