{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T08:08:49Z","timestamp":1773216529996,"version":"3.50.1"},"reference-count":47,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T00:00:00Z","timestamp":1773100800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institute of Mental Health of the National Institutes of Health","award":["R44MH116827"],"award-info":[{"award-number":["R44MH116827"]}]},{"name":"NYU CTSI Pilot NIH\/NCATS","award":["UL1TR001445"],"award-info":[{"award-number":["UL1TR001445"]}]},{"name":"Parekh Center for Interdisciplinary Neurology (PCIN) Pilot Research Grant"},{"name":"American Epilepsy Society Junior Investigator Award"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J. Imaging"],"abstract":"<jats:p>Three-dimensional (3D) ex vivo imaging of cleared tissue from intact brains from animal models, human brain surgical specimens, and large postmortem human and non-human primate brain specimens is essential for understanding physiological neural connectivity and pathological alterations underlying neurological and neuropsychiatric disorders. Contemporary light-sheet microscopy enables rapid, high-resolution imaging of large, cleared samples but is limited by the orthogonal arrangement of illumination and detection optics, which constrains specimen size. Light-sheet theta microscopy (LSTM) overcomes this limitation by employing two oblique illumination paths while maintaining a perpendicular detection geometry. Here, we report the development of a next-generation, fully integrated and user-friendly LSTM system that enables uniform subcellular-resolution imaging (with subcellular resolution determined by the lateral performance of the system) throughout large specimens without constraining lateral (XY) dimensions. The system provides a seamless workflow encompassing image acquisition, data storage, pre- and post-processing, enhancement and quantitative analysis. Performance is demonstrated by high-resolution 3D imaging of intact mouse brains and human brain samples, including complete downstream analyses such as digital neuron tracing, vascular reconstruction and design-based stereological analysis. This enhanced and accessible LSTM implementation enables rapid quantitative mapping of molecular and cellular features in very large biological specimens.<\/jats:p>","DOI":"10.3390\/jimaging12030118","type":"journal-article","created":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T09:55:38Z","timestamp":1773136538000},"page":"118","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["ClearScope: A Fully Integrated Light-Sheet Theta Microscope for Sub-Micron-Resolution Imaging Without Lateral Size Constraints"],"prefix":"10.3390","volume":"12","author":[{"given":"Matthew G.","family":"Fay","sequence":"first","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-1876-4104","authenticated-orcid":false,"given":"Peter J.","family":"Lang","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David S.","family":"Denu","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5928-8692","authenticated-orcid":false,"given":"Nathan J.","family":"O\u2019Connor","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Benjamin","family":"Haydock","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jeffrey","family":"Blaisdell","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicolas","family":"Roussel","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alissa","family":"Wilson","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sage R.","family":"Aronson","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Veronica","family":"Pessino","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul J.","family":"Angstman","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0026-2006","authenticated-orcid":false,"given":"Cheng","family":"Gong","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA"},{"name":"Department of Biological Sciences, Columbia University, New York, NY 10027, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tanvi","family":"Butola","sequence":"additional","affiliation":[{"name":"Neuroscience Institute, New York University Langone Health, New York, NY 10016, USA"},{"name":"Department of Neuroscience and Physiology, New York University Grossman School of Medicine, New York, NY 10016, USA"},{"name":"Comprehensive Epilepsy Center, Department of Neurology, New York University Grossman School of Medicine, New York, NY 10016, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Orrin","family":"Devinsky","sequence":"additional","affiliation":[{"name":"Department of Neuroscience and Physiology, New York University Grossman School of Medicine, New York, NY 10016, USA"},{"name":"Comprehensive Epilepsy Center, Department of Neurology, New York University Grossman School of Medicine, New York, NY 10016, USA"},{"name":"Department of Psychiatry, New York University Grossman School of Medicine, New York, NY 10016, USA"},{"name":"Department of Neurosurgery, New York University Grossman School of Medicine, New York, NY 10016, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7559-0936","authenticated-orcid":false,"given":"Jayeeta","family":"Basu","sequence":"additional","affiliation":[{"name":"Neuroscience Institute, New York University Langone Health, New York, NY 10016, USA"},{"name":"Department of Neuroscience and Physiology, New York University Grossman School of Medicine, New York, NY 10016, USA"},{"name":"Center for Neural Science, New York University, New York, NY 10003, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raju","family":"Tomer","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA"},{"name":"Department of Biological Sciences, Columbia University, New York, NY 10027, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4463-207X","authenticated-orcid":false,"given":"Jacob R.","family":"Glaser","sequence":"additional","affiliation":[{"name":"MBF Bioscience, Williston, VT 