Bio-orthogonal\nGlycan Imaging of Cultured Cells and\nWhole Animal <i>C. elegans</i> with Expansion Microscopy

Complex carbohydrates called glycans play crucial roles\nin regulating\ncell and tissue physiology, but how they map to nanoscale anatomical\nfeatures must still be resolved. Here, we present the first nanoscale\nmap of mucin-type <i>O</i>-glycans throughout the entirety\nof the <i>Caenorhabditis elegans</i> model organism. We\nconstructed a library of multifunctional linkers to probe and anchor\nmetabolically labeled glycans in expansion microscopy (ExM). A flexible\nstrategy was demonstrated for the chemical synthesis of linkers with\na broad inventory of bio-orthogonal functional groups, fluorophores,\nanchorage chemistries, and linker arms. Employing <i>C. elegans</i> as a test bed, metabolically labeled <i>O</i>-glycans\nwere resolved on the gut microvilli and other nanoscale anatomical\nfeatures. Transmission electron microscopy images of <i>C. elegans</i> nanoanatomy validated the fidelity and isotropy of gel expansion.\nWhole organism maps of <i>C. elegans O</i>-glycosylation\nin the first larval stage revealed <i>O</i>-glycan “hotspots”\nin unexpected anatomical locations, including the body wall furrows.\nBeyond <i>C. elegans</i>, we validated ExM protocols for\nnanoscale imaging of metabolically labeled glycans on cultured mammalian\ncells. Together, our results suggest the broad applicability of the\nmultifunctional reagents for imaging glycans and other metabolically\nlabeled biomolecules at enhanced resolutions with ExM.