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

Significance Hydrogen peroxide is a ubiquitous reactive oxygen species (ROS) with diverse signaling and stress contributions, but its transient and mobile nature makes it challenging to study in living systems. Here we report a tandem activity-based sensing and labeling strategy for capture and permanent recording of localized H 2 O 2 fluxes by fluorescence imaging. Application of this technology enables direct visualization of ROS transport in cell-to-cell communication using a microglia–neuron coculture model to monitor cell-specific elevations in H 2 O 2 levels. In addition to revealing a fundamental contribution of ROS to transcellular signaling, this work presages further opportunities to combine dual chemical sensing and labeling approaches to probe biology with improved spatial fidelity.