PhotoFiTT: A Quantitative Framework for Assessing Phototoxicity in Live-Cell Microscopy Experiments

PhotoFiTT: A Quantitative Framework for Assessing Phototoxicity in Live-Cell Microscopy Experiments | 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 Brief Communication PhotoFiTT: A Quantitative Framework for Assessing Phototoxicity in Live-Cell Microscopy Experiments Ricardo Henriques, Mario del Rosario, Estibaliz Gómez-de-Mariscal, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4809905/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Phototoxicity in live-cell fluorescence microscopy can compromise experimental outcomes, yet quantitative methods to assess its impact remain limited. Here we present PhotoFiTT (Phototoxicity Fitness Time Trial), an integrated framework combining a standardised experimental protocol with advanced image analysis to quantify light-induced cellular stress in label-free settings. PhotoFiTT leverages machine learning and cell cycle dynamics to analyse mitotic timing, cell size changes, and overall cellular activity in response to controlled light exposure. Using adherent mammalian cells, we demonstrate PhotoFiTT's ability to detect wavelength- and dose-dependent effects, showcasing that near-UV light induces significant mitotic delays at doses as low as 0.6 J/cm 2 , while longer wavelengths require higher doses for comparable effects. PhotoFiTT enables researchers to establish quantitative benchmarks for acceptable levels of photodamage, facilitating the optimisation of imaging protocols that balance image quality with sample health. Biological sciences/Cell biology Biological sciences/Biological techniques/Microscopy phototoxicity microscopy machine learning cell division cellular activity Full Text Additional Declarations There is NO Competing Interest. Supplementary Files supplementary.pdf SupplementaryVideo1.mp4 Supplementary Video 1 SupplementaryVideo2.mp4 Supplementary Video 2 SupplementaryVideo3.mov Supplementary Video 3 Cite Share Download PDF Status: Published Journal Publication published 13 Dec, 2025 Read the published version in Nature Communications → 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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