Photocontrollable Fluorescence Imaging of Mitochondrial Peroxynitrite during Ferroptosis with High Fidelity

Ferroptosis, a new regulatory cell death modality, underlies the pathogenesis of a broad range of disorders. Although much efforts have been made to uncover the molecular mechanisms, some mechanistic details of ferroptosis still remain poorly understood. Particularly, the functional relevance of mitochondrial reactive oxygen species (ROS) in ferroptosis is still highly controversial, which is partially due to the fact that it still remains puzzled how the mitochondrial ROS level varies during ferroptosis. The conventional mitochondria-targeted probes may react with cytosolic ROS and show fluorescence variation before entering mitochondria, thus probably giving a false result on the mitochondrial ROS level and leading to the misjudgment on its biofunction. To circumvent this issue, we rationally designed a photocontrollable and mitochondria-targeted fluorescent probe to in situ visualize the mitochondrial peroxynitrite (ONOO^-), which is the ROS member and mediator of ferroptosis. The photoactivated probe was endowed with a highly specific and sensitive fluorescence response to ONOO^-. Notably, the response activity could be artificially regulated with light irradiation, which ensured that all the probe molecules passed through the cytosol in the locked status and were then photoactivated after reaching mitochondria. This photocontrolled fluorescence imaging strategy eliminated the interference of ONOO^- outside the mitochondria, thus potentially afforded improved fidelity for mitochondrial ONOO^- bioimaging in live cells and animal models. With this probe, for the first time, we revealed the mitochondrial ONOO^- flux and its probable biological source during erastin-induced ferroptosis. These results suggest a tight correlation between mitochondrial ONOO^-/ROS and ferroptotic progression, which will further facilitate the comprehensive exploration and manipulation of ferroptosis.