Excitation-encoded single-emission shortwave infrared lanthanide fluorophore palette for real-time in vivo multispectral imaging

Multiplexed fluorescence imaging provides valuable biological insights from the cellular to the tissue level but remains limited in live-mammal studies by the lack of a fluorescent palette capable of overcoming photon scattering and autofluorescence noise for real-time, multiplexed in vivo imaging. Here we present a fluorophore palette engineered from erbium(III)-phthalocyanine complexes, termed the lanthanide rainbow (Lanbow), which offers tunable near-infrared absorption and a unified 1,530 nm emission with brightness surpassing that of existing molecular dyes. Lanbow uses excitation-encoded and efficient single-band detection in the 1,500–1,900 nm shortwave infrared subregion, where tissue scattering and autofluorescence are minimized, enabling up to nine-colour imaging in deep tissues. We also demonstrate fluorescence-guided surgery featuring multiparametric anatomical identification and functional assessment, with deep-learning networks automating real-time analysis for intraoperative guidance. This study establishes a transformative platform for real-time, highly multiplexed imaging in live mammals.