An Activatable Ratiometric Photoacoustic/Near-Infrared-II Fluorescence Dual-Modal Probe for in vivo Imaging of pH

Tumor acidity is a key driver of cancer progression and a promising target for early diagnosis and therapy. However, existing approaches remain limited in their ability to sensitively and specifically visualize and quantify tumor acidity in vivo. Here, we report a pH-sensitive dual-modal molecular probe, 5-HP-IR, that integrates ratiometric photoacoustic (PA) and near-infrared-II (NIR-II) fluorescence (FL) imaging within a single small-molecule scaffold. The probe consists of an NIR-II organic dye as the optical core and a piperazine molecule as a pH-responsive switch. Under acidic pH, protonation of the piperazine results in enhanced PA signals at 1064 nm and reduced PA signals at 680 nm, yielding a 34.5-fold increase in the PA ratio (PA 1064 /PA 680 ) from pH 7.4 to 3.0 and enabling imaging of tumor-associated acidity in vivo. Concurrently, NIR-II FL at 1066 nm is activated 174-fold from pH 8.0 to 3.0, providing high-resolution in vivo imaging. Together, the simultaneous activation mechanisms of the two imaging modalities afford built-in cross-validation, substantially improving diagnostic reliability. Unlike complex nanoparticle-based systems for pH imaging that often rely on coencapsulated internal reference and responsive molecules for ratiometric detection and suffer from molecular leakage and limited turn-on ratio, this probe features a well-defined structure, straightforward synthesis, and eliminates the need for an internal reference, thereby enhancing detection sensitivity. By synergizing ratiometric PA quantification with NIR-II FL visualization, this work establishes a molecular platform for noninvasive and real-time imaging of tumor-associated acidity, offering broad implications for tumor microenvironment characterization and precision cancer diagnosis.