Two-Photon Cellular and Intravital Imaging of Hypochlorous Acid by Fluorescent Probes That Exhibit a Synergistic Excited-State Intramolecular Proton Transfer-Intramolecular Charge Transfer Mechanism Enabling Near-Infrared Emission with a Large Stokes Shift.

To develop highly effective molecular tools for intravital imaging of hypochlorous acid (HOCl), in this study, we initially designed two-photon hybrid fluorophores, and , by conjugating the classical dye 2-(2'-hydroxyphenyl)benzothiazole with the two-photon hydroxylphenyl-butadienylpyridinium fluorophore. The designed fluorophores exhibit a synergistic interaction between excited-state intramolecular proton transfer and intramolecular charge transfer mechanisms, enabling near-infrared (NIR) emission and significant Stokes shifts. Subsequently, using these fluorophores, we developed two HOCl fluorescent probes, and , by further incorporating ,-dimethylthiocarbamate as a specific recognition group for HOCl. Toward HOCl, both and demonstrate an ultrafast response (less than 3 s), NIR emission at wavelengths of 714 and 682 nm, and remarkable Stokes shifts of 303 and 271 nm, respectively. Leveraging these advantages in conjunction with their ability to cross the blood-brain barrier, the probes find successful application in two-photon cellular and intravital imaging of HOCl. This includes visualizing endogenous generation of HOCl in cellular models related to inflammation, hyperglycemia, and ferroptosis, as well as mapping generation of HOCl within the brain and abdominal cavity using a murine model of systemic inflammation.