Lee Chia-Hung, Wallace Douglas C, Burke Peter J
Department of Biomedical Engineering, University of California, Irvine, CA, 92697, United States.
Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and Department of Pediatrics, Division of Human Genetics, University of Pennsylvania, Philadelphia, PA, 19104, United States.
Mitochondrial Commun. 2024;2:38-47. doi: 10.1016/j.mitoco.2024.03.001. Epub 2024 Mar 16.
Photobleaching and phototoxicity can induce detrimental effects on cell viability and compromise the integrity of collected data, particularly in studies utilizing super-resolution microscopes. Given the involvement of multiple factors, it is currently challenging to propose a single set of standards for assessing the potential of phototoxicity. The objective of this paper is to present empirical data on the effects of photobleaching and phototoxicity on mitochondria during super-resolution imaging of mitochondrial structure and function using Airyscan and the fluorescent structure dyes Mitotracker green (MTG), 10-N-nonyl acridine orange (NAO), and voltage dye Tetramethylrhodamine, Ethyl Ester (TMRE). We discern two related phenomena. First, phototoxicity causes a transformation of mitochondria from tubular to spherical shape, accompanied by a reduction in the number of cristae. Second, phototoxicity impacts the mitochondrial membrane potential. Through these parameters, we discovered that upon illumination, NAO is much more phototoxic to mitochondria compared to MTG or TMRE and that these parameters can be used to evaluate the relative phototoxicity of various mitochondrial dye-illumination combinations during mitochondrial imaging.
光漂白和光毒性可对细胞活力产生有害影响，并损害所收集数据的完整性，尤其是在使用超分辨率显微镜的研究中。鉴于涉及多种因素，目前提出一套单一的评估光毒性潜力的标准具有挑战性。本文的目的是提供关于在使用Airyscan以及荧光结构染料线粒体绿（MTG）、10-N-壬基吖啶橙（NAO）和电压染料四甲基罗丹明乙酯（TMRE）对线粒体结构和功能进行超分辨率成像期间，光漂白和光毒性对线粒体影响的实验数据。我们识别出两个相关现象。第一，光毒性导致线粒体从管状转变为球形，同时嵴的数量减少。第二，光毒性影响线粒体膜电位。通过这些参数，我们发现光照时，与MTG或TMRE相比，NAO对线粒体的光毒性要强得多，并且这些参数可用于评估线粒体成像期间各种线粒体染料 - 光照组合的相对光毒性。
