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Bio-orthogonal Glycan Imaging of Cultured Cells and Whole Animal <italic toggle="yes">C. elegans</italic> with Expansion Microscopy Complex carbohydrates called glycans play crucial roles in regulating cell and tissue physiology, but how they map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O-glycans throughout the entirety of the Caenorhabditis elegans model organism. We constructed a library of multifunctional linkers to probe and anchor metabolically labeled glycans in expansion microscopy (ExM). A flexible strategy was demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, metabolically labeled O-glycans were resolved on the gut microvilli and other nanoscale anatomical features. Transmission electron microscopy images of C. elegans nanoanatomy validated the fidelity and isotropy of gel expansion. Whole organism maps of C. elegans O-glycosylation in the first larval stage revealed O-glycan “hotspots” in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans, we validated ExM protocols for nanoscale imaging of metabolically labeled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labeled biomolecules at enhanced resolutions with ExM. https://pubs.acs.org/doi/10.1021/acscentsci.4c01061