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Hydrogen peroxide (H 2 O 2 ) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H 2 O 2 dynamics in live cells within a limited field of view. The visualization of H 2 O 2 within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H 2 O 2 concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with H 2 O 2 , but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from E . coli. When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (p K a  of 5.9 vs. ≥7.6) and oxidized states (p K a  of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to H 2 O 2 and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to H 2 O 2 when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to H 2 O 2 and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to H 2 O 2 upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of H 2 O 2 with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H 2 O 2 indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism.

Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide