{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T18:55:10Z","timestamp":1784141710109,"version":"3.55.0"},"reference-count":31,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,2,7]],"date-time":"2026-02-07T00:00:00Z","timestamp":1770422400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,2,7]],"date-time":"2026-02-07T00:00:00Z","timestamp":1770422400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R21AG090967"],"award-info":[{"award-number":["R21AG090967"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01NS126448"],"award-info":[{"award-number":["R01NS126448"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000052","name":"NIH Office of the Director","doi-asserted-by":"publisher","award":["1S10OD032204"],"award-info":[{"award-number":["1S10OD032204"]}],"id":[{"id":"10.13039\/100000052","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cytoskeleton"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>We describe a method for determining the ultrastructural organization of axons and varicosities of cultured dorsal root ganglion (DRG) neurons using cryogenic electron microscopy (cryo\u2010EM). Cryo\u2010EM reveals the dimensions, proximity, and overall organization of biological specimens in a near\u2010native state, avoiding artifacts of fixation and heavy metal staining employed in classic thin section ultramicroscopy. Cryo\u2010EM excels with thin specimens, and the axons of cryo\u2010preserved cultured DRG neurons are the ideal thickness for high\u2010resolution cryo\u2010electron tomography. DRG neurons are a particularly interesting neuronal preparation because they can be isolated from animals of any age, providing a unique resource to examine age\u2010related changes in axonal morphology. We provide a detailed, step\u2010by\u2010step protocol from DRG isolation and culturing, through cryo\u2010preservation and data acquisition and analysis. We also provide a description for data processing in batch and how implementing deep\u2010learning strategies can facilitate taking tilt\u2010series data through the process of semi\u2010automated tomographic segmentation required for quantitative descriptions of ultrastructural features. We use segmentations focused on the cytoskeletal elements of axons and varicosities of young and old cultured DRG neurons to highlight the approach.<\/jats:p>","DOI":"10.1002\/cm.70100","type":"journal-article","created":{"date-parts":[[2026,2,7]],"date-time":"2026-02-07T12:01:13Z","timestamp":1770465673000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Investigating the Cytoskeleton of\n                    <scp>DRGs<\/scp>\n                    Using Cryo\u2010Electron Microscopy and Deep Learning"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4801-1190","authenticated-orcid":false,"given":"M. Neal","family":"Waxham","sequence":"first","affiliation":[{"name":"Department of Neurobiology and Anatomy University of Texas Health Science Center  Houston Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0100-6589","authenticated-orcid":false,"given":"Mihir","family":"Relan","sequence":"additional","affiliation":[{"name":"Department of Neurobiology and Anatomy University of Texas Health Science Center  Houston Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8147-208X","authenticated-orcid":false,"given":"Matthew T.","family":"Swulius","sequence":"additional","affiliation":[{"name":"Department of Molecular and Precision Medicine Penn State College of Medicine  Hershey Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4073-4565","authenticated-orcid":false,"given":"Andrea K. H.","family":"Stavoe","sequence":"additional","affiliation":[{"name":"Department of Neurobiology and Anatomy University of Texas Health Science Center  Houston Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,2,7]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1390"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2209823119"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1920323117"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2011.12.017"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/J.BPJ.2016.02.001"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.2006169"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.202103154"},{"key":"e_1_2_12_9_1","doi-asserted-by":"crossref","DOI":"10.1093\/acprof:oso\/9780195182187.001.0001","volume-title":"Three\u2010Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State","author":"Frank J.","year":"2006"},{"key":"e_1_2_12_10_1","doi-asserted-by":"publisher","DOI":"10.3791\/64435"},{"key":"e_1_2_12_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010022\u201030116\u2010x"},{"key":"e_1_2_12_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2021.102959"},{"key":"e_1_2_12_13_1","doi-asserted-by":"publisher","DOI":"10.1093\/micmic\/ozad125"},{"key":"e_1_2_12_14_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.05793"},{"key":"e_1_2_12_15_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u20100716\u20100966\u20108_3"},{"key":"e_1_2_12_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2015.07.012"},{"key":"e_1_2_12_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth1014"},{"key":"e_1_2_12_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592\u2010019\u20100675\u20105"},{"key":"e_1_2_12_19_1","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.abe6204"},{"key":"e_1_2_12_20_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2308088120"},{"key":"e_1_2_12_21_1","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.e03\u201009\u20100704"},{"key":"e_1_2_12_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2024.102864"},{"key":"e_1_2_12_23_1","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0813\u201009.2009"},{"key":"e_1_2_12_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2016.07.010"},{"key":"e_1_2_12_25_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592\u2010023\u201002113\u20105"},{"key":"e_1_2_12_26_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0197886"},{"key":"e_1_2_12_27_1","doi-asserted-by":"publisher","DOI":"10.3791\/50783"},{"key":"e_1_2_12_28_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13104\u2010016\u20101915\u20108"},{"key":"e_1_2_12_29_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365\u20102818.1979.tb00170.x"},{"key":"e_1_2_12_30_1","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.adk6285"},{"key":"e_1_2_12_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2013.08.006"},{"key":"e_1_2_12_32_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.yjsbx.2022.100067"}],"container-title":["Cytoskeleton"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cm.70100","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cm.70100","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cm.70100","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,7]],"date-time":"2026-02-07T12:01:15Z","timestamp":1770465675000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/cm.70100"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,7]]},"references-count":31,"alternative-id":["10.1002\/cm.70100"],"URL":"https:\/\/doi.org\/10.1002\/cm.70100","archive":["Portico"],"relation":{},"ISSN":["1949-3584","1949-3592"],"issn-type":[{"value":"1949-3584","type":"print"},{"value":"1949-3592","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,7]]},"assertion":[{"value":"2025-08-28","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-01-14","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-02-07","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"cm.70100"}}