Adaptation of Iterative Ultrastructure Expansion Microscopy (iU-ExM) enables high-resolution imaging of the cytoskeleton and subcellular architecture of Trypanosoma cruzi

Kinetoplastid parasites, including Trypanosoma cruzi, possess a highly organized subpellicular microtubule network essential for maintaining cell shape, organelle positioning, and dividing. Here we describe results obtained using immunofluorescence microscopy of cells processed using conventional ultrastructure expansion microscopy (U-ExM), that improved visualization of the cytoskeletal structures, with an ~4.5-fold expansion, as well as the recently described and iterative ultrastructure expansion microscopy (iU-ExM) with about 6.2-fold and 18-fold expansion of epimastigotes of T. cruzi. The optimized protocol involves the formation of an initial hydrogel crosslinked with N,N'(1,2-dihydroxyethylene) bisacrylamide (DHEBA), a temperature- and alkaline pH-sensitive cleavable crosslinker, followed by thermal denaturation at 85 degrees Celsius, intermediate immunostaining, neutral gel re-embedding, and the formation of a second hydrogel crosslinked with N,N' methylenebisacrylamide (BIS). Subsequent dissolution of the first hydrogel under alkaline conditions enables a second round of expansion. This approach achieved approximately 6.2-fold expansion during the first round and a final linear expansion of approximately 18-fold. Using conventional confocal microscopy, iU-ExM enabled high-resolution visualization of individual subpellicular microtubules and specialized internal microtubule arrangements, including those associated with the basal body, flagellar pocket, Golgi complex, and cytopharynx. Furthermore, the enhanced spatial resolution facilitated the examination of cytoskeletal reorganization at different stages of the cell cycle, revealing changes in microtubule organization associated with mitotic spindle formation and cytokinesis. Together, these findings establish iU-ExM as an accessible high-resolution imaging approach for investigating the complex subcellular architecture of T. cruzi, providing new opportunities to explore cytoskeletal organization and remodeling during parasite cell division using conventional confocal microscopy.