Abstract
Optical imaging of tumor biomarkers provides key diagnostic information about tumor status. Light-induced afterglow (photoafterglow) imaging provides a higher signal to background ratio than typical fluorescence imaging; however, both modalities face challenges in detecting biomarkers in deep tissues owing to the limited penetration depth of light. Here we provide instructions for synthesizing ultrasound-induced afterglow (sonoafterglow) nanoprobes (SNAP) for the deep-tissue imaging of peroxynitrite (ONOO−), a biomarker specific for M1 macrophages and a proinflammatory tumor microenvironment. SNAPs are coassembled from initiators, afterglow substrates and amphiphilic polymers via the film rehydration method, a generic and facile approach that enables their rapid nanoconstruction (in 10 min), with high reproducibility, while also providing control over the nanoprobe concentration, which overcomes limitations of traditional nanoconstruction methods including solvent injection and emulsion–solvent evaporation. Following ultrasound stimulation, SNAPs emit sonoafterglow with bright near-infrared emission (peaking at 780 nm), with a long half-life (~2 min), and can be detected through biological tissues twice deeper than photoafterglow. We further develop SNAP into SNAP-M, which can be switched on only in the presence of ONOO−, allowing the real-time in vivo imaging of a proinflammatory tumor microenvironment at an unprecedented tissue depth for optical imaging. This Protocol can be implemented by users with expertise in material science in 1 week for nanoprobe construction and characterization, 1–2 week for cell assays and 3–4 weeks for animal experiments.
Key points
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The Protocol uses the film rehydration method for the formation of a thin film containing the sonoafterglow initiator, substrate and amphiphilic stabilizer, which are rehydrated to a monodispersed nanoparticle solution. The nanoparticles produced emit long-lasting luminescence following stimulation with ultrasound for the detection of deep-tissue peroxynitrite.
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Alternative methods are time-consuming and suffer from interbatch variations, whereas the film rehydration method is efficient, reproducible and applicable to various solvents.
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Data availability
All relevant data supporting the findings of this study are available within the article and its supplementary information or from the corresponding authors on reasonable request. Source data are provided with this paper.
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Acknowledgements
K.P. thanks the Singapore National Research Foundation (grant no. NRF-NRFI07-2021-0005) and the Singapore Ministry of Education Academic Research Fund Tier2 (grant nos. MOE-T2EP30220-0010 and MOE-T2EP30221-0004) for financial support.
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K.P. and C.X. conceived and developed the Protocol. C.X. conducted in vitro and in vivo assays and drafted the manuscript. J.H. conducted the synthesis. K.P. contributed to the discussion and manuscript review. All authors contributed to the writing of this manuscript.
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Key references
Xu, C. et al. Nat. Biomed. Eng. 7, 298–312 (2023): https://doi.org/10.1038/s41551-022-00978-z
Xu, C. et al. Adv. Mater. 35, e2211651 (2023): https://doi.org/10.1002/adma.202211651
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Xu, C., Huang, J. & Pu, K. Sonoafterglow nanoprobes for deep-tissue imaging of peroxynitrite. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01202-3
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DOI: https://doi.org/10.1038/s41596-025-01202-3
