All-optical spatiotemporal mapping of ROS dynamics across mitochondrial microdomains in situ

Hydrogen peroxide (H₂O₂) functions as a second messenger to signal metabolic distress through highly compartmentalized production in mitochondria. The dynamics of reactive oxygen species (ROS) generation and diffusion between mitochondrial compartments and into the cytosol govern oxidative stress responses and pathology, though these processes remain poorly understood. Here, we couple the H₂O₂ biosensor, HyPer7, with optogenetic stimulation of the ROS-generating protein KillerRed targeted into multiple mitochondrial microdomains. Single mitochondrial photogeneration of H₂O₂ demonstrates the spatiotemporal dynamics of ROS diffusion and transient hyperfusion of mitochondria due to ROS. This transient hyperfusion phenotype required mitochondrial fusion but not fission machinery. Measurement of microdomain-specific H₂O₂ diffusion kinetics reveals directionally selective diffusion through mitochondrial microdomains. All-optical generation and detection of physiologically-relevant concentrations of H₂O₂ between mitochondrial compartments provide a map of mitochondrial H₂O₂ diffusion dynamics in situ as a framework to understand the role of ROS in health and disease.