Monitoring in real time and far-red imaging of H₂O₂ dynamics with subcellular resolution

Monitoring H₂O₂ dynamics in conjunction with key biological interactants is critical for elucidating the physiological outcome of cellular redox regulation. Optogenetic hydrogen peroxide sensor with HaloTag with JF635 (oROS-HT₆₃₅) allows fast and sensitive chemigenetic far-red H₂O₂ imaging while overcoming drawbacks of existing red fluorescent H₂O₂ indicators, including oxygen dependency, high pH sensitivity, photoartifacts and intracellular aggregation. The compatibility of oROS-HT₆₃₅ with blue-green-shifted optical tools allows versatile optogenetic dissection of redox biology. In addition, targeted expression of oROS-HT₆₃₅ and multiplexed H₂O₂ imaging enables spatially resolved imaging of H₂O₂ targeting the plasma membrane and neighboring cells. Here we present multiplexed use cases of oROS-HT₆₃₅ with other green fluorescence reporters by capturing acute and real-time changes in H₂O₂ with intracellular redox potential and Ca²⁺ levels in response to auranofin, an inhibitor of antioxidative enzymes, via dual-color imaging. oROS-HT₆₃₅ enables detailed insights into intricate intracellular and intercellular H₂O₂ dynamics, along with their interactants, through spatially resolved, far-red H₂O₂ imaging in real time.