PhotoFiTT: a quantitative framework for assessing phototoxicity in live-cell microscopy experiments
Nat Commun. 2025 Dec 13. doi: 10.1038/s41467-025-66209-6. Online ahead of print.
Published on December 13, 2025
Abstract
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/cm2, 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.
PMID:41390466 | DOI:10.1038/s41467-025-66209-6