{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T05:52:32Z","timestamp":1781848352083,"version":"3.54.5"},"reference-count":24,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T00:00:00Z","timestamp":1778716800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T00:00:00Z","timestamp":1778716800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Microscopy"],"published-print":{"date-parts":[[2026,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Spatial transcriptomics and in situ hybridisation techniques have become essential for contextualising single\u2010cell RNA sequencing (scRNA\u2010seq) data by mapping cell type\u2013 and state\u2013specific mRNA expression within intact tissues. Beyond validating scRNA\u2010seq predictions, these spatial methods provide critical insight into the localisation of specific cell types and their spatial relationships with neighbouring cells. However, current approaches rely on light microscopy (LM), which, despite advances, lacks the resolution needed to resolve detailed cell morphology or subcellular RNA localisation in situ. While correlative imaging methods can enhance spatial context, RNA detection remains limited by the optical constraints of LM. To overcome these challenges, we developed a three\u2010dimensional RNA\u2010based correlated light and electron microscopy (RCLEM) workflow. This method integrates RNA labelling with serial block\u2010face scanning electron microscopy (SBF\u2010SEM), allowing high\u2010resolution 3D visualisation of RNA molecules within their ultrastructural environment. We optimised the RNA labelling protocol for thick, whole\u2010mount tissues by fine\u2010tuning detergent conditions to preserve ultrastructure while maintaining probe accessibility. This RCLEM approach offers several advantages over protein\u2010based correlative methods, including robust detection with nucleotide probes and independence from antibody optimisation or fixation\u2010sensitive epitopes. Applied to choroid plexus (ChP) and liver tissue, this workflow enabled precise 3D correlation between mRNA expression and EM features, supporting detailed morphological characterisation of rare scRNA\u2010seq\u2013defined cell subtypes. This protocol is broadly applicable to other complex tissues and provides a powerful platform for integrative, high\u2010resolution spatial transcriptomic analyses that bridge gene expression with ultrastructure.<\/jats:p>\n                  <jats:p>\n                    <jats:bold>LAY DESCRIPTION<\/jats:bold>\n                    : To overcome the resolution limits of current spatial transcriptomics, we developed a 3D RNA\u2010based correlated light and electron microscopy (RCLEM) workflow. By integrating RNA labelling with scanning electron microscopy, this method enables high\u2010resolution 3D visualisation of mRNA within its ultrastructural environment in thick tissues. Validated in choroid plexus and liver, our antibody\u2010independent RCLEM approach provides a broadly applicable platform bridging gene expression and precise cellular morphology for advanced spatial analyses.\n                  <\/jats:p>","DOI":"10.1111\/jmi.70105","type":"journal-article","created":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T08:45:13Z","timestamp":1778748313000},"page":"120-131","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["RNA\u2010based CLEM (RCLEM) bridging RNA localisation and ultrastructural mapping in 3D"],"prefix":"10.1111","volume":"303","author":[{"given":"Daan","family":"Verhaege","sequence":"first","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"Department of Biomedical Molecular Biology Ghent University  Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lore","family":"Van Acker","sequence":"additional","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"Department of Biomedical Molecular Biology Ghent University  Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Camille","family":"Wagner","sequence":"additional","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"Department of Biomedical Molecular Biology Ghent University  Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4893-6225","authenticated-orcid":false,"given":"Yentl","family":"Van den Berghe","sequence":"additional","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"Department of Mathematics Computer Science and Statistics Ghent University Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00e9r\u00e9my","family":"Verbeke","sequence":"additional","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"VIB BioImaging Core VIB  Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin","family":"Guilliams","sequence":"additional","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"Department of Biomedical Molecular Biology Ghent University  Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4613-1060","authenticated-orcid":false,"given":"Anna","family":"Kremer","sequence":"additional","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"VIB BioImaging Core VIB  Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lien","family":"Van Hoecke","sequence":"additional","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"Department of Biomedical Molecular Biology Ghent University  Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roosmarijn E","family":"Vandenbroucke","sequence":"additional","affiliation":[{"name":"VIB\u2010UGent Center for Inflammation Research VIB  Ghent Belgium"},{"name":"Department of Biomedical Molecular Biology Ghent University  Ghent Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,14]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.micron.2014.01.009"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1111\/jmi.12211"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2005.01.003"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978\u20100\u201012\u2010407761\u20104.00022\u20101"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1093\/bfgp\/ell036"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.0020329"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2006.03.006"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tice.2018.08.011"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mcb.2019.04.002"},{"key":"e_1_2_6_11_1","doi-asserted-by":"publisher","DOI":"10.1111\/jmi.12967"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2024.07.002"},{"key":"e_1_2_6_13_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1252945"},{"key":"e_1_2_6_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2019.08.017"},{"key":"e_1_2_6_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhepr.2021.100278"},{"key":"e_1_2_6_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010018\u201006318\u20107"},{"key":"e_1_2_6_17_1","doi-asserted-by":"crossref","unstructured":"Deerinck T. J. Bushong E. A. Ellisman M. H. &Thor A.(2022).Preparation of biological tissues for serial block face scanning electron microscopy (SBEM) V.2.https:\/\/doi.org\/10.17504\/protocols.io.36wgq7je5vk5\/2","DOI":"10.17504\/protocols.io.36wgq7je5vk5\/v2"},{"key":"e_1_2_6_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mcb.2023.01.011"},{"key":"e_1_2_6_19_1","unstructured":"Bogovic J. A. H P. Wong A. &Saalfeld S. in2016 IEEE 13th International Symposium on Biomedical Imaging (ISBI)(2016)."},{"key":"e_1_2_6_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41593\u2010025\u201002188\u20107"},{"key":"e_1_2_6_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.12.018"},{"key":"e_1_2_6_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1622"},{"key":"e_1_2_6_23_1","doi-asserted-by":"crossref","unstructured":"Van den Berghe Y. Kremer A. Verhaege D. 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