Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of

Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms.