Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Bright sub-20-nm cathodoluminescent nanoprobes for electron microscopy

Nature Nanotechnology volume 14, pages 420–425 (2019)Cite this article

Subjects

Abstract

Electron microscopy has been instrumental in our understanding of complex biological systems. Although electron microscopy reveals cellular morphology with nanoscale resolution, it does not provide information on the location of different types of proteins. An electron-microscopy-based bioimaging technology capable of localizing individual proteins and resolving protein–protein interactions with respect to cellular ultrastructure would provide important insights into the molecular biology of a cell. Here, we synthesize small lanthanide-doped nanoparticles and measure the absolute photon emission rate of individual nanoparticles resulting from a given electron excitation flux (cathodoluminescence). Our results suggest that the optimization of nanoparticle composition, synthesis protocols and electron imaging conditions can lead to sub-20-nm nanolabels that would enable high signal-to-noise localization of individual biomolecules within a cellular context. In ensemble measurements, these labels exhibit narrow spectra of nine distinct colours, so the imaging of biomolecules in a multicolour electron microscopy modality may be possible.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: CL microscopy concept.
Fig. 2: CL imaging of single NaGdF4:5% Eu3+.
Fig. 3: Variability of CL brightness and SNR.
Fig. 4: Multicolour imaging of lanthanide-based nanoprobes.

Similar content being viewed by others

Data availability

The data sets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Roth, J., Bendayan, M. & Orci, L. Ultrastructural localization of intracellular antigens by the use of protein A–gold complex. J. Histochem. Cytochem. 26, 1074–1081 (1978).

    Article  CAS  Google Scholar 

  2. Martell, J. D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

    Article  CAS  Google Scholar 

  3. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    Article  CAS  Google Scholar 

  4. Adams, S. R. et al. Multicolor electron microscopy for simultaneous visualization of multiple molecular species. Cell Chem. Biol. 23, 1417–1427 (2016).

    Article  CAS  Google Scholar 

  5. Sochacki, Ka, Shtengel, G., van Engelenburg, S. B., Hess, H. F. & Taraska, J. W. Correlative super-resolution fluorescence and metal-replica transmission electron microscopy. Nat. Methods 11, 305–308 (2014).

    Article  CAS  Google Scholar 

  6. Carter, S. D. et al. Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells. J. Struct. Biol. 201, 15–25 (2018).

    Article  CAS  Google Scholar 

  7. Fisher, P. J., Wessels, W. S., Dietz, A. B. & Prendergast, F. G. Enhanced biological cathodoluminescence. Opt. Commun. 281, 1901–1908 (2008).

    Article  CAS  Google Scholar 

  8. Mahfoud, Z. et al. Cathodoluminescence in a scanning transmission electron microscope: a nanometer-scale counterpart of photoluminescence for the study of II–VI quantum dots. J. Phys. Chem. Lett. 4, 4090–4094 (2013).

    Article  CAS  Google Scholar 

  9. Zhang, H., Glenn, D. R., Schalek, R., Lichtman, J. W. & Walsworth, R. L. Efficiency of cathodoluminescence emission by nitrogen-vacancy color centers in nanodiamonds. Small 13, 1700543 (2017).

  10. Glenn, D. R. et al. Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours. Sci. Rep. 2, 1–6 (2012).

    Article  Google Scholar 

  11. Niioka, H. et al. Multicolor cathodoluminescence microscopy for biological imaging with nanophosphors. Appl. Phys. Express 4, 2402 (2011).

    Article  Google Scholar 

  12. Cai, E. et al. Stable small quantum dots for synaptic receptor tracking on live neurons. Angew. Chem. Int. Ed. 53, 12484–12488 (2014).

    CAS  Google Scholar 

  13. Yokota, S. Effect of particle size on labeling density for catalase in protein A–gold immunocytochemistry. J. Histochem. Cytochem. 36, 107–109 (1988).

    Article  CAS  Google Scholar 

  14. Kijanka, M. et al. A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy. J. Struct. Biol. 199, 1–11 (2017).

    Article  CAS  Google Scholar 

  15. Giepmans, B. N. G., Deerinck, T. J., Smarr, B. L., Jones, Y. Z. & Ellisman, M. H. Correlated light and electron microscopic imaging of multiple endogenous proteins using quantum dots. Nat. Methods 2, 743–749 (2005).

    Article  CAS  Google Scholar 

  16. Bischak, C. G. et al. Cathodoluminescence-activated nanoimaging: noninvasive near-field optical microscopy in an electron microscope. Nano Lett. 15, 3383–3390 (2015).

