High-Speed Wide-Field Fluorescence Lifetime Imaging for Intraoperative Tumor Visualization and In Vivo Multiplexing

High-Speed Wide-Field Fluorescence Lifetime Imaging for Intraoperative Tumor Visualization and In Vivo Multiplexing | 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 Research Article High-Speed Wide-Field Fluorescence Lifetime Imaging for Intraoperative Tumor Visualization and In Vivo Multiplexing Murali Krishnamoorthy, Rahul Pal, Steven Hou, Hak Soo Choi, Brian J. Bacskai, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6579067/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 We present a technique for fast wide-field fluorescence lifetime (FLT) imaging using simultaneous steady-state and time- (or frequency-) domain measurements acquired in a single shot, and exploiting the theoretical dependence of fluorescence intensity on FLT. Using theory, simulations and in-vitro experiments, we show that the single-shot FLT (S-FLT) method can provide absolute FLTs at a higher signal-to-noise ratio compared to other high speed FLT imaging techniques, without the need for sample-dependent system calibration or model training. We demonstrate that S-FLT can provide accurate tumor delineation in real-time during surgical resections in preclinical tumor models in vivo and in clinical patient specimens ex vivo . We also extend S-FLT for real-time multiplexing and demonstrate this technique for quantifying dynamic FLT changes in vitro and for multiplexing of organ targeted NIR fluorophores in-vivo . Our results suggest that S-FLT can enable real-time quantitative high throughput studies in preclinical applications and accurate tumor margin delineation in real-time during surgeries. Biomedical Engineering Full Text Additional Declarations The authors declare no competing interests. Supplementary Files VideoV1.mp4 VideoV2.mp4 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. 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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-6579067","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451148355,"identity":"94c3d1c9-495e-4129-8079-9948144cf048","order_by":0,"name":"Murali Krishnamoorthy","email":"","orcid":"","institution":"Department of Otolaryngology – Head and Neck Surgery, Massachusetts Eye and Ear Hospital, Harvard Medical School, Boston, MA, USA","correspondingAuthor":false,"prefix":"","firstName":"Murali","middleName":"","lastName":"Krishnamoorthy","suffix":""},{"id":451148356,"identity":"c8e9f156-72e6-4f85-94bb-5408a0a0bc65","order_by":1,"name":"Rahul Pal","email":"","orcid":"","institution":"Department of Otolaryngology – Head and Neck Surgery, Massachusetts Eye and Ear Hospital, Harvard Medical School, Boston, MA, USA","correspondingAuthor":false,"prefix":"","firstName":"Rahul","middleName":"","lastName":"Pal","suffix":""},{"id":451149110,"identity":"24c15d85-eb42-4af7-8f8f-65e6feb835aa","order_by":2,"name":"Steven Hou","email":"","orcid":"","institution":"Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Hou","suffix":""},{"id":451149111,"identity":"eb083b82-88a4-4454-9881-05eadd31157f","order_by":3,"name":"Hak Soo Choi","email":"","orcid":"","institution":"Gordon Center for Medical Imaging, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA","correspondingAuthor":false,"prefix":"","firstName":"Hak","middleName":"Soo","lastName":"Choi","suffix":""},{"id":451149112,"identity":"a6baf31d-7e2f-403e-bb8d-7f9534c5802c","order_by":4,"name":"Brian J. 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