Deep tissue imaging in the second near-infrared (NIR-II) window holds great promise for physiological studies and biomedical applications1,2,3,4,5,6. However, inhomogeneous signal attenuation in biological matter7,8 hampers the application of multiple-wavelength NIR-II probes to multiplexed imaging. Here, we present lanthanide-doped NIR-II nanoparticles with engineered luminescence lifetimes for in vivo quantitative imaging using time-domain multiplexing. To achieve this, we have devised a systematic approach based on controlled energy relay that creates a tunable lifetime range spanning three orders of magnitude with a single emission band. We consistently resolve selected lifetimes from the NIR-II nanoparticle probes at depths of up to 8 mm in biological tissues, where the signal-to-noise ratio derived from intensity measurements drops below 1.5. We demonstrate that robust lifetime coding is independent of tissue penetration depth, and we apply in vivo multiplexing to identify tumour subtypes in living mice. Our results correlate well with standard ex vivo immunohistochemistry assays, suggesting that luminescence lifetime imaging could be used as a minimally invasive approach for disease diagnosis.

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This work was supported by the National Key R&D Program of China (2017YFA0207303), the National Natural Science Fund for Distinguished Young Scholars (21725502), the Key Basic Research Program of Science and Technology Commission of Shanghai Municipality (17JC1400100), the China Postdoctoral Science Foundation (KLH1615151), the Australian Research Council Discovery Early Career Researcher Award (DE170100821) and the Centre of Excellence for Nanoscale BioPhotonics (CE140100003).

Author information


  1. Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and iChem, Fudan University, Shanghai, China

    • Yong Fan
    • , Peiyuan Wang
    • , Rui Wang
    • , Lei Zhou
    • , Xiaomin Li
    •  & Fan Zhang
  2. ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Department of Physics and Astronomy, Macquarie University, Sydney, New South Wales, Australia

    • Yiqing Lu
    • , Xianlin Zheng
    •  & James A. Piper


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F.Z., Y.F. and Y.L. designed the project. Y.F. and R.W. synthesized the nanoparticles. Y.L., Y.F. and X.Z. built the optical system. P.W. and L.Z. conducted the animal experiments. Y.F. was primarily responsible for data collection. Y.F., Y.L. and F.Z. analysed the results, and prepared the manuscript, figures and Supplementary Information. All authors contributed to the discussion and editing of the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Yiqing Lu or Fan Zhang.

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  1. Supplementary Information

    Supplementary Discussion, Supplementary Methods, Supplementary Tables 1–9, Supplementary Figures 1–22, Supplementary References

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