ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058 Basel, Switzerland and
ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058 Basel, Switzerland and.
G3 (Bethesda). 2020 Dec 3;10(12):4373-4385. doi: 10.1534/g3.120.401465.
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.
使用多个荧光报告基因对活细胞进行延时成像，是研究单细胞内分子过程的重要工具。然而，即使是中等剂量的可见光激发光的照射，也会干扰细胞生理学并改变所研究细胞的定量行为。在这里，我们着手制定指南，以避免多色长期成像中激发光的混杂影响。我们使用宽场荧光显微镜来测量在酵母中施加的激发光对生长速率（这里称为光病变）的影响。我们发现光病变取决于每个波长的累积光剂量，但与激发光的施加方式无关。重要的是，光病变具有一个可检测到无影响的阈值光剂量（NOEL）。我们发现，荧光蛋白在各自的激发光 NOEL 下用于活细胞成像的适用性同样由细胞自发荧光和荧光蛋白亮度决定。最后，我们表明，多个波长的光病变是可加的，并且可以预测无光病变的成像条件。我们的发现使研究人员能够找到对生理学影响最小的成像条件，并为如何在其他生物体中处理光病变提供了框架。
