Mitochondria-ActivatedWash-Free Fluorescent Probefor Visualizing Single-Cell Photodamage

Cell viability assessment is essential in biological and medical research. There is a growing demand for fluorescent probes that can rapidly, reliably, and wash-free evaluate cell viability in complex scenarios. Electron transport chain (ETC) is one of the earliest indicators of cellular distress and holds great potential for assessing cell viability. In this study, we introduce <b>Rhodalive</b>, a mitochondria-targeted ETC-activated fluorescence turn-on probe for single-cell viability assessment. <b>Rhodalive</b> is specifically activated by free electrons leaked from the ETC and localized to the mitochondria, enabling wash-free, spatiotemporal, and super-resolution fluorescence imaging of active mitochondria. Moreover, <b>Rhodalive</b> can effectively distinguish live cells from fixed cells, quantify H₂O₂-induced cell damage, and visualize single-cell photodamage induced by localized blue light exposure and photodynamic therapy. <b>Rhodalive</b> provides a convenient and reliable tool for dynamically assessing single-cell viability with promising applications in evaluating early mitochondrial dysfunction and advancing drug discovery.