Synchronized Imaging of Hydrogen Peroxide and Hydroxyl Radical in Pyroptosis and Epilepsy

ABSTRACT Real‐time visualization of oxidative stress in living systems poses a formidable challenge in epilepsy research, primarily due to the scarcity of tools capable of selectively distinguishing structurally similar reactive oxygen species (ROS) with high specificity. Here, we present a dual‐functional fluorescent probe, HH , capable of independently detecting hydrogen peroxide (H ₂ O ₂ ) and hydroxyl radicals (•OH) through distinct recognition mechanisms, yielding spectrally resolved signals with high sensitivity, selectivity, and biocompatibility. HH enables high‐resolution, interference‐free imaging of redox dynamics in both gasdermin‐mediated pyroptosis and pentylenetetrazol (PTZ)‐induced epileptic cell models. Furthermore, HH was successfully employed in PTZ‐induced zebrafish and kainic acid (KA)‐induced mouse epilepsy models, revealing elevated ROS levels under seizure‐like conditions. Notably, ROS‐targeted therapeutic interventions significantly modulated oxidative profiles, demonstrating HH ’s potential for therapeutic evaluation. This work not only fills a critical methodological gap in ROS biology and epilepsy research but also provides a versatile platform for studying redox regulation in neurological and inflammatory diseases and enabling mechanistic and therapeutic investigations.