Selective Lysosomal H2O2 – ROS Imaging with a Naphthalimide Probe Forming Hydroxylamine without Overoxidation

Hydrogen peroxide (H ₂ O ₂ ) plays a central role in oxidative stress, signaling, and pathophysiology, yet its highly dynamic and compartmentalized distribution in living cells makes precise monitoring a major analytical challenge. Here, we present LysoH ₂ O ₂ , a naphthalimide-derived fluorescent probe specifically designed for lysosomal H ₂ O ₂ detection and imaging. The probe exploits a selective transformation of an amine group into a hydroxylamine moiety upon reaction with H ₂ O ₂ , leading to a pronounced fluorescence enhancement. This molecular design integrates a lysosome-targeting unit to ensure subcellular specificity, enabling real-time visualization of H ₂ O ₂ fluctuations in the lysosomal microenvironment. Confocal fluorescence microscopy demonstrates the ability of LysoH ₂ O ₂ to track endogenous and exogenous H ₂ O ₂ with high sensitivity and minimal cytotoxicity in living cells. Distinct from conventional boronate-based probes, LysoH ₂ O ₂ employs a novel, biocompatible chemical transformation to achieve sensitive and selective monitoring of H ₂ O ₂ within lysosomes.