An Organelle-Orthogonal Dual-Response Probe for In Situ Monitoring of Mitochondrial H ₂ O ₂ and Lipid Droplet Polarity in Ferroptosis

Ferroptosis, an iron-dependent form of regulated cell death, is characterized by organelle dysfunction and disrupted communication due to reactive oxygen species (ROS). A major obstacle has been the lack of methods for real-time, simultaneous monitoring of key organellar parameters, which has kept the spatiotemporal role of H 2 O 2 in organelle interactions unclear. Here, we designed a dual-function near-infrared fluorescent probe, PSi-NXZD/HCy-H 2 O 2, that enables synchronous, dual-channel tracking of lipid droplet polarity and mitochondrial H 2 O 2 in living cells. Application of this probe unveiled that an H 2 O 2 burst specifically drives a decrease in lipid droplet abundance coupled with a rise in polarity during ferroptosis. Thus, our study delivers a unique imaging tool for probing organelle communication and offers pivotal spatiotemporal insights into the mechanism of ferroptosis.