Compressive Fourier-Domain Intensity Coupling (C-FOCUS) enables near-millimeter deep imaging in the intact mouse brain in vivo

Compressive Fourier-Domain Intensity Coupling (C-FOCUS) enables near-millimeter deep imaging in the intact mouse brain in vivo | 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 Compressive Fourier-Domain Intensity Coupling (C-FOCUS) enables near-millimeter deep imaging in the intact mouse brain in vivo Yi Xue, Renzhi He, Yucheng Li, Brianna Urbina, Jiandi Wan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7104566/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Two-photon microscopy is a powerful tool for in vivo imaging, but its imaging depth is typically limited to a few hundred microns due to tissue scattering, even with existing scattering correction techniques. Moreover, most active scattering correction methods are restricted to small regions by the optical memory effect. Here, we introduce compressive Fourier-domain intensity coupling for scattering correction (C-FOCUS), an active scattering correction approach that integrates Fourier-domain intensity modulation with compressive sensing for two-photon microscopy. Using C-FOCUS, we demonstrate high-resolution imaging of YFP labeled neurons and FITC-labeled blood vessels at depths exceeding 900 µm in the intact mouse brain in vivo. Furthermore, we achieve transcranial imaging of YFP-labeled dendritic structures through the intact adult mouse skull. C-FOCUS enables high-contrast fluorescence imaging at depths previously inaccessible using two-photon microscopy with 1035 nm excitation, enhancing fluorescence intensity by over 20-fold compared to uncorrected imaging. C-FOCUS provides a broadly applicable strategy for rapid, deep-tissue optical imaging in vivo. Biological sciences/Biological techniques/Microscopy/Multiphoton microscopy Biological sciences/Biological techniques/Imaging/Fluorescence imaging Two-photon microscopy Active scattering correction Compressive sensing Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryVideoFigure1dcompareThy1YFPHS1RGB.avi Figure 1d Video 3D stack Comparison SupplementaryVideoFigure1dwithcorrectionThy1YFPHS1.mp4 Figure 1d Video volume view with correction SupplementaryVideoFigure3ewithcorrectionThy1YFPH.mp4 Figure 3e Video volume view with correction SupplementaryVideoFigure3ewithoutcorrectionThy1YFPH.mp4 Figure 3e Video volume view without correction SupplementaryVideoFigure3ecompareThy1YFPHRGB.avi Figure 3e Video 3D stack Comparison SupplementaryVideoFigure4awithcorrectionFITCWT.mp4 Figure 4a Video volume view with correction SupplementaryVideoFigure4awithcorrectionFITCWTRGB.avi Figure 4a Video 3D stack with correction SupplementaryVideoFigure5awithoutcorrectionThy1YFPHwithskull.mp4 Figure 5a Video volume view without correction SupplementaryVideoFigure5awithcorrectionThy1YFPHwithskull.mp4 Figure 5a Video volume view with correction SupplementaryVideoFigure5acompareThy1YFPHwithskullRGB.avi Figure 5a Video 3D stack Comparison CFOCUSSI.pdf Cite Share Download PDF Status: Under Review 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-7104566","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":490859580,"identity":"c85f9330-ab97-42c3-b829-6f0030f3f79d","order_by":0,"name":"Yi Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYNCCCiA+AEYMjA3EaTlDshbGNogWBqK0GNzIMZP4OO+wPN/xM4YHfzDYyG44QEjLmWNpkjO3HTaceSbH4DAPQ5oxYS3Hm49J8247nGBwIC3hMAPD4UTCWg4ztkn/nQPUcv5ZAtBh/4nQArKFsQGo5UbygQM8DAcIa5E8cyzZsudYuuHMG48PHOYxSDaeSUgL340cwxs/aqzl+c4nNn/8UWEn20dIiwKqAgMCykFAvoEIRaNgFIyCUTDCAQC4ck29gRio3wAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2622-083X","institution":"University of California, Davis","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Xue","suffix":""},{"id":490859581,"identity":"5b8b0388-89e6-4957-ab03-f9d1ded3c8eb","order_by":1,"name":"Renzhi He","email":"","orcid":"https://orcid.org/0000-0003-1837-4062","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Renzhi","middleName":"","lastName":"He","suffix":""},{"id":490859582,"identity":"3b4a1e7f-93b5-48ff-b613-385f0a88e3e3","order_by":2,"name":"Yucheng