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Noninvasive in vivo microscopy of single neutrophils in the mouse brain via NIR-II fluorescent nanomaterials

Nature Protocols (2024)Cite this article

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Abstract

In vivo microscopy of single cells enables following pathological changes in tissues, revealing signaling networks and cell interactions critical to disease progression. However, conventional intravital microscopy at visible and near-infrared wavelengths <900 nm (NIR-I) suffers from attenuation and is typically performed following the surgical creation of an imaging window. Such surgical procedures cause the alteration of the local vasculature and induce inflammation in skin, muscle and skull, inevitably altering the microenvironment in the imaging area. Here, we detail the use of near-infrared fluorescence (NIR-II, 1,000–1,700 nm) for in vivo microscopy to circumvent attenuation in living tissues. This approach enables the noninvasive visualization of cell migration in deep tissues by labeling specific cells with NIR-II lanthanide downshifting nanoparticles exhibiting high physicochemical stability and photostability. We further developed a NIR-II fluorescence microscopy setup for in vivo imaging through the intact skull with high spatiotemporal resolution, which we use for the real-time dynamic visualization of single-neutrophil behavior in the deep brain of a mouse model of ischemic stroke. The labeled downshifting nanoparticle synthesis takes 5–6 d, the imaging system setup takes 1–2 h, the in vivo cell labeling takes 1–3 h, the in vivo NIR-II microscopic imaging takes 3–5 h and the data analysis takes 3–8 h. The procedures can be performed by users with standard laboratory training in nanomaterials research and appropriate animal handling.

Key points

  • Near-infrared fluorescence (NIR-II, 1,000–1,700 nm) microscopy enables noninvasive visualization of neutrophil dynamics in vivo, demonstrated by visualizing neutrophil recruitment to inflamed tissue, through the intact skull.

  • The procedure covers preparation of lanthanide downshifting nanoparticles, the custom-built NIR-II microscopic imaging setup, induction of acute inflammation models in mice, in vivo labeling of neutrophils and real-time image acquisition of single-cell dynamics.

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Fig. 1: Workflow of in vivo NIR-II fluorescence microscopic imaging in this protocol.
Fig. 2: NIR-II fluorescent DSNPs preparation.
Fig. 3: The custom-built NIR-II microscopy system.
Fig. 4: In vivo imaging of neutrophils in the inflammatory vessel.
Fig. 5: In vivo imaging of neutrophils chemotaxis in the inflammatory vessel.
Fig. 6: In vivo imaging of cell extravasation.
Fig. 7: In vivo imaging of activated neutrophils in the inflamed vessels.

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Data availability

The authors declare that the main data discussed in this protocol are available in the supporting primary research papers (https://doi.org/10.1038/s41565-023-01422-2 and https://doi.org/10.1038/s41563-021-01063-7). The raw datasets are too large to be publicly shared but are available for research purposes from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by the National Key R&D Program of China（2023YFB3507100), National Natural Science Foundation of China (grant nos. 22088101, 21725502, 51961145403), New Cornerstone Science Foundation through the XPLORER PRIZE and the Research Program of Science, Innovation Program of Shanghai Municipal Education Commission and the Research Program of Science and Technology Commission of Shanghai Municipality (grant nos. 20JC1411700, 21142201000, 22JC1400400).

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  1. These authors contributed equally: Ying Chen, Yiwei Yang.

Authors and Affiliations

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

    Ying Chen, Yiwei Yang & Fan Zhang

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Contributions

F.Z. and Y.C., conceived and initiated the project. Y.C. and Y.Y. contributed to the experimental work shown in this protocol. Y.C., Y.Y. and F.Z. wrote the protocol. F.Z. supervised the study and the manuscript preparation. All authors reviewed and edited the manuscript and approved the final draft.

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Correspondence to Fan Zhang.

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Nature Protocols thanks Xiaoyuan Chen, Dan Ding and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol:

Yang, Y. et al. Nat. Nanotechnol. 18, 1195–1204 (2023): https://doi.org/10.1038/s41565-023-01422-2

Wang, T. et al. Nat. Mater. 20, 1571–1578, (2021): https://doi.org/10.1038/s41563-021-01063-7

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Chen, Y., Yang, Y. & Zhang, F. Noninvasive in vivo microscopy of single neutrophils in the mouse brain via NIR-II fluorescent nanomaterials. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00983-3

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