05495, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1404","DOI":"10.1126\/science.1191776","article-title":"Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain","volume":"330","author":"Li","year":"2010","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1016\/j.neuroimage.2015.12.017","article-title":"Large-scale functional connectivity networks in the rodent brain","volume":"127","author":"Gozzi","year":"2016","journal-title":"NeuroImage"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s11307-016-0988-z","article-title":"Whole-brain microscopy meets in vivo neuroimaging: Techniques, benefits, and limitations","volume":"19","author":"Aswendt","year":"2017","journal-title":"Mol. Imaging Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1038\/nmeth.4224","article-title":"A guide to light-sheet fluorescence microscopy for multiscale imaging","volume":"14","author":"Power","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"508","DOI":"10.1038\/nmeth.2481","article-title":"CLARITY for mapping the nervous system","volume":"10","author":"Chung","year":"2013","journal-title":"Nat. Methods"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1038\/nature12107","article-title":"Structural and molecular interrogation of intact biological systems","volume":"497","author":"Chung","year":"2013","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1682","DOI":"10.1038\/nprot.2014.123","article-title":"Advanced CLARITY for rapid and high-resolution imaging of intact tissues","volume":"9","author":"Tomer","year":"2014","journal-title":"Nat. Protoc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3506","DOI":"10.1038\/s41596-019-0240-9","article-title":"Advanced CUBIC tissue clearing for whole-organ cell profiling","volume":"14","author":"Matsumoto","year":"2019","journal-title":"Nat. Protoc."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"896","DOI":"10.1016\/j.cell.2014.10.010","article-title":"iDISCO: A simple, rapid method to immunolabel large tissue samples for volume imaging","volume":"159","author":"Renier","year":"2014","journal-title":"Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1038\/nmeth.3964","article-title":"Shrinkage-mediated imaging of entire organs and organisms using uDISCO","volume":"13","author":"Pan","year":"2016","journal-title":"Nat. Methods"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1154","DOI":"10.1038\/nn.3447","article-title":"SeeDB: A simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction","volume":"16","author":"Ke","year":"2013","journal-title":"Nat. Neurosci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1481","DOI":"10.1038\/nn.2928","article-title":"Scale: A chemical approach for fluorescence imaging and reconstruction of transparent mouse brain","volume":"14","author":"Hama","year":"2011","journal-title":"Nat. Neurosci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1007\/s13770-024-00658-w","article-title":"High-speed clearing and high-resolution staining for analysis of various markers for neurons and vessels","volume":"21","author":"Park","year":"2024","journal-title":"Tissue Eng. Regen. Med."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Migliori, B., Datta, M.S., Dupre, C., Apak, M.C., Asano, S., Gao, R., Boyden, E.S., Hermanson, O., Yuste, R., and Tomer, R. (2018). Light sheet theta microscopy for rapid high-resolution imaging of large biological samples. BMC Biol., 16.","DOI":"10.1186\/s12915-018-0521-8"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Gong, C., Affatato, P., Fay, M., Guttikonda, S.R., O\u2019Connor, N.J., Noble, E., Heal, M., Haydock, B., Mapa, R., and De La Cruz, E.D. (2025). Hybrid solid\u2013liquid optics enable scalable, high-resolution, multi-immersion light-sheet microscopy. bioRxiv.","DOI":"10.1101\/2025.11.02.685475"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"404","DOI":"10.1038\/nmeth.3323","article-title":"CIDRE: An illumination-correction method for optical microscopy","volume":"12","author":"Smith","year":"2015","journal-title":"Nat. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1109\/JPROC.1998.659497","article-title":"Communication in the presence of noise","volume":"86","author":"Shannon","year":"1998","journal-title":"Proc. IEEE"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41592-019-0501-0","article-title":"BigStitcher: Reconstructing high-resolution image datasets of cleared and expanded samples","volume":"16","author":"Preusser","year":"2019","journal-title":"Nat. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Butola, T., Robert, V., Kim, B., Doyle, W., Hussain, F., Gong, C., Leibner, Y., Bilash, O., O\u2019Neil, K., and Peng, L. (2025). Enhanced distal signaling in human hippocampal neurons despite lower intrinsic excitability. Res Sq., rs.3.rs-7820914.","DOI":"10.21203\/rs.3.rs-7820914\/v1"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1007\/BF02057902","article-title":"Localization of Thy-1 expression during postnatal development of the mouse cerebellar cortex","volume":"15","author":"Bolin","year":"1986","journal-title":"J. Neurocytol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2200","DOI":"10.1002\/cne.24635","article-title":"Automatic navigation system for the mouse brain","volume":"527","author":"Tappan","year":"2019","journal-title":"J. Comp. Neurol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"936","DOI":"10.1016\/j.cell.2020.04.007","article-title":"The Allen Mouse Brain Common Coordinate Framework: A 3D reference atlas","volume":"181","author":"Wang","year":"2020","journal-title":"Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1016\/j.neuroscience.2004.08.050","article-title":"Design-based stereology in neuroscience","volume":"130","author":"Schmitz","year":"2005","journal-title":"Neuroscience"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1038\/s41592-022-01468-5","article-title":"A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues","volume":"19","author":"Glaser","year":"2022","journal-title":"Nat. Methods"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1111\/j.1365-2818.1993.tb03346.x","article-title":"Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens","volume":"170","author":"Voie","year":"1993","journal-title":"J. Microsc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1126\/science.1100035","article-title":"Optical sectioning deep inside live embryos by selective plane illumination microscopy","volume":"305","author":"Huisken","year":"2004","journal-title":"Science"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2807","DOI":"10.1016\/j.bpj.2015.05.013","article-title":"Deconvolution-free subcellular imaging with axially swept light sheet microscopy","volume":"108","author":"Dean","year":"2015","journal-title":"Biophys. J."