Zoomable dual-channel NIR-I/II fluorescence mesoscopy for in vivo characterization

In vivo fluorescence imaging is constrained by the detector-limited incompatibility between NIR-I (high-resolution superficial imaging) and NIR-II (deep-tissue penetration) windows, alongside an inherent tradeoff between centimeter-scale whole-organ field of view and micrometer-scale spatial resolution. Therefore, there is an urgent need for an in vivo imaging system capable of balancing field of view and resolution, while synergistically integrating the complementary advantages of different NIR imaging windows. This study aims to develop a zoomable dual-channel NIR-I/II fluorescence mesoscopic imaging system (ZD-FMI) that bridges macroscopic and microscopic scales via seamless zooming, and to validate its utility for comprehensive hepatobiliary disease diagnosis using a cholestatic liver injury model. We constructed a ZD-FMI system integrating coaxial NIR-I and NIR-II channels with an adjustable field of view. System performance was evaluated using resolution targets and vessel phantoms. In a mouse model of cholestatic liver injury, we performed cross-scale imaging using IR780 (a hepatobiliary-metabolized probe) and ICG (a biliary-excreted tracer) to acquire macroscopic quantification, microstructural morphology, and metabolic functional data. The ZD-FMI system enabled seamless switching from wide-field organ localization (centimeter scale) to high-resolution inspection (micrometer scale) without spatial misregistration. In cholestatic liver injury models, the system concurrently provided macroscopic fluorescence quantification, microstructural details of liver and bile ducts, and hepatobiliary metabolic functional assessment, offering complementary multi-perspective diagnostic information. These results demonstrate that the ZD-FMI system effectively overcomes the scale-resolution and band-specific limitations of conventional fluorescence imaging. The integrated coaxial design ensures cross-scale spatial matching. Its validated performance in cholestatic liver injury diagnosis, together with drift-free zoom capability, supports its substantial translational potential for surgical navigation and optical biopsy beyond hepatobiliary applications.