Fluorescence illumination can cause phototoxicity that negatively affects living samples. This study demonstrates that much of the phototoxicity and photobleaching experienced with live-cell fluorescence imaging occurs as a result of 'illumination overhead' (IO). This occurs when a sample is illuminated but fluorescence emission is not being captured by the microscope camera. Several technological advancements have been developed, including fast-switching LED lamps and transistor-transistor logic (TTL) circuits, to diminish phototoxicity caused by IO. These advancements are not standard features on most microscopes and many biologists are unaware of their necessity for live-cell imaging. IO is particularly problematic when imaging rapid processes that require short exposure times. This study presents a workflow to optimize imaging conditions for measuring both slow and dynamic processes while minimizing phototoxicity on any standard microscope. The workflow includes a guide on how to (1) determine the maximum image exposure time for a dynamic process, (2) optimize excitation light intensity and (3) assess cell health with mitochondrial markers.This article has an associated First Person interview with the first author of the paper.

中文翻译：

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优化活细胞荧光成像条件以尽量减少光毒性。

荧光照明会导致光毒性，对活样品产生负面影响。这项研究表明，活细胞荧光成像所经历的大部分光毒性和光漂白都是由“照明开销”(IO) 造成的。当样品被照亮但荧光发射未被显微镜相机捕获时，就会发生这种情况。已经开发出多项技术进步，包括快速开关 LED 灯和晶体管-晶体管逻辑 (TTL) 电路，以减少 IO 引起的光毒性。这些进步并不是大多数显微镜的标准功能，许多生物学家没有意识到它们对于活细胞成像的必要性。当对需要短曝光时间的快速过程进行成像时，IO 的问题尤其严重。这项研究提出了一个工作流程，可优化成像条件，以测量缓慢和动态过程，同时最大限度地减少任何标准显微镜上的光毒性。该工作流程包括关于如何 (1) 确定动态过程的最大图像曝光时间、(2) 优化激发光强度以及 (3) 使用线粒体标记评估细胞健康状况的指南。本文有相关的第一人称采访论文第一作者。