Project Summary Reactive oxygen species (ROS) are a family of small-molecules in living systems that serve vital roles in both signaling and stress. Hydrogen peroxide (H2O2), superoxide (O2•-), and hypochlorous acid (HOCl), among others, are all examples of ROS that have been traditionally viewed as sources of oxidative stress and damage. Aberrant ROS production contributes to a multitude of pathologies such as neurodegeneration, cancer, and cardiovascular disorders. However, ROS are also critical for maintaining metabolic homeostasis through activation of multiple classes of proteins. This signal-stress dichotomy, coupled with the small and transient nature of ROS, presents a challenge when attempting to decode the complex landscape of cellular redox homeostasis. Fluorescent probes are frequently employed to visualize ROS in living systems through fluorescence microscopy, however these probes are prone to diffusion after ROS detection. This leads to inaccurate determination of ROS localization and poor signal-to-noise responses. As such, there is a need to create probes amenable to the permanent recording of ROS via fluorescence imaging. We hypothesize that activity-based cell-trappable fluorescent probes can be used as a platform to gain further understanding of ROS-mediated inter- and intra- cellular signaling. We propose three specific aims to test this hypothesis. First, we will synthesize fluorophores caged with activity-based triggers and proximal fluoromethyl groups to serve as latent equivalents of quinone methide upon ROS sensing. ROS responsive uncaging will allow for the fluorescent labeling of adjacent biomolecules. Second, we will apply our probes across multiple model live cell lines to monitor ROS fluxes. We will also map cell-to-cell communication mediated by ROS using microglia-neuron co-culture as a biological model. This system will allow us to probe transcellular redox signaling as microglia can be selectively activated in the presence of neurons thereby dispatching H2O2 to nearby neurons. The third aim involves developing a fluorescent polymer amplification strategy to increase signal-to-noise responses of tandem activity-based sensing/labeling probes and will primarily be carried out in the R00 phase. Small- molecule polymer initiators will be caged in a similar manner to the previously described fluorescent probes. After ROS sensing and biomolecule labeling, polymerization will be performed to generate fluorescent polymers from biomolecule surfaces thus enabling signal amplification and visualization. This strategy will be carried over into live cell lines described above. This research fits into the applicant’s goal of establishing a program which uses polymer chemistry to probe fundamental questions in biological systems.