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Continuous-wave near-infrared stimulated-emission depletion microscopy using downshifting lanthanide nanoparticles

Abstract

Stimulated-emission depletion (STED) microscopy has profoundly extended our horizons to the subcellular level1,2,3. However, it remains challenging to perform hours-long, autofluorescence-free super-resolution imaging in near-infrared (NIR) optical windows under facile continuous-wave laser depletion at low power4,5. Here we report downshifting lanthanide nanoparticles that enable background-suppressed STED imaging in all-NIR spectral bands (λexcitation = 808 nm, λdepletion = 1,064 nm and λemission = 850–900 nm), with a lateral resolution of below 20 nm and zero photobleaching. With a quasi-four-level configuration and long-lived (τ > 100 μs) metastable states, these nanoparticles support near-unity (98.8%) luminescence suppression under 19 kW cm−2 saturation intensity. The all-NIR regime enables high-contrast deep-tissue (~50 μm) imaging with approximately 70 nm spatial resolution. These lanthanide nanoprobes promise to expand the application realm of STED microscopy and pave the way towards high-resolution time-lapse investigations of cellular processes at superior spatial and temporal dimensions.

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Fig. 1: All-NIR quasi-four-level CW STED microscopy.
Fig. 2: Optical switching features of neodymium-activated NIR STED nanoprobes.
Fig. 3: Mechanistic investigation of the neodymium-activated nanocrystals for superior STED.
Fig. 4: Low-power full-NIR CW STED imaging of subcellular structures and deep-tissue super-resolution imaging.

Data availability

Source data are provided with this paper. The data that support the findings of this study are available within the article and its Supplementary Information. Additional data are available from the corresponding authors upon request.

References

  1. 1.

    Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    CAS  Article  Google Scholar 

  2. 2.

    Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    CAS  Article  Google Scholar 

  3. 3.

    Vicidomini, G. et al. Sharper low-power STED nanoscopy by time gating. Nat. Methods 8, 571–573 (2011).

    CAS  Article  Google Scholar 

  4. 4.

    Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).

    CAS  Article  Google Scholar 

  5. 5.

    Willig, K. I., Harke, B., Medda, R. & Hell, S. W. STED microscopy with continuous wave beams. Nat. Methods 4, 915–918 (2007).

    CAS  Article  Google Scholar 

  6. 6.

    Chen, B.-C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

    Article  Google Scholar 

  7. 7.

    Bates, M., Huang, B., Dempsey, G. T. & Zhuang, X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).

    CAS  Article  Google Scholar 

  8. 8.

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    CAS  Article  Google Scholar 

  9. 9.

    Fölling, J. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat. Methods 5, 943–945 (2008).

    Article  Google Scholar 

  10. 10.

    Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217–224 (2020).

    CAS  Article  Google Scholar 

  11. 11.

    Klar, T. A. & Hell, S. W. Subdiffraction resolution in far-field fluorescence microscopy. Opt. Lett. 24, 954–956 (1999).

    CAS  Article  Google Scholar 

  12. 12.

    Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2008).

    Article  Google Scholar 

  13. 13.

    Vicidomini, G., Bianchini, P. & Diaspro, A. STED super-resolved microscopy. Nat. Methods 15, 173–182 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    Hoebe, R. et al. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging. Nat. Biotechnol. 25, 249–253 (2007).

    CAS  Article  Google Scholar 

  15. 15.

    An, Z. et al. Stabilizing triplet excited states for ultralong organic phosphorescence. Nat. Mater. 14, 685–690 (2015).

    CAS  Article  Google Scholar 

  16. 16.

    Bünzli, J.-C. G., Chauvin, A.-S., Kim, H. K., Deiters, E. & Eliseeva, S. V. Lanthanide luminescence efficiency in eight-and nine-coordinate complexes: role of the radiative lifetime. Coord. Chem. Rev. 254, 2623–2633 (2010).

    Article  Google Scholar 

  17. 17.

    Malta, O. Mechanisms of non-radiative energy transfer involving lanthanide ions revisited. J. Non Cryst. Solids 354, 4770–4776 (2008).

    CAS  Article  Google Scholar 

  18. 18.

    O’Brien, J. J. & O’Brien, J. F. The Laporte selection rule in electronic absorption spectroscopy. J. Coll. Sci. Teach. 29, 138–140 (1999).

    Google Scholar 

  19. 19.

    Wisser, M. D. et al. Strain-induced modification of optical selection rules in lanthanide-based upconverting nanoparticles. Nano Lett. 15, 1891–1897 (2015).

    CAS  Article  Google Scholar 

  20. 20.

    Jackson, S. D. Towards high-power mid-infrared emission from a fibre laser. Nat. Photonics 6, 423–431 (2012).

    CAS  Article  Google Scholar 

  21. 21.

    Fernandez-Bravo, A. et al. Continuous-wave upconverting nanoparticle microlasers. Nat. Nanotechnol. 13, 572–577 (2018).

    CAS  Article  Google Scholar 

  22. 22.

    Chen, X. et al. Confining energy migration in upconversion nanoparticles towards deep ultraviolet lasing. Nat. Commun. 7, 10304 (2016).

    CAS  Article  Google Scholar 

  23. 23.

