Two-beam Interference Microscopy: high-resolution multi-scale light-sheet imaging from subcellular structures to whole organs

Two-beam Interference Microscopy: high-resolution multi-scale light-sheet imaging from subcellular structures to whole organs | 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 Two-beam Interference Microscopy: high-resolution multi-scale light-sheet imaging from subcellular structures to whole organs Peng Fei, Yao Zhou, Xinyi Guo, Shiqi Mao, Jianchao Liu, Meng Zhang, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4531286/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 Despite offering rapid and gentle 3D imaging of biological samples, the revolution by light-sheet fluorescence microscopy faces challenges due to optical constraints and limited geometry flexibility, hindering its widespread application. Here we present Two-beam Interference Microscopy (TIM), which sweeps the interference pattern of two symmetric laser sheets to achieve horizontal plane illumination and orthogonal wide-field detection for the first time using a single objective. The geometry-unconstrained TIM yields adaptive optical sectioning of samples with various sizes and mounting approaches. It enables high-speed (over 150 megavoxels/s), adaptive-resolution (~200 nm to ~2500 nm), nanoscale-to-mesoscale 3D imaging ranging from single organelles within a live cell (×100), to a whole mouse brain containing tens of millions of neurons (×4 tile stitching). TIM exhibits remarkably high versatility while achieving superior spatiotemporal resolution. With feedback-controlled imaging strategies, intelligent TIM demonstrates in situ trans-scale visualization from entire brain slices to single dendritic spines, and efficient event-triggered imaging of CAR-T/tumor cells’ cytotoxic interactions. Biological sciences/Biological techniques/Microscopy/Light-sheet microscopy Biological sciences/Biological techniques/Imaging/Fluorescence imaging Biological sciences/Cell biology/Organelles Full Text Additional Declarations There is NO Competing Interest. Supplementary Files TIMSI.pdf SupplementaryVideo1.mp4 Supplementary Video 1 SupplementaryVideo2.mp4 Supplementary Video 2 SupplementaryVideo3.mp4 Supplementary Video 3 SupplementaryVideo4.mp4 Supplementary Video 4 SupplementaryVideo5.mp4 Supplementary Video 5 SupplementaryVideo6.mp4 Supplementary Video 6 SupplementaryVideo7.mp4 Supplementary Video 7 SupplementaryVideo8.mp4 Supplementary Video 8 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. 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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-4531286","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":312703379,"identity":"bf3adfb0-6e45-4531-bc2c-b0583dc82337","order_by":0,"name":"Peng 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