Highly Doped Tm³⁺ Nanoparticles with Efficient 1632 nm Emission Enable High-Fidelity Multiplexing In Vivo Bioimaging

Optical imaging serves as a powerful tool for real-time visualization of biological processes, yet high-fidelity multiplexed and quantitative imaging in deep tissue remains challenging. In the second near-infrared (NIR-II, 1000-2000 nm) window, especially the long-wavelength subregion (NIR-II-L, 1500-1900 nm) with minimized photon scattering and an enhanced signal-to-noise ratio, multiplexed luminescence probes are highly desirable for dynamic imaging and quantitative sensing. Herein, we report the first dual-excitation (690 and 785 nm) ratiometric Tm³⁺ nanoparticles (TmNPs) with enhanced emission at 1632 nm via Yb³⁺ doping. This design enables robust ratiometric quantification with minimal coefficients of variation (<10%), allowing real-time blood oxygen quantification in ischemia and reperfusion. Moreover, it facilitates wide-field multiplexed vasculature and lymphatic imaging without filter switching or refocusing, achieving consistent spatial resolution across channels. This work provides a novel probe platform for high-fidelity dynamic sensing and multiplexing in the NIR-II-L window, advancing preclinical diagnostics and biomedical research.