Development of ratiometric imaging techniques to quantitatively measure hypoxia levels in vivo is essential in predicting the effectiveness of cancer treatments and diagnostics. With complementary advantages of near-infrared fluorescent (NIRF) and photoacoustic (PA) imaging, NIRF/PA dual-ratiometric probes are imperative for precisely visualizing tumor hypoxia levels. However, because the absorbed excitation energy is fixed in a single chromophore, its NIRF and PA effects are always competitive, which greatly thwarts the development of NIRF/PA dual-ratiometric probes. Herein, for the first time, we describe an energy balance strategy between fluorescence and photoacoustic effects by sulfur substitution to transform existing hemicyanine dyes (Cy) into optimized NIRF/PA dual ratiometric scaffolds. Based on this optimized scaffold, we reported the first dual-ratio response of nitroreductase probe AS-Cy-NO 2 which allows quantitative visualization of tumor hypoxia in vivo . AS-Cy-NO 2 , composed of a new NIRF/PA scaffold thioxanthene-hemicyanine (AS-Cy) and a 4-nitrobenzene moiety, showed a 10-fold ratiometric NIRF enhancement (I 773 /I 733 ) and 2.4-fold ratiometric PA enhancement (PA 730 /PA 670 ) upon activation by a biomarker (nitroreductase, NTR) associated with tumor hypoxia. Moreover, the dual ratiometric NIRF/PA imaging accurately quantified hypoxia extent with high sensitivity and high imaging depth in xenograft breast cancer models. More importantly, the 3D maximal intensity projection (MIP) PA images of the probe can precisely differentiate the highly heterogeneous oxygen distribution in solid tumor. Thus, this study not only presents the first dual ratiometric NIRF and PA probe for quantitatively and precisely monitoring of tumor hypoxia levels but also provides a promising NIRF/PA scaffold that may be generalized for applying in dual ratiometric imaging of other disease-relevant biomarkers.

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