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Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.<\/jats:p>","DOI":"10.1101\/2023.05.20.540091","type":"posted-content","created":{"date-parts":[[2023,5,21]],"date-time":"2023-05-21T05:00:11Z","timestamp":1684645211000},"source":"Crossref","is-referenced-by-count":5,"title":["Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1409-7419","authenticated-orcid":false,"given":"Xiaomeng","family":"Han","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8575-5394","authenticated-orcid":false,"given":"Xiaotang","family":"Lu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6193-4454","authenticated-orcid":false,"given":"Peter H.","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5186-4157","authenticated-orcid":false,"given":"Shuohong","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2450-1718","authenticated-orcid":false,"given":"Richard","family":"Schalek","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1946-8012","authenticated-orcid":false,"given":"Yaron","family":"Meirovitch","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4176-8035","authenticated-orcid":false,"given":"Zudi","family":"Lin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6247-7475","authenticated-orcid":false,"given":"Jason","family":"Adhinarta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9677-6932","authenticated-orcid":false,"given":"Daniel","family":"Berger","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0075-1237","authenticated-orcid":false,"given":"Yuelong","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tao","family":"Fang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elif Sevde","family":"Meral","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shadnan","family":"Asraf","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1090-6071","authenticated-orcid":false,"given":"Hidde","family":"Ploegh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3620-2582","authenticated-orcid":false,"given":"Hanspeter","family":"Pfister","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2329-5484","authenticated-orcid":false,"given":"Donglai","family":"Wei","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1488-3505","authenticated-orcid":false,"given":"Viren","family":"Jain","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6117-3912","authenticated-orcid":false,"given":"James S.","family":"Trimmer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0208-3212","authenticated-orcid":false,"given":"Jeff W.","family":"Lichtman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023071112400662000_2023.05.20.540091v4.1","doi-asserted-by":"publisher","DOI":"10.1038\/nature06293"},{"key":"2023071112400662000_2023.05.20.540091v4.2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2450"},{"key":"2023071112400662000_2023.05.20.540091v4.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.1260088"},{"key":"2023071112400662000_2023.05.20.540091v4.4","doi-asserted-by":"crossref","first-page":"18212","DOI":"10.1021\/jacs.2c03743","article-title":"Lipid Expansion Microscopy","volume":"144","year":"2022","journal-title":"J. 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