Li","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Yucheng","middleName":"","lastName":"Li","suffix":""},{"id":490859583,"identity":"5ca0faaf-6e61-443f-9414-4d7804a9acb0","order_by":3,"name":"Brianna Urbina","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Brianna","middleName":"","lastName":"Urbina","suffix":""},{"id":490859584,"identity":"21ad7407-9b7b-4ced-aeb8-1b45db7bc627","order_by":4,"name":"Jiandi Wan","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Jiandi","middleName":"","lastName":"Wan","suffix":""}],"badges":[],"createdAt":"2025-07-11 21:25:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7104566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7104566/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87693917,"identity":"eabc3039-6ac4-41a9-94e2-83bca5342cfc","added_by":"auto","created_at":"2025-07-28 05:37:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5261473,"visible":true,"origin":"","legend":"","description":"","filename":"CFOCUS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1_covered_f8190e04-aad5-4a9f-a0ff-24f02e0dbeea.pdf"},{"id":87692659,"identity":"da92467e-e9fb-409e-bc8b-5157fd1d9a9d","added_by":"auto","created_at":"2025-07-28 05:13:31","extension":"avi","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36252912,"visible":true,"origin":"","legend":"Figure 1d Video 3D stack Comparison","description":"","filename":"SupplementaryVideoFigure1dcompareThy1YFPHS1RGB.avi","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/7f532cc58eb78625bc285b58.avi"},{"id":87692656,"identity":"1cfc3298-c076-41d8-aa1a-9fb508f21d0a","added_by":"auto","created_at":"2025-07-28 05:13:30","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5510221,"visible":true,"origin":"","legend":"Figure 1d Video volume view with correction","description":"","filename":"SupplementaryVideoFigure1dwithcorrectionThy1YFPHS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/08a417f4339a4e3f1396915e.mp4"},{"id":87692655,"identity":"ce13551b-ff43-48c3-bd96-caabf6ff1a84","added_by":"auto","created_at":"2025-07-28 05:13:30","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9060797,"visible":true,"origin":"","legend":"Figure 3e Video volume view with correction","description":"","filename":"SupplementaryVideoFigure3ewithcorrectionThy1YFPH.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/b4ac6012244317785993b778.mp4"},{"id":87692663,"identity":"4fe09129-16ba-4fa8-94fc-c7905537890d","added_by":"auto","created_at":"2025-07-28 05:13:31","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12703661,"visible":true,"origin":"","legend":"Figure 3e Video volume view without correction","description":"","filename":"SupplementaryVideoFigure3ewithoutcorrectionThy1YFPH.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/7241c67d990f52118628f257.mp4"},{"id":87692665,"identity":"12d9d43c-8a6f-491d-87b1-594ee9242275","added_by":"auto","created_at":"2025-07-28 05:13:31","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":45293724,"visible":true,"origin":"","legend":"Figure 3e Video 3D stack Comparison","description":"","filename":"SupplementaryVideoFigure3ecompareThy1YFPHRGB.avi","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/3d476e75f6d18e34457b4f70.avi"},{"id":87692666,"identity":"aa6688a7-1e2a-49cf-8dad-b0ba48fce3d5","added_by":"auto","created_at":"2025-07-28 05:13:31","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17632127,"visible":true,"origin":"","legend":"Figure 4a Video volume view with correction","description":"","filename":"SupplementaryVideoFigure4awithcorrectionFITCWT.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/6b840b622894d6d1977ea177.mp4"},{"id":87692661,"identity":"5c4e72a3-c68c-4d10-91e5-54b37b756c20","added_by":"auto","created_at":"2025-07-28 05:13:31","extension":"avi","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":20497668,"visible":true,"origin":"","legend":"Figure 4a Video 3D stack with correction","description":"","filename":"SupplementaryVideoFigure4awithcorrectionFITCWTRGB.avi","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/2f4c4808362b7f2be4eac757.avi"},{"id":87692660,"identity":"fd902722-b00b-4950-956a-6447f5b57310","added_by":"auto","created_at":"2025-07-28 05:13:31","extension":"mp4","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":6454220,"visible":true,"origin":"","legend":"Figure 5a Video volume view without correction","description":"","filename":"SupplementaryVideoFigure5awithoutcorrectionThy1YFPHwithskull.