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

Authors: Mario Del Rosario, Estibaliz Gómez-de-Mariscal, Leonor Morgado, Raquel Portela, Guillaume Jacquemet, Pedro M. Pereira, Ricardo Henriques
Technologies: CARE, DL4MicEverywhere, NanoJ-SRRF, PhotoFiTT and ZeroCostDL4Mic
Preprint published in bioRxiv, July 2024
Publisher: Cold Spring Harbor Laboratory

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 0.6J/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.