Deciphering the role of mtH₂O₂ in hepatic ischemia-reperfusion injury mechanisms with low-background super-resolution fluorogenic probe

Hepatic ischemia-reperfusion injury (HIRI) is characterized by mitochondrial dysfunction and oxidative stress. Monitoring mitochondrial hydrogen peroxide (mtH₂O₂) levels in real-time through super-resolution imaging is crucial for elucidating its distribution in live cells and its mechanism of action during HIRI. However, low-background fluorogenic probes have been overlooked in the context of super-resolution imaging. In this study, we developed a low-background fluorogenic probe (Mito-WG) with the potential for super-resolution morphology-correlated mitochondrial identification to track the fluctuates of mtH₂O₂ in HIRI. Activation of the desirable fluorescence properties of the probe by mtH₂O₂ was confirmed using structural illumination microscopy (SIM), enabling high-quality mitochondrial imaging with exceptional specificity and sensitivity. Fluctuations in mtH₂O₂ levels were successfully observed in both cellular and rat models of HIRI. Furthermore, we associated the decline in mitochondrial redox homeostasis with accelerated mtH₂O₂ production during HIRI, which triggered mitophagy deficiency and led to cell death. In conclusion, Mito-WG possesses excellent photophysical and low-background properties for SIM imaging, making it a promising tool for mtH₂O₂ tracking in HIRI research and clinical diagnosis.