},{"key":"ref_29","unstructured":"Tomer, R. (2022). Imaging Instrument Having Objective Axis and Light Sheet or Light Beam Projector Axis Intersecting at less than 90 Degrees. (11,506,877), U.S. Patent."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1038\/nmeth.2507","article-title":"OpenSPIM: An open-access light-sheet microscopy platform","volume":"10","author":"Pitrone","year":"2013","journal-title":"Nat. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e2101182","DOI":"10.1002\/adbi.202101182","article-title":"Time to upgrade: A new OpenSPIM guide to build and operate advanced OpenSPIM configurations","volume":"6","author":"Girstmair","year":"2022","journal-title":"Adv. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1105","DOI":"10.1038\/s41592-019-0554-0","article-title":"The mesoSPIM initiative: Open-source light-sheet microscopes for imaging cleared tissue","volume":"16","author":"Voigt","year":"2019","journal-title":"Nat. Methods"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"113491","DOI":"10.1016\/j.celrep.2023.113491","article-title":"Whole-brain mapping reveals the divergent impact of ketamine on the dopamine system","volume":"42","author":"Datta","year":"2023","journal-title":"Cell Rep."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Dayton, H., Kiss, J., Wei, M., Chauhan, S., LaMarre, E., Cornell, W.C., Morgan, C.J., Janakiraman, A., Min, W., and Tomer, R. (2024). Cellular arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms. PLoS Biol., 22.","DOI":"10.1371\/journal.pbio.3002205"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Scholler, J., Jonsson, J., Jord\u00e1-Siquier, T., Gantar, I., Batti, L., Cheeseman, B., Pages, S., Sbalzarini, I., and Lamy, C. (2023). Efficient image analysis for large-scale next generation histopathology using pAPRica. bioRxiv.","DOI":"10.1101\/2023.01.27.525687"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Jord\u00e0-Siquier, J., Scholler, J., Gantar, I., Policet-B\u00e9tend, H., Batti, L., Pages, S., K\u00f6rvari, E., and Lamy, C. (2025). CleLight: A scalable 3D histology pipeline for mapping neurodegenerative and psychiatric pathology in archival human brains. bioRxiv.","DOI":"10.64898\/2025.12.10.693496"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"502","DOI":"10.1038\/s42003-023-04857-4","article-title":"Light-sheets and smart microscopy, an exciting future is dawning","volume":"6","author":"Daetwyler","year":"2023","journal-title":"Commun. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"32880","DOI":"10.1038\/srep32880","article-title":"\u03c0SPIM: High-NA high-resolution isotropic light-sheet imaging in cell culture dishes","volume":"6","author":"Theer","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1385","DOI":"10.1038\/s41587-021-01042-y","article-title":"Volumetric interferometric lattice light-sheet imaging","volume":"39","author":"Cao","year":"2021","journal-title":"Nat. Biotechnol."},{"key":"ref_40","first-page":"Unit14.20","article-title":"Computer control of microscopes using \u00b5Manager","volume":"14","author":"Edelstein","year":"2010","journal-title":"Curr. Protoc. Mol. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1038\/s41592-021-01087-6","article-title":"Pycro-Manager: Open-source software for customized and reproducible microscope control","volume":"18","author":"Pinkard","year":"2021","journal-title":"Nat. Methods"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Tosi, S., Llad\u00f3, A., Bardia, L., Rebollo, E., Godo, A., Stockinger, P., and Colombelli, J. (2021). AutoScanJ: A suite of ImageJ scripts for intelligent microscopy. Front. Bioinform., 1.","DOI":"10.3389\/fbinf.2021.627626"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3394","DOI":"10.21105\/joss.03394","article-title":"ImSwitch: Generalizing microscope control in Python","volume":"6","author":"Moreno","year":"2021","journal-title":"J. Open Source Softw."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2199","DOI":"10.1038\/s41467-022-29888-z","article-title":"Enabling reactive microscopy with MicroMator","volume":"13","author":"Fox","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1967","DOI":"10.1038\/s41592-024-02413-4","article-title":"Navigate: An open-source platform for smart light-sheet microscopy","volume":"21","author":"Marin","year":"2024","journal-title":"Nat. Methods"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"jcs258955","DOI":"10.1242\/jcs.258955","article-title":"Python-Microscope: A new open-source Python library for the control of microscopes","volume":"134","author":"Phillips","year":"2021","journal-title":"J. Cell Sci."},{"key":"ref_47","unstructured":"(2025, August 12). Pybind11. Available online: https:\/\/github.com\/pybind\/pybind11."}],"container-title":["Journal of Imaging"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2313-433X\/12\/3\/118\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T10:22:16Z","timestamp":1773138136000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2313-433X\/12\/3\/118"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,10]]},"references-count":47,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["jimaging12030118"],"URL":"https:\/\/doi.org\/10.3390\/jimaging12030118","relation":{},"ISSN":["2313-433X"],"issn-type":[{"value":"2313-433X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,10]]}}}