Spatiotemporal analysis of reactive oxygen species using specific activity-based sensing fluorescent imaging probes.

Oxidative stress, driven by reactive oxygen species (ROS), is implicated in numerous diseases. ROS are vital for redox signalling and homeostasis but can damage proteins, lipids and nucleic acids, compromising cellular health and contributing to disease progression. ROS functions are governed by their spatiotemporal distribution within cells and organisms; however, their mechanisms of action remain insufficiently understood, highlighting the compelling need for accurate ROS detection methods. In this context, fluorescence imaging has emerged as a powerful tool for real-time ROS detection. Effective fluorescence probes for ROS feature high specificity and sensitivity, enable imaging within subcellular organelles, support dynamic and reversible imaging and/or allow simultaneous detection of multiple molecules. Despite many advances, ROS imaging remains challenging. This Review provides guidelines for developing reliable fluorescent probes for ROS detection in cells and in vivo, using representative examples of activity-based sensing to aid researchers in improving ROS monitoring to advance research across biology and medicine.