Depth-Variant Deconvolution Applied to Widefield Microscopy for Rapid Large-Volume Tissue Imaging

Depth-Variant Deconvolution Applied to Widefield Microscopy for Rapid Large-Volume Tissue Imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Depth-Variant Deconvolution Applied to Widefield Microscopy for Rapid Large-Volume Tissue Imaging Daniel Lee, Kevin Telfer, Mark Koenis, Yim Lee, Kevin Namink, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6710731/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Innovations in 3D tissue imaging have revolutionized research, but limitations stemming from lengthy protocols and equipment accessibility persist. Widefield microscopy is fast and accessible but often excluded from 3D imaging workflows due to its lack of optical sectioning. Here we combine tissue clearing with a depth-variant deconvolution approach customized for large-volume widefield imaging to achieve subnuclear axial resolution in tissues to a depth of 500 µm. We illustrate the utility of this method in a model of ileitis and to gain a 3D perspective in thick brain slices from a model of cerebral amyloid angiopathy, where we resolved amyloid deposits along small blood vessels, attaining resolution that compared favorably to confocal microscopy. Finally, we leveraged our approach for richer pathological evaluation of human kidney biopsies. Our approach produced hundreds of consecutive z-planes in five minutes of imaging for 3D visualization of winding arterioles entering glomeruli. This perspective afforded straightforward identification of atrophic tubes in kidney biopsies prepared in 2 hours to simulate donor kidney evaluation before transplant. Having achieved subnuclear z-resolution in sections hundreds of microns thick, widefield microscopy coupled to robust deconvolution now emerges as an accessible and viable method to gain 3D insight in research or clinical evaluations. Biological sciences/Biological techniques/Imaging/Fluorescence imaging Biological sciences/Biological techniques/Imaging/3-D reconstruction Health sciences/Neurology/Neurological disorders/Neurovascular disorders Health sciences/Nephrology/Kidney Full Text Additional Declarations Yes there is potential Competing Interest. Some of the authors have filed a provisional patent for the ADAPT-3D clearing method used in this manuscript. Supplementary Files Video1.mp4 Video 1 related to Figure 3. Multi-tile 3D imaging using epifluorescent widefield microscopy followed by deconvolution with depth-variant point spread functions. Video2.mp4 Video 2 related to Figure 3. Deconvolved widefield (5X, NA 0.16) overview of CAA along leptomeningeal vessels across the entire dorsal surface of an intact 5XE4 brain hemisphere. Video3.mp4 Video 3 related to Figure 3. Widefield 3D volume of entire mouse cortex contained in a 500 µm sagittal section deconvolved to achieve high axial resolution. Video4.mp4 Video 4 related to Figure 4. 3D imaging by epifluorescent widefield microscopy and deconvolution allows for rapid, continuous axial examination of human kidney wedge biopsies. Cite Share Download PDF Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6710731","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":466512642,"identity":"0ba175b0-89f4-4724-a38f-9fe1514aa630","order_by":0,"name":"Daniel Lee","email":"","orcid":"","institution":"Department of Pathology \u0026 Immunology, Washington University School of Medicine, Saint Louis, MO, USA.","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Lee","suffix":""},{"id":466512643,"identity":"01db5d9e-b4f2-4f00-8d76-a10db4dd294a","order_by":1,"name":"Kevin 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Deconvolved widefield (5X, NA 0.16) overview of CAA along leptomeningeal vessels across the entire dorsal surface of an intact 5XE4 brain hemisphere.","description":"","filename":"Video2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6710731/v1/63bc86efae66b225cf2da760.mp4"},{"id":84039783,"identity":"c1aa4c04-7feb-4cd8-af5a-abcefa835ca5","added_by":"auto","created_at":"2025-06-06 05:32:15","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":54231194,"visible":true,"origin":"","legend":"\u003cp\u003eVideo 3 related to Figure 3. 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