Speed-Up Phase Resolved Fluorescence Lifetime Imaging Microscopy (SUPER-FLIM) for Real-Time Microenvironmental Sensing

Speed-Up Phase Resolved Fluorescence Lifetime Imaging Microscopy (SUPER-FLIM) for Real-Time Microenvironmental Sensing | 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 Speed-Up Phase Resolved Fluorescence Lifetime Imaging Microscopy (SUPER-FLIM) for Real-Time Microenvironmental Sensing Thomas Kellerer, Lukas Moser, Judith A. Müllerer, Joachim O. Rädler, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6724982/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 Imaging cell biological processes while simultaneously sensing the microenvironment at high spatial and temporal resolution is a key goal in modern live-cell microscopy. However, adding parameters such as fluorescence lifetime compromises acquisition speed. To address this challenge, we introduce S peed- U p P has E R esolved F luorescence L ifetime I maging M icroscopy ( SUPER-FLIM ), an ultra-fast fluorescence lifetime imaging method that enables 3D live-imaging at unprecedented speed. Our approach combines digital lock-in detection with laser scanning excitation, allowing the simultaneous capture of fluorescence intensity, lifetime, and phasor data in real-time. With an acquisition time of 100 ns, SUPER-FLIM is ten times faster than state of the art FLIM techniques and is, for the first time, integrated with resonant galvo-scanners. SUPER-FLIM is a major step forward in live imaging as it provides high spatial resolution in 3D that cannot be achieved with camera-based frequency domain FLIM. As a proof of concept, we demonstrate its utility in observing lipid-nanoparticle acidification within living cells. Beyond imaging, SUPER-FLIM’s rapid lifetime acquisition unlocks new applications, including multi-lifetime fluorescence correlation spectroscopy (ml-FCS), a novel FCS mode that sorts and correlates fluorescence bursts from diffusing particles based on lifetime characteristics. The technique is compatible with virtually any modulated light source and can be easily integrated into any laser scanning microscope using our open-source software. Biophysics Drug Delivery Photonics/optics Fluorescence Lifetime Imaging Microscopy Fluorescence Correlation Spectroscopy Two-Photon Excitation Resonant Galvo Scanner Lipid Nanoparticles Endosomes Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SUPERFLIMSupplementary.pdf SUPER-FLIM Supplementary Document SM1RhodamineB.mp4 SM1 - Rhodamine B SM2CellTimelaps.mp4 SM2 - Cell Timelaps SM3BeadDiffusion.mp4 SM3 - Bead Diffusion 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-6724982","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":460443475,"identity":"8cf0b77b-17b3-4f54-9456-372a6d0f1bc6","order_by":0,"name":"Thomas Kellerer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACCQhlwMDO3ABiyBmASB48OnjgWpgZwVqMSdeSuIGQFnvp5qMbfvxhMOZnZmx8XFFxL3272AHGB2/b8NgicyztZm8bg5lkM2Oz4Zkzxbk7ZycwG87Fp0Uix+wGbwODjcFhxjbJxraE3A23E9ikeQloufnnD4ON/WHG9p+N/xLSDW4nsP8mpOU2DxuDmQEzYxtjY0NCAlALGzNeLTfS0m7LtkkYSxxmbJZsOJZguOF2YrPknHO4tbDPSD52880fG8P+9uaDHxtqEuQNbicf/PCmDLcWKJBA5kAiaBSMglEwCkYBBQAAi95OvnZbleYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1660-1738","institution":"Multiphoton Imaging Lab, Munich University of Applied Sciences, 80335 Munich, Germany","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Kellerer","suffix":""},{"id":460444521,"identity":"851bcf24-ce8f-4803-b80d-9100759118c6","order_by":1,"name":"Lukas Moser","email":"","orcid":"","institution":"Multiphoton Imaging Lab, Munich University of Applied Sciences, 80335 Munich, Germany","correspondingAuthor":false,"prefix":"","firstName":"Lukas","middleName":"","lastName":"Moser","suffix":""},{"id":460444522,"identity":"7278ca8f-adac-4a52-87e8-47cfbecfc906","order_by":2,"name":"Judith A. 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