Optimizing Tissue Clearing Protocols for Whole-Organoid Imaging of Human Cortical and Retinal iPSC-Derived Models

Optimizing Tissue Clearing Protocols for Whole-Organoid Imaging of Human Cortical and Retinal iPSC-Derived Models | 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 Method Article Optimizing Tissue Clearing Protocols for Whole-Organoid Imaging of Human Cortical and Retinal iPSC-Derived Models Erica Debbi, Chiara D’Antoni, Lorenza Mautone, Federica Cordella, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6734606/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Three-dimensional brain organoids derived from human induced pluripotent stem cells provide valuable in vitro systems for studying human brain development and neurological disorders. However, their dense and heterogeneous structure presents significant challenges for high-resolution imaging, particularly when using conventional histological sectioning, which disrupts native cellular architecture. Improved methodologies are needed to preserve the three-dimensional integrity of organoids while enabling detailed visualization of internal structures and cell types. Results: We present a combined methodological approach that integrates solvent-based tissue clearing protocols with high-resolution volumetric imaging using commonly accessible microscopy platforms. Specifically, we applied two clearing techniques—iDISCO plus and Visikol HISTO—to cortical and retinal organoids, followed by imaging with spinning disk confocal microscopy, laser scanning confocal microscopy, and structured illumination microscopy. This strategy enabled deep tissue penetration and preservation of structural features, such as neural rosettes, cortical plate-like zones, and astrocytic networks. Immunostaining with markers for radial glial progenitors, adherent junctions, and mature neuronal and glial elements revealed fine morphological details and spatial organization without the need for sectioning. Minimal necrosis was observed in central regions of organoids, supporting the effectiveness of the clearing protocols in preserving tissue quality. Importantly, our results demonstrate that conventional imaging platforms can achieve sufficient resolution and depth for comprehensive three-dimensional analysis when paired with optimized clearing methods. Conclusions: Our combined protocol for tissue clearing and volumetric imaging offers an accessible, scalable, and effective approach for three-dimensional visualization of brain organoids. By avoiding the use of more complex light-sheet microscopy, this methodology broadens access to high-content imaging of organoids in standard laboratory settings. This platform can facilitate detailed investigation of neurodevelopmental processes, disease phenotypes, and therapeutic responses in organoid models, supporting a wide range of applications in developmental biology and translational research. cortical organoids retinal organoids tissue clearing induced pluripotent stem cells neuronal rosette neurodevelopment neurons astrocytes confocal imaging microphysiological constructs Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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. 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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-6734606","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":477141764,"identity":"3a792f86-dfe2-4b0c-b1e5-f1e6af400088","order_by":0,"name":"Erica Debbi","email":"","orcid":"","institution":"Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Erica","middleName":"","lastName":"Debbi","suffix":""},{"id":477141767,"identity":"67550c53-a95d-40e0-9fca-31debb55fc3a","order_by":1,"name":"Chiara D’Antoni","email":"","orcid":"","institution":"Sapienza University of 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Angelantonio","email":"data:image/png;base64,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","orcid":"","institution":"Sapienza University of Rome","correspondingAuthor":true,"prefix":"","firstName":"Silvia","middleName":"Di","lastName":"Angelantonio","suffix":""}],"badges":[],"createdAt":"2025-05-23 16:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6734606/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6734606/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93060370,"identity":"016c6af6-3462-4247-9477-3cc2a0644f31","added_by":"auto","created_at":"2025-10-08 15:46:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1446654,"visible":true,"origin":"","legend":"","description":"","filename":"DebbietalBMCBiol.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6734606/v1_covered_aa509e51-3628-4730-8bbf-092e3473db47.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimizing Tissue Clearing Protocols for Whole-Organoid Imaging of Human Cortical and Retinal iPSC-Derived 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neurodevelopment, neurons, astrocytes, confocal imaging, microphysiological constructs","lastPublishedDoi":"10.21203/rs.3.rs-6734606/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6734606/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThree-dimensional brain organoids derived from human induced pluripotent stem cells provide valuable in vitro systems for studying human brain development and neurological disorders. However, their dense and heterogeneous structure presents significant challenges for high-resolution imaging, particularly when using conventional histological sectioning, which disrupts native cellular architecture. Improved methodologies are needed to preserve the three-dimensional integrity of organoids while enabling detailed visualization of internal structures and cell types.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe present a combined methodological approach that integrates solvent-based tissue clearing protocols with high-resolution volumetric imaging using commonly accessible microscopy platforms. Specifically, we applied two clearing techniques—iDISCO plus and Visikol HISTO—to cortical and retinal organoids, followed by imaging with spinning disk confocal microscopy, laser scanning confocal microscopy, and structured illumination microscopy. This strategy enabled deep tissue penetration and preservation of structural features, such as neural rosettes, cortical plate-like zones, and astrocytic networks. Immunostaining with markers for radial glial progenitors, adherent junctions, and mature neuronal and glial elements revealed fine morphological details and spatial organization without the need for sectioning. Minimal necrosis was observed in central regions of organoids, supporting the effectiveness of the clearing protocols in preserving tissue quality. Importantly, our results demonstrate that conventional imaging platforms can achieve sufficient resolution and depth for comprehensive three-dimensional analysis when paired with optimized clearing methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur combined protocol for tissue clearing and volumetric imaging offers an accessible, scalable, and effective approach for three-dimensional visualization of brain organoids. By avoiding the use of more complex light-sheet microscopy, this methodology broadens access to high-content imaging of organoids in standard laboratory settings. This platform can facilitate detailed investigation of neurodevelopmental processes, disease phenotypes, and therapeutic responses in organoid models, supporting a wide range of applications in developmental biology and translational research.\u003c/p\u003e","manuscriptTitle":"Optimizing Tissue Clearing Protocols for Whole-Organoid Imaging of Human Cortical and Retinal iPSC-Derived Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 13:17:52","doi":"10.21203/rs.3.rs-6734606/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7185cbea-c0a7-4c76-9d8c-4d380f6f199a","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-08T15:38:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-01 13:17:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6734606","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6734606","identity":"rs-6734606","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}