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In vivo non-invasive confocal fluorescence imaging beyond 1,700 nm using superconducting nanowire single-photon detectors

Abstract

Light scattering by biological tissues sets a limit to the penetration depth of high-resolution optical microscopy imaging of live mammals in vivo. An effective approach to reduce light scattering and increase imaging depth is to extend the excitation and emission wavelengths to the second near-infrared window (NIR-II) at >1,000 nm, also called the short-wavelength infrared window. Here we show biocompatible core–shell lead sulfide/cadmium sulfide quantum dots emitting at ~1,880 nm and superconducting nanowire single-photon detectors for single-photon detection up to 2,000 nm, enabling a one-photon excitation fluorescence imaging window in the 1,700–2,000 nm (NIR-IIc) range with 1,650 nm excitation—the longest one-photon excitation and emission for in vivo mouse imaging so far. Confocal fluorescence imaging in NIR-IIc reached an imaging depth of ~1,100 μm through an intact mouse head, and enabled non-invasive cellular-resolution imaging in the inguinal lymph nodes of mice without any surgery. We achieve in vivo molecular imaging of high endothelial venules with diameters as small as ~6.6 μm, as well as CD169 + macrophages and CD3 + T cells in the lymph nodes, opening the possibility of non-invasive intravital imaging of immune trafficking in lymph nodes at the single-cell/vessel-level longitudinally.

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Fig. 1: Lead sulfide QDs and superconducting nanowire single-photon detectors enabling fluorescence imaging beyond 1,700 nm.
Fig. 2: Fluorescence imaging in NIR-IIb and NIR-IIc windows.
Fig. 3: Non-invasive in vivo confocal microscopy of intact mouse head in NIR-IIc window.
Fig. 4: Non-invasive in vivo NIR-II confocal microscopy of mouse iLNs.

Data availability

Source data are provided with this paper. All data that support the findings of this study are presented in the main text and the Supplementary Information. Source Data are provided with this paper.

References

  1. Horton, N. G. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nat. Photon. 7, 205–209 (2013).

    CAS  Article  Google Scholar 

  2. Yildirim, M., Sugihara, H., So, P. T. C. & Sur, M. Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy. Nat. Commun. 10, 177 (2019).

    Article  Google Scholar 

  3. Kobat, D., Horton, N. & Xu, C. In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. J. Biomed. Opt. 16, 106014 (2011).

    Article  Google Scholar 

  4. Miller, M. J., Wei, S. H., Parker, I. & Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).

    CAS  Article  Google Scholar 

  5. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

    CAS  Article  Google Scholar 

  6. Svoboda, K. & Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50, 823–839 (2006).

    CAS  Article  Google Scholar 

  7. Horton, N. G. & Xu, C. Dispersion compensation in three-photon fluorescence microscopy at 1,700 nm. Biomed. Opt. Exp. 6, 1392–1397 (2015).

    Article  Google Scholar 

  8. Wang, T. et al. Three-photon imaging of mouse brain structure and function through the intact skull. Nat. Methods 15, 789–792 (2018).

    Article  Google Scholar 

  9. Yang, Q. et al. Donor engineering for NIR-II molecular fluorophores with enhanced fluorescent performance. J. Am. Chem. Soc. 140, 1715–1724 (2018).

    CAS  Article  Google Scholar 

  10. Li, Y. et al. Design of AIEgens for near-infrared IIb imaging through structural modulation at molecular and morphological levels. Nat. Commun. 11, 1255 (2020).

    Article  Google Scholar 

  11. Welsher, K. et al. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nat. Nanotechnol. 4, 773–780 (2009).

    CAS  Article  Google Scholar 

  12. Zhang, M. et al. Bright quantum dots emitting at 1,600 nm in the NIR-IIb window for deep tissue fluorescence imaging. Proc. Natl Acad. Sci. USA 115, 6590–6595 (2018).

