A Tandem Activity‐Based Sensing and Labeling Strategy Enables Imaging of Transcellular Hydrogen Peroxide Signaling

Hydrogen Peroxide (H 2 O 2 ) is one of many reactive oxygen species (ROS) that plays vital roles in intracellular signaling and stress processes, but its transient nature has made deciphering its roles in transcellular signaling a significant challenge. We have designed boronate caged small‐molecule probes bearing a fluoromethylene pendant group that serves as a latent quinone methide source that can undergo a tandem activity‐based sensing and biomolecule labeling reaction cascade to produce a spatially localized fluorescence signal upon exposure to H 2 O 2 . This strategy enables us to capture and record spatial information on ROS fluxes and has enabled us to move beyond studying intracellular H 2 O 2 signaling to identifying and mapping cell‐to‐cell H 2 O 2 communication in a microglia‐neuron co‐culture cell model, where selective activation of microglia for ROS production increases H 2 O 2 in nearby neurons.