    Lee, C. et al. Giant nonlinear optical responses from photon-avalanching nanoparticles. Nature 589, 230–235 (2021).

    CAS  Article  Google Scholar 

  24. 24.

    Lando, M., Kagan, J., Linyekin, B. & Dobrusin, V. A solar-pumped Nd:YAG laser in the high collection efficiency regime. Opt. Commun. 222, 371–381 (2003).

    CAS  Article  Google Scholar 

  25. 25.

    Wang, F., Deng, R. & Liu, X. Preparation of core–shell NaGdF4 nanoparticles doped with luminescent lanthanide ions to be used as upconversion-based probes. Nat. Protoc. 9, 1634–1644 (2014).

    CAS  Article  Google Scholar 

  26. 26.

    Liu, Y. et al. Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy. Nature 543, 229–233 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Rittweger, E., Han, K. Y., Irvine, S. E., Eggeling, C. & Hell, S. W. STED microscopy reveals crystal colour centres with nanometric resolution. Nat. Photonics 3, 144–147 (2009).

    CAS  Article  Google Scholar 

  28. 28.

    Han, K. Y., Kim, S. K., Eggeling, C. & Hell, S. W. Metastable dark states enable ground state depletion microscopy of nitrogen vacancy centers in diamond with diffraction-unlimited resolution. Nano Lett. 10, 3199–3203 (2010).

    CAS  Article  Google Scholar 

  29. 29.

    Hanne, J. et al. STED nanoscopy with fluorescent quantum dots. Nat. Commun. 6, 7127 (2015).

    CAS  Article  Google Scholar 

  30. 30.

    Gao, P., Prunsche, B., Zhou, L., Nienhaus, K. & Nienhaus, G. U. Background suppression in fluorescence nanoscopy with stimulated emission double depletion. Nat. Photonics 11, 163–169 (2017).

    CAS  Article  Google Scholar 

  31. 31.

    Koechner, W. Solid-State Laser Engineering 38–101 (Springer, 2006).

  32. 32.

    White, J. O. Parameters for quantitative comparison of two-, three-, and four-level laser media, operating wavelengths, and temperatures. IEEE J. Quantum Electron. 45, 1213–1220 (2009).

    CAS  Article  Google Scholar 

  33. 33.

    Rehor, I. & Cigler, P. Precise estimation of HPHT nanodiamond size distribution based on transmission electron microscopy image analysis. Diam. Relat. Mater. 46, 21–24 (2014).

    CAS  Article  Google Scholar 

  34. 34.

    Han, K. Y. et al. Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light. Nano Lett. 9, 3323–3329 (2009).

    CAS  Article  Google Scholar 

  35. 35.

    Gu, Y. et al. High-sensitivity imaging of time-domain near-infrared light transducer. Nat. Photonics 13, 525–531 (2019).

    CAS  Article  Google Scholar 

  36. 36.

    Chen, C. et al. Multi-photon near-infrared emission saturation nanoscopy using upconversion nanoparticles. Nat. Commun. 9, 3290 (2018).

    Article  Google Scholar 

  37. 37.

    Jin, D. et al. Nanoparticles for super-resolution microscopy and single-molecule tracking. Nat. Methods 15, 415–423 (2018).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work is supported by the National Key R&D Program of China (no. 2018YFA0902600 and no. 2018YFB1107200), the National Natural Science Foundation of China (21635002, 21771135, 21871071 and 61975123), the Ministry of Education, Singapore (MOE2017-T2-2-110), the Agency for Science, Technology and Research (A*STAR) (grant no. A1883c0011 and no. A1983c0038), National Research Foundation, the Prime Minister’s Office of Singapore under its NRF Investigatorship Programme (award no. NRF-NRFI05-2019-0003), the National Basic Research Program of China (973 Program, grant no. 2015CB932200), Zhangjiang National Innovation Demonstration Zone (ZJ-2019-ZD-005) and the Guangdong Provincial Innovation and Entrepreneurship Project (grant no. 2016ZT06D081). We thank X. Zhao, J. Chen and Z. Mu for technical assistance with the TEM imaging and NIR spectral measurements. We also thank J. Hong for absorption spectra measurements.

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L.L. and X. Liu conceived and designed the project. X. Liu, X. Li, M.G. and B.X. supervised the project and led the collaboration efforts. L.L. synthesized the nanocrystals and conducted the numerical simulations with contribution from L.Z., Q.Z. and Z.F. Optical experiments and super-resolution imaging were conducted by L.L., Z.F. and Y.W. The preparation of mouse-brain slices and cell labelling was the responsibility of Z.Y., M.J.Y.A., T.D.C. and H.F. The density functional theory calculations were conducted by X.Q. The manuscript was written by L.L., Z.F. and X. Liu. All authors participated in the discussion and analysis of the manuscript.

Corresponding authors

Correspondence to Min Gu or Xiangping Li or Xiaogang Liu.

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Liang, L., Feng, Z., Zhang, Q. et al. Continuous-wave near-infrared stimulated-emission depletion microscopy using downshifting lanthanide nanoparticles. Nat. Nanotechnol. 16, 975–980 (2021). https://doi.org/10.1038/s41565-021-00927-y

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