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However, the application of cryo\u2010EM for EV sample characterization has been limited by a combination of low imaging throughput and complex image analysis of the structurally diverse EVs. To address these challenges, we developed a workflow combining automated cryo\u2010EM image acquisition with supervised machine learning (sML)\u2010assisted particle detection and classification. Automated image acquisition facilitates the routine acquisition of thousands of cryo\u2010EM images with consistent quality, enabling the imaging of hundreds of EVs. sML\u2010assisted particle detection enabled efficient and reproducible identification, size measurement, and structural classification of EVs. Furthermore, using sML we are able to differentiate EVs from non\u2010EV particles, such as lipoproteins and protein aggregates, which might co\u2010isolate due to overlapping physical properties or by physical association with EVs. In mixed EV\u2010lipoprotein samples, we demonstrate that our pipeline can distinguish EVs and differentiate between high\u2010density (HDL), low\u2010density (LDL), and very low\u2010density (VLDL) lipoproteins. Our automated cryo\u2010EM and sML workflow overcomes key limitations of EV characterization using cryo\u2010EM by increasing imaging throughput and enabling reproducible EV characterization. Furthermore, this method provides a tool for analysing EV heterogeneity, sample purity, and co\u2010isolated contaminants, advancing the field of EV research.<\/jats:p>","DOI":"10.1002\/jev2.70273","type":"journal-article","created":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T09:58:49Z","timestamp":1776160729000},"source":"Crossref","is-referenced-by-count":3,"title":["Automated Cryo\u2010EM and Supervised Machine Learning Enable Reproducible Characterization of Extracellular Vesicles and Co\u2010Isolating Particles"],"prefix":"10.1002","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9856-9160","authenticated-orcid":false,"given":"Agustin","family":"Enciso\u2010Martinez","sequence":"first","affiliation":[{"name":"Oncode Institute and Department of Cell and Chemical Biology Leiden University Medical Center  Leiden The Netherlands"},{"name":"Biomedical Engineering &amp; Physics; Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam Amsterdam UMC, University of Amsterdam  Amsterdam The Netherlands"},{"name":"Laboratory of Experimental Clinical Chemistry Laboratory Specialized Diagnostics &amp; Research Department of Laboratory Medicine Amsterdam UMC, University of Amsterdam  Amsterdam The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frank G. A.","family":"Faas","sequence":"additional","affiliation":[{"name":"Electron Microscopy Facility, Department of Cell and Chemical Biology Leiden University Medical Center  Leiden The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anja W. M.","family":"de Jong","sequence":"additional","affiliation":[{"name":"Electron Microscopy Facility, Department of Cell and Chemical Biology Leiden University Medical Center  Leiden The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ton G.","family":"van Leeuwen","sequence":"additional","affiliation":[{"name":"Laboratory of Experimental Clinical Chemistry Laboratory Specialized Diagnostics &amp; Research Department of Laboratory Medicine Amsterdam UMC, University of Amsterdam  Amsterdam The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5671-3400","authenticated-orcid":false,"given":"Rienk","family":"Nieuwland","sequence":"additional","affiliation":[{"name":"Laboratory of Experimental Clinical Chemistry Laboratory Specialized Diagnostics &amp; Research Department of Laboratory Medicine Amsterdam UMC, University of Amsterdam  Amsterdam The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edwin","family":"van der Pol","sequence":"additional","affiliation":[{"name":"Biomedical Engineering &amp; Physics; Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam Amsterdam UMC, University of Amsterdam  Amsterdam The Netherlands"},{"name":"Laboratory of Experimental Clinical Chemistry Laboratory Specialized Diagnostics &amp; Research Department of Laboratory Medicine Amsterdam UMC, University of Amsterdam  Amsterdam The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7234-342X","authenticated-orcid":false,"given":"Peter","family":"ten Dijke","sequence":"additional","affiliation":[{"name":"Oncode Institute and Department of Cell and Chemical Biology Leiden University Medical Center  Leiden The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6736-7147","authenticated-orcid":false,"given":"Roman I.","family":"Koning","sequence":"additional","affiliation":[{"name":"Electron Microscopy Facility, Department of Cell and Chemical Biology Leiden University Medical Center  Leiden The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,4,14]]},"reference":[{"key":"e_1_2_10_2_1","unstructured":"Abadi M. 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