Autophagy is a vital cytoprotective pathway against oxidative stress, and the regulatory function of reactive oxygen species (ROS) in autophagy has been thoroughly investigated. However, nitric oxide (NO), a representative reactive nitrogen species, exerts dual contradictory effects on autophagy, and the underlying reason for such discrepancy remains ambiguous. Existing studies indicate that the biological activity of NO is highly dependent on mitochondrial microenvironmental pH; the pH fluctuation during autophagy further modulates NO-mediated protein modification and downstream autophagic signaling. Nevertheless, conventional fluorescent probes can only separately detect NO or mitochondrial pH, lacking the capacity to synchronously track the two parameters in mitochondria, which severely restricts the exploration of the coupling relationship between NO and pH during autophagy. Herein, we constructed a benzindolium-functionalized mitochondria-targeted ratiometric fluorescent probe for simultaneous visualization of mitochondrial NO and pH. The probe possesses donor-acceptor structures whose spectral properties are modulated by ambient pH via protonation and deprotonation, and it only responds to pH variation in the presence of NO, guaranteeing the specificity of dual-parameter imaging. Spectral characterizations confirm the distinct ratiometric fluorescence shifts triggered by NO and pH co-stimulation. Cellular imaging experiments based on rapamycin-induced and ischemia-reperfusion-induced autophagy models demonstrate that autophagy activation is accompanied by upregulated mitochondrial NO and synchronous mitochondrial pH alternation. Distinct from single-analyte probes reported previously, this probe enables real-time synchronous ratiometric imaging of mitochondrial NO and pH in living cells. This work provides a reliable visual tool to monitor the dynamic coupling of NO and mitochondrial pH during autophagy, laying a foundation for further dissecting the disparate regulatory effects of NO on autophagy under varied pathological microenvironments.
