PhotoFiTT: a quantitative framework for assessing phototoxicity in live-cell microscopy experiments
Year: 2025
Type: article
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.
Source: Nature Communications
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,990
Funders: European Molecular Biology Organization, European Commission, Academy of Finland, Syöpäjärjestöt, Åbo Akademi, Fundación Bancaria Caixa d'Estalvis i Pensions de Barcelona, HORIZON EUROPE Framework Programme, Fundação para a Ciência e a Tecnologia, European Regional Development Fund, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa
Awards: G118516010, G1255325603, G1332930224, G1500452600, G1516346500, G1763632427, Junior Leader la Caixa Postdoctoral Fellowship Programme: Shaping the new generation of leaders in research, G198227700, G2195151728, G2427613718, InFLAMES: Innovation Ecosystem based on the Immune System, G274581676, G2792792013, G2921923652, G2964751290, G2994560175, G3309579038, G3340144160, G3415473929, InFLAMES: Innovation Ecosystem based on the Immune System, G3660205263, G3840324753, G4046065984, G434337789, G4439699107, G4504718640, G4516981482, G4724859719, G473357141, G4783475982, G4941345466, G5065677113, G5132640529, G5224927334, Investigação dos mecanismos subjacentes à interacção da imunidade autónoma com a divisão e persistência intracelular de Staphylococcus aureus., Ciências da Vida para um Futuro Saudável e Sustentável, G5677667711, G5955501538, Microbiologia Molecular, Estrutural e Celular - – Instituto de Tecnologia Química e Biológica António Xavier, G6361605312, G6376227556, InFLAMES: Innovation Ecosystem based on the Immune System, G6997658380, Artificial Intelligence for Image Data Analysis in the Life Sciences, G7315488542, G7426977871, G7533282544, G7685134749, G7731791180, G77786263, PERSISTAUREUS - Unravelling the mechanisms of Staphylococcus aureus antibiotic induced persistence during intracellular host-cell infection., Microbiologia Molecular, Estrutural e Celular - – Instituto de Tecnologia Química e Biológica António Xavier, G7965899483, Filopodia-mediated communication in cancer progression (FILOCOM), InFLAMES: Innovation Ecosystem based on the Immune System, G841604482, G862925997, G886374
OpenAlex ID: W4417321450