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In vivo ultrasound-induced luminescence imaging via trianthracene derivatives nanomaterials

Nature Protocols (2025)Cite this article

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Abstract

Photoluminescence imaging is valuable for elucidating biological processes and diagnosing diseases, but its tissue penetration is limited. We developed an imaging technique that utilizes ultrasound to activate the piezoelectric effect of a molecular probe, transforming ultrasound energy into chemical energy. The chemical energy is then converted into light emission through the chemiluminescence effect, improving penetration depth and overcoming traditional photoluminescence imaging constraints. Here we describe how to build two kinds of ultrasound-induced luminescence imaging systems. We introduce a procedure for the synthesis of trianthracene derivative (TD) nanoparticles with ultrasound-induced luminescence properties. The TDs are converted into water-soluble nanoparticles by a simple nanoprecipitation method. Utilizing the constructed ultrasound-induced luminescence imaging systems, TD nanoparticles can be stimulated to exhibit a luminescence spectrum peaking between 625 and 650 nm. Under optimized ultrasound excitation time and excitation power density parameters, the imaging quality and tissue penetration depth are effectively enhanced. Notably, our procedure enables the detection of both subcutaneous tumor models and challenging deep-tissue orthotopic gliomas. This ultrasound-mediated approach represents an important advancement over conventional photoluminescence imaging methods, enabling high-fidelity in vivo tumor imaging with superior signal quality. Establishment of the ultrasound-induced luminescence imaging systems requires ~2 h, the synthesis of TD molecules requires ~4 d, nanoparticle preparation requires ~1 d, ex vivo characterization requires ~1 d, investigation of the ultrasound-induced luminescence of TD nanoparticles requires ~3 d and ultrasound-induced luminescence imaging takes ~1 d. These steps can be performed by operators trained in chemical synthesis, nanomaterial synthesis standards and qualified in relevant animal experiments.

Key points

  • A technique for ultrasound-induced luminescence imaging, which uses a dual-stage energy conversion mechanism to enhance the performance of luminescence.

  • Alternative methods include photoluminescence imaging such as fluorescence imaging, chemiluminescence or bioluminescence, which suffer from signal attenuation, or Cerenkov luminescence, which lacks resolution.

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Fig. 1: Delayed ultrasound-induced luminescence imaging system and Real-time ultrasound-induced luminescence imaging system.
Fig. 2: Preparation and characterization of TD nanoparticles.
Fig. 3: Investigation of the ultrasound-induced luminescence of TD nanoparticles in the delayed ultrasound-induced luminescence imaging system.
Fig. 4: Investigation of the ultrasound-induced luminescence of TD nanoparticles in the real-time ultrasound-induced luminescence imaging system.
Fig. 5: Ultrasound-induced luminescence imaging and photoluminescence imaging penetration depth test.
Fig. 6: Ultrasound-induced luminescence imaging in vivo.

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Data availability

The main data discussed in this protocol are available in the supporting primary research papers (https://doi.org/10.1038/s41566-024-01387-1 and https://doi.org/10.1038/s41551-024-01274-8). The raw datasets are provided in the Source Data file. The online version also contains a Supplementary Information PDF file. All other data are available for research purposes from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key R&D Program of China (grant no. 2019YFA0210100 (to X.-B.Z.)) and the National Natural Science Foundation of China (grant nos. U21A20287 (to G.S.) and 22234003 (to X.-B.Z.)).

Author information

Author notes

  1. These authors contributed equally: Xinyu Xu, Youjuan Wang.

Authors and Affiliations

  1. State Key Laboratory of Chemo and Biosensing, College of Chemistry and Chemical Engineering, Hunan University, Changsha, China

    Xinyu Xu, Youjuan Wang, Zhe Li, Xiao-Bing Zhang & Guosheng Song

Authors
  1. Xinyu Xu
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Contributions

G.S. and X.-B.Z. designed the study. X.X., Y.W. and Z.L. performed experiments, collected and analyzed data and wrote the manuscript.

Corresponding authors

Correspondence to Xiao-Bing Zhang or Guosheng Song.

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Nature Protocols thanks Roman A. Barmin, Wenfeng Xia and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Wang, Y. et al. Nat. Photon. 18, 334–343 (2024): https://doi.org/10.1038/s41566-024-01387-1

Wang, Y. et al. Nat. Biomed. Eng. 9, 656–670 (2025): https://doi.org/10.1038/s41551-024-01274-8

Wang, Y. et al. J. Am. Chem. Soc. 146, 6252–6265 (2024): https://doi.org/10.1021/jacs.3c14387

Supplementary information

Supplementary Information

Supplementary Figs. 1–14, Table 1 and Procedure.

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Source data

Source Data Fig. 2

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Source Data Fig. 3

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Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

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Xu, X., Wang, Y., Li, Z. et al. In vivo ultrasound-induced luminescence imaging via trianthracene derivatives nanomaterials. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01246-5

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