HBT-Fl-BnB is a fluorescent probe used for the detection of ONOO- both in vitro and in vivo. It consists of an HBT core with Fl groups at the ortho and para positions, which responds to the zwitterionic excited-state intramolecular proton transfer (zwitterionic ESIPT) process. The boronic pinacol ester serves a dual function, blocking zwitterionic ESIPT and identifying ONOO-.

HBT-Fl-BnB solutions at varying concentrations (0, 5, 10, 15, 20, 30, 50 μM) are added to Raw264.7 cells in a cell culture incubator. After 24 hours of incubation, cells treated with HBT-Fl-BnB are imaged using a confocal microscope with dual channels: the first channel (430ch) λ em = 415−515 nm, and the second channel (583ch) λ ex = 405 nm, λ em = 550-650 nm.

Eight-week-old female C57BL/6 mice were selected. Thirty minutes prior to imaging, HBT-Fl-BnB (50 μM; 200 μL) was administered via tail vein injection. The mice were subsequently imaged using the PerkinElmer IVIS Lumina XR and the Fluorescence Navigator 360I systems. Imaging of HBT-Fl-BnB-treated mice was conducted using a small animal in vivo dual-channel imaging system: the first channel (430ch) with λ em = 415-515 nm, and the second channel (583ch) with λ ex = 405 nm and λ em = 550-650 nm.