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Hydrogen peroxide (H 2 O 2 ) is important in the regulation of a variety of biological processes and is involved in various diseases. Quantitative measurement of H 2 O 2  levels at the subcellular level is important for understanding its positive and negative effects on biological processes. Herein, a two‐photon ratiometric fluorescent probe (SHP‐Cyto) with a boronate‐based carbamate leaving group as the H 2 O 2  reactive trigger and 6‐(benzo[ d ]thiazol‐2′‐yl)‐2‐( N , N ‐dimethylamino) naphthalene (BTDAN) as the fluorophore was synthesized and examined for its ability to detect cytosolic H 2 O 2  in situ. This probe, based on the specific reaction between boronate and H 2 O 2 , displayed a fluorescent color change (455 to 528 nm) in response to H 2 O 2  in the presence of diverse reactive oxygen species in a physiological medium. In addition, ratiometric two‐photon microscopy (TPM) images with SHP‐Cyto revealed that H 2 O 2  levels gradually increased from brain to kidney, skin, heart, lung, and then liver tissues. SHP‐Cyto was successfully applied to the imaging of endogenously produced cytosolic H 2 O 2  levels in live cells and various rat organs by using TPM.

A Two‐Photon Ratiometric Fluorescent Probe for Imaging of Hydrogen Peroxide Levels in Rat Organ Tissues