Modern fluorescence-imaging methods promise to unveil organelle dynamics in live cells. Phototoxicity, however, has become a prevailing issue when boosted illumination applies. Mitochondria are representative organelles whose research heavily relies on optical imaging, yet these membranous hubs of bioenergy are exceptionally vulnerable to photodamage. We report that cyclooctatetraene-conjugated cyanine dyes (PK Mito dyes), are ideal mitochondrial probes with remarkably low photodynamic damage for general use in fluorescence cytometry. In contrast, the nitrobenzene conjugate of Cy3 exhibits enhanced photostability but unaffected phototoxicity compared to parental Cy3. PK Mito Red, in conjunction with Hessian-structural illumination microscopy, enables 2000-frame time-lapse imaging with clearly resolvable crista structures, revealing rich mitochondrial dynamics. In a rigorous stem cell sorting and transplantation assay, PK Mito Red maximally retains the stemness of planarian neoblasts, exhibiting excellent multifaceted biocompatibility. Resonating with the ongoing theme of reducing photodamage using optical approaches, this work advocates the evaluation and minimization of phototoxicity when developing imaging probes.

中文翻译：

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环辛酸酯与共轭的花青线粒体探针可最大程度地减少荧光和纳米成像中的光毒性

现代荧光成像方法有望揭示活细胞中的细胞器动力学。然而，当应用增强照明时，光毒性已经成为普遍的问题。线粒体是代表性的细胞器，其研究主要依赖于光学成像，但这些生物能的膜状集线器极易受到光损伤。我们报告说，环辛酸酯与共轭的花菁染料（PK水户染料）是理想的线粒体探针，具有显着较低的光动力损伤，通常用于荧光细胞计数。相反，与亲本Cy3相比，Cy3的硝基苯共轭物表现出增强的光稳定性，但未受影响的光毒性。PK Mito Red与Hessian结构照明显微镜相结合，可实现2000帧延时成像，并具有清晰可分辨的crista结构，揭示丰富的线粒体动力学。在严格的干细胞分选和移植测定中，PK Mito Red最大程度地保留了涡虫成骨细胞的干性，表现出出色的多方面生物相容性。与使用光学方法减少光损害这一持续主题相呼应，这项工作提倡在开发成像探针时评估并最大程度地降低光毒性。