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Aberrant glycosylation is implicated in numerous diseases, including neurodegenerative disorders and cancer. While glycan organization at the cell surface is studied extensively, the nanoscale spatial arrangement of glycans inside cells and within organelles remains largely unexplored. Here, super\u2010resolution imaging with fluorophore\u2010labeled lectins, combined with a dedicated multiplexing strategy (Glyco\u2010STORM), was used in thin neuronal tissue sections and permeabilized cells to systematically map glycosylation across multiple organelles and cellular compartments. Lectin markers were identified that enabled the visualization of nanodomains within the endoplasmic reticulum, subdomains along the Golgi axes, diverse glycosylation states in the endolysosomal system, and a polarized lysosomal clathrin coat. At neuronal synapses, mature glycans were found to demarcate the synaptic cleft and subsynaptic tubules adjacent to the postsynaptic density. In summary, Glyco\u2010STORM establishes one of the first comprehensive nanoscale maps of intracellular glycosylation and provides entry points into health\u2010 and disease\u2010related studies.<\/jats:p>","DOI":"10.1002\/advs.202506731","type":"journal-article","created":{"date-parts":[[2025,11,11]],"date-time":"2025-11-11T05:40:47Z","timestamp":1762839647000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Nanoscale Mapping of the Subcellular Glycosylation Landscape"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-0497-5268","authenticated-orcid":false,"given":"Helene Gregoria","family":"Schroeter","sequence":"first","affiliation":[{"name":"Department of Functional Neuroanatomy Institute for Anatomy and Cell Biology Heidelberg University  Im Neuenheimer Feld 307 69120 Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8939-1084","authenticated-orcid":false,"given":"Steffen","family":"Sass","sequence":"additional","affiliation":[{"name":"Department of Functional Neuroanatomy Institute for Anatomy and Cell Biology Heidelberg University  Im Neuenheimer Feld 307 69120 Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9821-3578","authenticated-orcid":false,"given":"Mike","family":"Heilemann","sequence":"additional","affiliation":[{"name":"Department of Functional Neuroanatomy Institute for Anatomy and Cell Biology Heidelberg University  Im Neuenheimer Feld 307 69120 Heidelberg Germany"},{"name":"Institute of Physical and Theoretical Chemistry Goethe\u2010University Frankfurt  Max\u2010von\u2010Laue\u2010Str. 7 60438 Frankfurt Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1896-9031","authenticated-orcid":false,"given":"Thomas","family":"Kuner","sequence":"additional","affiliation":[{"name":"Department of Functional Neuroanatomy Institute for Anatomy and Cell Biology Heidelberg University  Im Neuenheimer Feld 307 69120 Heidelberg Germany"},{"name":"German Center for Lung Research (DZL), Partner Site Heidelberg (TLRC)  69120 Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4041-6102","authenticated-orcid":false,"given":"Maja","family":"Klevanski","sequence":"additional","affiliation":[{"name":"Department of Functional Neuroanatomy Institute for Anatomy and Cell Biology Heidelberg University  Im Neuenheimer Feld 307 69120 Heidelberg Germany"},{"name":"German Center for Lung Research (DZL), Partner Site Heidelberg (TLRC)  69120 Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,11,10]]},"reference":[{"key":"e_1_2_9_1_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrg1894"},{"key":"e_1_2_9_2_1","volume-title":"Essentials of Glycobiology","author":"Varki A.","year":"2009"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrn1349"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-1154-7_17"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2015.03.015"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.RA119.001896"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-neuro-071714-034019"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrc3982"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.3389\/fonc.2019.00380"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1093\/jnci\/djv326"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms23116312"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1002\/ar.21311"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.brainres.2015.12.040"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-025-56390-z"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201406045"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.devcel.2019.04.035"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41565-025-01966-5"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-025-07626-7"},{"key":"e_1_2_9_19_1","author":"Joseph D. 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