    CAS  Article  Google Scholar 

  13. Bruns, O. T. et al. Next-generation in vivo optical imaging with short-wave infrared quantum dots. Nat. Biomed. Eng. 1, 0056 (2017).

    CAS  Article  Google Scholar 

  14. Zhong, Y. et al. In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles. Nat. Biotechnol. 37, 1322–1331 (2019).

    CAS  Article  Google Scholar 

  15. Fan, Y. et al. Lifetime-engineered NIR-II nanoparticles unlock multiplexed in vivo imaging. Nat. Nanotechnol. 13, 941–946 (2018).

    CAS  Article  Google Scholar 

  16. Naczynski, D. J. et al. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nat. Commun. 4, 2199 (2013).

    CAS  Article  Google Scholar 

  17. Zhu, S. et al. 3D NIR-II molecular imaging distinguishes targeted organs with high-performance NIR-II bioconjugates. Adv. Mater. 30, 1705799 (2018).

    Article  Google Scholar 

  18. Wang, F. et al. Light-sheet microscopy in the near-infrared II window. Nat. Methods 16, 545–552 (2019).

    Article  Google Scholar 

  19. Wang, F. et al. In vivo NIR-II structured-illumination light-sheet microscopy. Proc. Natl Acad. Sci. USA 118, e2023888118 (2021).

    CAS  Article  Google Scholar 

  20. Wan, H. et al. A bright organic NIR-II nanofluorophore for three-dimensional imaging into biological tissues. Nat. Commun. 9, 1–9 (2018).

    Article  Google Scholar 

  21. Golovynskyi, S. et al. Optical windows for head tissues in near-infrared and short-wave infrared regions: approaching transcranial light applications. J. Biophoton. 11, e201800141 (2018).

    Article  Google Scholar 

  22. Hong, G. et al. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nat. Photon. 8, 723–730 (2014).

    CAS  Article  Google Scholar 

  23. Burrows, P. E. et al. Lymphatic abnormalities are associated with RASA1 gene mutations in mouse and man. Proc. Natl Acad. Sci. USA 110, 8621–8626 (2013).

    CAS  Article  Google Scholar 

  24. Diao, S. et al. Fluorescence imaging in vivo at wavelengths beyond 1,500 nm. Angew. Chem. Int. Ed. 54, 14758–14762 (2015).

    CAS  Article  Google Scholar 

  25. Pasko, J., Shin, S. & Cheung, D. Epitaxial HgCdTe/CdTe Photodiodes For The 1 to 3 pm Spectral Region 0282 TSE (SPIE, 1981).

  26. Ren, F., Zhao, H., Vetrone, F. & Ma, D. Microwave-assisted cation exchange toward synthesis of near-infrared emitting PbS/CdS core/shell quantum dots with significantly improved quantum yields through a uniform growth path. Nanoscale 5, 7800–7804 (2013).

    CAS  Article  Google Scholar 

  27. Ma, Z. et al. Cross‐link‐functionalized nanoparticles for rapid excretion in nanotheranostic applications. Angew. Chem. 132, 20733–20741 (2020).

    Article  Google Scholar 

  28. Zichi, J. et al. Optimizing the stoichiometry of ultrathin NbTiN films for high-performance superconducting nanowire single-photon detectors. Opt. Exp. 27, 26579–26587 (2019).

    CAS  Article  Google Scholar 

  29. Wang, L., Jacques, S. L. & Zheng, L. MCML—Monte Carlo modeling of light transport in multi-layered tissues. Comput. Meth. Prog. Biomed. 47, 131–146 (1995).

    CAS  Article  Google Scholar 

  30. Herisson, F. et al. Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration. Nat. Neurosci. 21, 1209–1217 (2018).

    CAS  Article  Google Scholar 

  31. Pereira, E. R. et al. Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice. Science 359, 1403–1407 (2018).

    CAS  Article  Google Scholar 

  32. Sewald, X. et al. Retroviruses use CD169-mediated trans-infection of permissive lymphocytes to establish infection. Science 350, 563–567 (2015).

