PhotoFiTT: a quantitative framework for assessing phototoxicity in live-cell microscopy experiments
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<sup>2</sup>, while longer wavelengths require higher doses for comparable deleterious 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.
Authors
- Guillaume Jacquemet (ORCID: https://orcid.org/0000-0002-9286-920X)
- Leonor Morgado (ORCID: https://orcid.org/0000-0002-3760-5180)
- Mario Del Rosario (ORCID: https://orcid.org/0000-0002-0430-1463)
- Raquel Portela (ORCID: https://orcid.org/0000-0002-5559-9554)
- Estibaliz Gómez‐de‐Mariscal (ORCID: https://orcid.org/0000-0003-2082-3277)
- Pedro M. Pereira (ORCID: https://orcid.org/0000-0002-1426-9540)
- Ricardo Henriques (ORCID: https://orcid.org/0000-0002-2043-5234)
Institutions
- Åbo Akademi University (FI)
- University of Turku (FI)
- Turku Centre for Computer Science (FI)
- MRC Laboratory for Molecular Cell Biology (GB)
- University College London (GB)
- Instituto Gulbenkian de Ciência (PT)
- Universidade Nova de Lisboa (PT)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-12-13
- DOI
- https://doi.org/10.1038/s41467-025-66209-6
- Citations
- 1
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 1.63
Funders
- European Commission
- Academy of Finland
- Syöpäjärjestöt
- 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