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/c976040346b06782df5011f4.mp4"},{"id":87692664,"identity":"45e2d264-8447-4dd2-9b36-cd0e3ca75c57","added_by":"auto","created_at":"2025-07-28 05:13:31","extension":"mp4","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":3819704,"visible":true,"origin":"","legend":"Figure 5a Video volume view with correction","description":"","filename":"SupplementaryVideoFigure5awithcorrectionThy1YFPHwithskull.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/6b10c4ffa6e05c47434cd4a9.mp4"},{"id":87692667,"identity":"401d62d4-3441-4a4e-a50f-8abae42f53c9","added_by":"auto","created_at":"2025-07-28 05:13:32","extension":"avi","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":31856148,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 5a Video 3D stack Comparison\u003c/p\u003e","description":"","filename":"SupplementaryVideoFigure5acompareThy1YFPHwithskullRGB.avi","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/8b4a0e114b9ddf06526aea28.avi"},{"id":87692657,"identity":"76a7a07f-2136-4bd2-96f8-60fa8fe9d9d9","added_by":"auto","created_at":"2025-07-28 05:13:31","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":492709,"visible":true,"origin":"","legend":"","description":"","filename":"CFOCUSSI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7104566/v1/8df476ad3d79c120f429f974.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Compressive Fourier-Domain Intensity Coupling (C-FOCUS) enables near-millimeter deep imaging in the intact mouse brain in vivo","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Two-photon microscopy, Active scattering correction, Compressive sensing","lastPublishedDoi":"10.21203/rs.3.rs-7104566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7104566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTwo-photon microscopy is a powerful tool for in vivo imaging, but its imaging depth is typically limited to a few hundred microns due to tissue scattering, even with existing scattering correction techniques. Moreover, most active scattering correction methods are restricted to small regions by the optical memory effect. Here, we introduce compressive Fourier-domain intensity coupling for scattering correction (C-FOCUS), an active scattering correction approach that integrates Fourier-domain intensity modulation with compressive sensing for two-photon microscopy. Using C-FOCUS, we demonstrate high-resolution imaging of YFP labeled neurons and FITC-labeled blood vessels at depths exceeding 900 µm in the intact mouse brain in vivo. Furthermore, we achieve transcranial imaging of YFP-labeled dendritic structures through the intact adult mouse skull. C-FOCUS enables high-contrast fluorescence imaging at depths previously inaccessible using two-photon microscopy with 1035 nm excitation, enhancing fluorescence intensity by over 20-fold compared to uncorrected imaging. C-FOCUS provides a broadly applicable strategy for rapid, deep-tissue optical imaging in vivo.\u003c/p\u003e","manuscriptTitle":"Compressive Fourier-Domain Intensity Coupling (C-FOCUS) enables near-millimeter deep imaging in the intact mouse brain in vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 05:13:26","doi":"10.21203/rs.3.rs-7104566/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"201f3389-fffa-4d8b-a2cc-ed8a20d9c0c2","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52122616,"name":"Biological sciences/Biological techniques/Microscopy/Multiphoton microscopy"},{"id":52122617,"name":"Biological sciences/Biological techniques/Imaging/Fluorescence imaging"}],"tags":[],"updatedAt":"2025-12-08T07:05:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 05:13:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7104566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7104566","identity":"rs-7104566","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}