    CAS  Article  Google Scholar 

  33. Milutinovic, S., Abe, J., Godkin, A., Stein, J. V. & Gallimore, A. The dual role of high endothelial venules in cancer progression versus immunity. Trends Cancer 7, 214–225 (2021).

    CAS  Article  Google Scholar 

  34. Hoshino, H. et al. Apical membrane expression of distinct sulfated glycans represents a novel marker of cholangiolocellular carcinoma. Lab. Invest. 96, 1246–1255 (2016).

    CAS  Article  Google Scholar 

  35. Fonsatti, E. & Maio, M. Highlights on endoglin (CD105): from basic findings towards clinical applications in human cancer. J. Transl. Med. 2, 18 (2004).

    Article  Google Scholar 

  36. Gaya, M. et al. Inflammation-induced disruption of SCS macrophages impairs B cell responses to secondary infection. Science 347, 667–672 (2015).

    CAS  Article  Google Scholar 

  37. Girard, J.-P., Moussion, C. & Förster, R. HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes. Nat. Rev. Immunol. 12, 762–773 (2012).

    CAS  Article  Google Scholar 

  38. Gu, M., Gan, X., Kisteman, A. & Xu, M. G. Comparison of penetration depth between two-photon excitation and single-photon excitation in imaging through turbid tissue media. Appl. Phys. Lett. 77, 1551–1553 (2000).

    CAS  Article  Google Scholar 

  39. Hu, C., Muller-Karger, F. E. & Zepp, R. G. Absorbance, absorption coefficient, and apparent quantum yield: a comment on common ambiguity in the use of these optical concepts. Limnol. Oceanogr. 47, 1261–1267 (2002).

    Article  Google Scholar 

  40. Wang, M. et al. Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain. Biomed. Opt. Exp. 9, 3534–3543 (2018).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This study was supported by the National Institutes of Health (NIH DP1-NS-105737, H.D.). We thank K. Taylor from JASCO who helped measuring the UV–vis–NIR absorbance spectrum of water using their V-770 Spectrophotometer. F.R. thanks the Fonds de recherche du Québec—Nature et technologies (FRQNT) for funding (F.R.). Single Quantum acknowledges support from the EIC SME Phase 2 project SQP (grant no. 848827 to R.G., J.W.L., A.F. and J.Q-.D.).

Author information

Authors and Affiliations

Authors

Contributions

H.D. and F.W. conceived and designed the experiments. H.D. and F.W. designed the optical system. F.W. set up the optical system. F.W., F.R. and Z.M. performed the experiments. F.R. synthesized the PbS/CdS QD. R.G., I.E.Z., J.W.N.L., A.F. and J.Q-.D. developed the SNSPD optimized in a 1,550–2,000 nm window. F.W., F.R., Z.M., L.Q., C.X., A.B., J.L. and H.D. analysed the data. F.W. and H.D. wrote the manuscript. All authors contributed to the general discussion and revision of the manuscript.

Corresponding author

Correspondence to Hongjie Dai.

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The following authors were employed by Single Quantum and may profit financially: R.G., J.W.L., A.F. and J.Q.D.

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Nature Nanotechnology thanks Eva Sevick-Muraca, Wei Zheng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Notes 1–3, Figs. 1–18 and Tables 1–3.

Reporting Summary

Supplementary Video

Animated mouse brain video from non-invasive in vivo NIR-IIc confocal microscopy images of vasculatures in intact mouse head.

Source data

Source Data Fig. 3

Statistical Source Data for Fig. 3e.

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Wang, F., Ren, F., Ma, Z. et al. In vivo non-invasive confocal fluorescence imaging beyond 1,700 nm using superconducting nanowire single-photon detectors. Nat. Nanotechnol. 17, 653–660 (2022). https://doi.org/10.1038/s41565-022-01130-3

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