A tandem activity-based sensing and labeling strategy enables imaging of transcellular hydrogen peroxide signaling

Reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂) are transient species that have broad actions in signaling and stress, but spatioanatomical understanding of their biology remains insufficient. Here, we report a tandem activity-based sensing and labeling strategy for H₂O₂ imaging that enables capture and permanent recording of localized H₂O₂ fluxes. Peroxy Green-1 Fluoromethyl (PG1-FM) is a diffusible small-molecule probe that senses H₂O₂ by a boronate oxidation reaction to trigger dual release and covalent labeling of a fluorescent product, thus preserving spatial information on local H₂O₂ changes. This unique reagent enables visualization of transcellular redox signaling in a microglia-neuron coculture cell model, where selective activation of microglia for ROS production increases H₂O₂ in nearby neurons. In addition to identifying ROS-mediated cell-to-cell communication, this work provides a starting point for the design of chemical probes that can achieve high spatial fidelity by combining activity-based sensing and labeling strategies.