Rational construction of a reversible arylazo-based NIR probe for cycling hypoxia imaging in vivo

Reversible NIR luminescent probes with negligible photocytotoxicity are required for long-term tracking of cycling hypoxia in vivo. However, almost all of the reported organic fluorescent hypoxia probes reported until now were irreversible. Here we report a reversible arylazo-conjugated fluorescent probe (HDSF) for cycling hypoxia imaging. HDSF displays an off-on fluorescence switch at 705 nm in normoxia-hypoxia cycles. Mass spectroscopic and theoretical studies confirm that the reversible sensing behavior is attributed to the two electron-withdrawing trifluoromethyl groups, which stabilizes the reduction intermediate phenylhydrazine and blocks the further reductive decomposition. Cycling hypoxia monitoring in cells and zebrafish embryos is realized by HDSF using confocal imaging. Moreover, hypoxic solid tumors are visualized and the ischemia-reperfusion process in mice is monitored in real-time. This work provides an effective strategy to construct organic fluorescent probes for cycling hypoxia imaging and paves the way for the study of cycling hypoxia biology.