The multispectral optoacoustic tomography (MSOT) technique can be used to perform high-resolution molecular imaging under deep tissues, which gives the technology significant prospective for clinical application. Here, we developed a superoxide anion (O2•-)-activated MSOT and fluorescence dual-modality imaging probe (APSA) for early diagnosis of drug-induced liver injury (DILI). APSA can respond quickly to O2•-, resulting in an absorption peak blueshift from 845 to 690 nm, which also leads to the photoacoustic (PA) signal at 690 nm and the fluorescence signal at 748 nm increases linearly with increasing O2•- concentration, which can be utilized to assess the extent of liver damage. The developed MSOT imaging method can eliminate background interference from hematopoietic tissue by collecting the PA signals excited at 680, 690, 740, 760, 800, 845, and 900 nm wavelengths to achieve noninvasive in situ visual diagnosis of DILI. The developed fluorescence imaging method can be used for the imaging of endogenous O2•- in living cells and anatomic diagnosis of liver injury. The developed probe has broad application prospects in the early diagnosis of DILI.

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