Chip-Based 3D Interferometric Nanoscopy

Chip-Based 3D Interferometric Nanoscopy | 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 Chip-Based 3D Interferometric Nanoscopy Pakorn Kanchanawong, Wei Wang, Hangfeng Li, Yilin Wang, Samuel Barnett, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6693948/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 Ultra-high resolution 3D single-molecule localization microscopy (SMLM) traditionally requires complex dual-objective lenses (4Pi) configurations to enhance axial (z) precision through interferometry. Here we present a streamlined chip-based alternative, Silicon-assisted interferometric Localization Microscopy (SiLM), which achieves comparable performance using a single-objective lens design. By combining tunable axial structured illumination field, arising from surface-generated excitation interference, with asynchronous interferometry, SiLM enhances axial localization precision to approximately twice that of the lateral (xy), comparable to 4Pi-based methods. Additionally, SiLM provides intrinsic axial self-referencing, offering dramatically improved robustness against mechanical drift. Our method is readily implementable on standard SMLM-capable microscopes and supports a broad range of applications including dual-color imaging, extended-depth imaging, and live-cell 3D single-molecule tracking. Using SiLM, we demonstrate accurate mapping of the stratified nanoscale architecture of integrin-based focal adhesions, establishing it as a powerful and accessible method for high-precision 3D structural cell biology. Biological sciences/Biotechnology/Nanobiotechnology/Nanostructures Physical sciences/Nanoscience and technology/Techniques and instrumentation/Imaging techniques Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Movie1.gif Raw SiLM dataset SupplementaryMaterials20250518.docx Supplementary Notes, Supplementary Figures, and Supplementary Tables 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-6693948","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":462300337,"identity":"620081bd-81b2-4755-9bf1-037bbf0d0cd1","order_by":0,"name":"Pakorn Kanchanawong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACA2YGZgaGCghHggQtZ0jSwgDUwthGihZzdubDxoXz7iTOb2A+eJuHoc6ugZAWy2a25OSZ254lbjjAlmzNw3A4maAWg8M8xod5tx1O3MDAYybNw3AgmaDDDA7zfz7MO+cw0GH834Ba6ojRwsOczNtwOLHhAA8bUAuzHUEtQL8YG884dth4w2E2Y8s5BocTCGox5z/8WLqg5rDs/PbmhzfeVNTZE9QCAswI0oAhsYF4LVBAnC2jYBSMglEwogAAufA2uHFVEWcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8765-3861","institution":"National University of Singapore","correspondingAuthor":true,"prefix":"","firstName":"Pakorn","middleName":"","lastName":"Kanchanawong","suffix":""},{"id":462300338,"identity":"45ba979f-f156-48ad-8e91-edff2a866e12","order_by":1,"name":"Wei Wang","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":462300339,"identity":"9b35423f-39b4-4992-b6fc-3244245825e0","order_by":2,"name":"Hangfeng Li","email":"","orcid":"https://orcid.org/0000-0001-9644-5052","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Hangfeng","middleName":"","lastName":"Li","suffix":""},{"id":462300340,"identity":"c1cd494b-94f3-4ebf-924b-2311834501e9","order_by":3,"name":"Yilin Wang","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Yilin","middleName":"","lastName":"Wang","suffix":""},{"id":462300341,"identity":"842c7587-4f03-4743-aec0-1ad32e0df61f","order_by":4,"name":"Samuel Barnett","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Barnett","suffix":""},{"id":462300342,"identity":"fbfdaffd-0b2e-4eec-9629-67a0d84a8bf1","order_by":5,"name":"Zengxin Huang","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Zengxin","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-05-19 00:30:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6693948/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6693948/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83548010,"identity":"21b2d73c-5910-451f-b085-7fc35e321f0a","added_by":"auto","created_at":"2025-05-28 09:34:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1236566,"visible":true,"origin":"","legend":"","description":"","filename":"prefinalNNano202505flat.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6693948/v1_covered_c965c0ac-5d14-433e-9b50-33435e53c885.pdf"},{"id":83546878,"identity":"da898254-be1b-4fef-a1f2-990997190557","added_by":"auto","created_at":"2025-05-28 09:18:16","extension":"gif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16179739,"visible":true,"origin":"","legend":"Raw SiLM dataset","description":"","filename":"Movie1.gif","url":"https://assets-eu.researchsquare.com/files/rs-6693948/v1/16eb438319ab46a499a6d329.gif"},{"id":83547875,"identity":"341bf748-77db-4352-9087-eed5505df744","added_by":"auto","created_at":"2025-05-28 09:26:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22259500,"visible":true,"origin":"","legend":"Supplementary Notes, Supplementary Figures, and Supplementary Tables","description":"","filename":"SupplementaryMaterials20250518.docx","url":"https://assets-eu.researchsquare.com/files/rs-6693948/v1/9ae15a09ced7e3e0e8cccaad.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Chip-Based 3D Interferometric Nanoscopy","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":"","lastPublishedDoi":"10.21203/rs.3.rs-6693948/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6693948/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUltra-high resolution 3D single-molecule localization microscopy (SMLM) traditionally requires complex dual-objective lenses (4Pi) configurations to enhance axial (z) precision through interferometry. Here we present a streamlined chip-based alternative, Silicon-assisted interferometric Localization Microscopy (SiLM), which achieves comparable performance using a single-objective lens design. By combining tunable axial structured illumination field, arising from surface-generated excitation interference, with asynchronous interferometry, SiLM enhances axial localization precision to approximately twice that of the lateral (xy), comparable to 4Pi-based methods. Additionally, SiLM provides intrinsic axial self-referencing, offering dramatically improved robustness against mechanical drift. Our method is readily implementable on standard SMLM-capable microscopes and supports a broad range of applications including dual-color imaging, extended-depth imaging, and live-cell 3D single-molecule tracking. Using SiLM, we demonstrate accurate mapping of the stratified nanoscale architecture of integrin-based focal adhesions, establishing it as a powerful and accessible method for high-precision 3D structural cell biology.\u003c/p\u003e","manuscriptTitle":"Chip-Based 3D Interferometric Nanoscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-28 09:02:10","doi":"10.21203/rs.3.rs-6693948/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-nanotechnology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nnano","sideBox":"Learn more about [Nature Nanotechnology](http://www.nature.com/nnano/)","snPcode":"","submissionUrl":"","title":"Nature Nanotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f0133cdf-e4fd-462a-b6d7-2085199d8de3","owner":[],"postedDate":"May 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49086791,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanostructures"},{"id":49086792,"name":"Physical sciences/Nanoscience and technology/Techniques and instrumentation/Imaging techniques"}],"tags":[],"updatedAt":"2025-07-09T11:55:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-28 09:02:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6693948","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6693948","identity":"rs-6693948","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}