Orthogonal Multiplexed NIR-II Imaging with Excitation-Selective Lanthanide-Based Nanoparticles

Multiplexed imaging in the second near-infrared (NIR-II, 1000-1700 nm) window, with much reduced tissue scattering and autofluorescence background noises, could offer comprehensive information for studying biological processes and accurate diagnosis. A critical requirement for harvesting the full potential of multiplexing is to develop fluorescent probes with emission profiles specifically tuned at distinct excitations toward their target applications. However, the lack of versatile probes with separated signals in this NIR-II window hinders the potential of in vivo multiplexed imaging. In this study, we designed three types of Nd³⁺-, Ho³⁺-, and Er³⁺-based down-shifting nanoparticles (DSNPs) with core-shell structures (csNd, csHo, and csEr). Excitation wavelengths of these nanoparticles were first screened and confirmed at 730, 915, and 655 nm. Under the new excitations, orthogonal three-color emissions in the NIR-II window (1060, 1180, and 1525 nm for csNd, csHo, and csEr, respectively) were efficiently achieved. These excitation-selective DSNPs were then demonstrated to be promising in encrypted anticounterfeiting applications with increased optical codes. By programmed administration of the DSNPs, anatomical rotation imaging can also be successfully performed to differentiate mouse bones, stomach, and blood vessels with high contrast and resolution in a fixed NIR-II channel (>1000 nm) by only switching the excitation wavelengths. This study suggests that the designed NIR-II excitation-selective DSNPs with orthogonal emissions may offer a powerful framework for spatially multiplexed imaging in biological and life sciences.