Fig. 1: Phototoxicity fitness time trial (PhotoFiTT) assay.

PhotoFiTT integrates a experimental imaging assay with an image analysis workflow, using cellular processes and mitotic cycles as natural timers to track consistent patterns of cell behaviour. It detects deviations in these patterns caused by light exposure, enabling the quantification of phototoxic effects in fluorescence microscopy. This is only the case where a dependence on light dose (or irradiance) has been demonstrated. The patterns can also be caused by other factors (temperature, media, cumulative damage, etc.) and thus, comparison against a control is also recommended. The workflow analyses three cellular features: a Mitosis: Identifies cell rounding (yellow squares), typically 30–50 min post-G2 exit. Phototoxicity alters rounding time distributions (orange: normal, blue: photodamaged). b Size: Tracks cell diameters (black arrowheads) through division. Mother cells (purple) transition to daughter cells (orange) ~60 min post-G2 exit. Phototoxicity delays this transition (light orange). c Activity: Quantifies observable cellular changes between frames (red outlines). Here cumulative cell activity represents the sum of activity in time. Normal cells show increased post-division activity (orange, around 60 min), while photodamaged cells (blue) exhibit reduced activity.