A highly sensitive NIR-II ratiometric photoacoustic probe activated rapidly by nitric oxide for accurate in vivo imaging of liver injury

Nitric oxide (NO) plays key roles as a signaling molecule and an inflammatory mediator in the pathophysiology of diseases. Real-time, precise visualization of NO dynamics in deep tissues is essential for understanding disease mechanisms and evaluating therapeutic efficacy of drugs. However, existing NO probes, particularly those operating in the first near-infrared (NIR-I) window or single-wavelength “turn-on”, have limitations such as shallow penetration depth, tissue scattering, slow response and low sensitivity. To address these limitations, we used an electron cloud density enhancement strategy to develop NO-activated second near-infrared (NIR-II) window ratiometric photoacoustic (PA) probe (TCF-BA) by replacing the cationic heptamethine cyanine scaffold with an anionic counterpart. This anion-modulated strategy significantly increases electron cloud density, thereby improving the reactivity and sensitivity toward NO. The TCF-BA probe exhibits a significant ratiometric PA response upon NO activation, enabling high-contrast, deep-tissue imaging. Leveraging the high spatiotemporal resolution of PA imaging, the TCF-BA probe was used to quantitatively monitor NO fluctuations in a mouse model of liver injury. Overall, this study provides a reliable molecular imaging tool to investigate NO-related pathological processes and offers a promising strategy for the design of activatable NIR-II ratiometric probes.