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

Lee J.D., Nguyen A, Gibbs C.E., Jin, Z.R.¹, Wang Y., Moghadasi A., Wait S.J., Choi H., Evitts K.M., Asencio A., Bremner S.B., Zuniga S., Chavan V., Pranoto I.K.A., Williams A., Smith A., Moussavi-Harami F., Regnier M., Baker D., Young J.E., Mack D., Nance E., Boyle P.M., Berndt A.* Monitoring in real time and far-red imaging of H₂O₂ dynamics with subcellular resolution. Nat. Chem. Biol. (2025). Doi: 10.1038/s41589-025-01891-7

Monitoring hydrogen peroxide (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.