    Article  CAS  Google Scholar 

  17. Feng, W., Zhu, X. & Li, F. Recent advances in the optimization and functionalization of upconversion nanomaterials for in vivo bioapplications. NPG Asia Mater. 5, e75–15 (2013).

    Article  CAS  Google Scholar 

  18. García De Abajo, F. J. Optical excitations in electron microscopy. Rev. Mod. Phys. 82, 209–275 (2010).

    Article  Google Scholar 

  19. 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).

  20. Liu, Q., Feng, W., Yang, T., Yi, T. & Li, F. Upconversion luminescence imaging of cells and small animals. Nat. Protoc. 8, 2033–2044 (2013).

    Article  CAS  Google Scholar 

  21. Li, Z. & Zhang, Y. An efficient and user-friendly method for the synthesis of hexagonal-phase NaYF4:Yb, Er/Tm nanocrystals with controllable shape and upconversion fluorescence. Nanotechnology 19, 345606 (2008).

    Article  Google Scholar 

  22. Ostrowski, A. D. et al. Controlled synthesis and single-particle imaging of bright, sub-10-nm lanthanide-doped upconverting nanocrystals. ACS Nano 6, 2686–2692 (2012).

    Article  CAS  Google Scholar 

  23. Dong, A. et al. A generalized ligand-exchange strategy enabling sequential surface functionalization of colloidal nanocrystals. J. Am. Chem. Soc. 133, 998–1006 (2011).

    Article  CAS  Google Scholar 

  24. Gargas, D. J. et al. Engineering bright sub-10-nm upconverting nanocrystals for single-molecule imaging. Nat. Nanotechnol. 9, 300–305 (2014).

    Article  CAS  Google Scholar 

  25. Feng, W., Sun, L. D., Zhang, Y. W. & Yan, C. H. Solid-to-hollow single-particle manipulation of a self-assembled luminescent NaYF4:Yb,Er nanocrystal monolayer by electron-beam lithography. Small 5, 2057–2060 (2009).

    Article  CAS  Google Scholar 

  26. Judd, B. R. Optical absorption intensities of rare-earth ions. Phys. Rev. 127, 750–761 (1962).

    Article  CAS  Google Scholar 

  27. Ofelt, G. S. Intensities of crystal spectra of rare‐earth ions. J. Chem. Phys. 37, 511–520 (1962).

    Article  CAS  Google Scholar 

  28. Chan, E. M. Combinatorial approaches for developing upconverting nanomaterials: high-throughput screening, modeling, and applications. Chem. Soc. Rev. 44, 1653–1679 (2015).

    Article  CAS  Google Scholar 

  29. Fischer, S., Johnson, N. J. J., Pichaandi, J., Goldschmidt, J. C. & van Veggel, F. C. J. M. Upconverting core–shell nanocrystals with high quantum yield under low irradiance: on the role of isotropic and thick shells. J. Appl. Phys. 118, 193105 (2015).

    Article  Google Scholar 

  30. Ma, C. et al. Optimal sensitizer concentration in single upconversion nanocrystals. Nano Lett. 17, 2858–2864 (2017).

    Article  CAS  Google Scholar 

  31. Lu, Y. et al. Tunable lifetime multiplexing using luminescent nanocrystals. Nat. Photon. 8, 32–36 (2013).

    Article  Google Scholar 

  32. Nawa, Y. et al. Dynamic autofluorescence imaging of intracellular components inside living cells using direct electron beam excitation. Biomed. Opt. Express 5, 378–386 (2014).

    Article  Google Scholar 

  33. Xu, C. S. et al. Enhanced FIB-SEM systems for large-volume 3D imaging. eLife 3, 1–36 (2017).

    CAS  Google Scholar 

  34. Zhang, Z., Kenny, S. J., Hauser, M., Li, W. & Xu, K. Ultrahigh-throughput resolved super-resolution microscopy. Nat. Methods 12, 935–938 (2015).

    Article  CAS  Google Scholar 

  35. DaCosta, M. V., Doughan, S., Han, Y. & Krull, U. J. Lanthanide upconversion nanoparticles and applications in bioassays and bioimaging: a review. Anal. Chim. Acta 832, 1–33 (2014).

    Article  CAS  Google Scholar 

  36. Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004).

  37. Hayworth, K. J. et al. Ultrastructurally smooth thick partitioning and volume stitching for large-scale connectomics. Nat. Methods 12, 319–322 (2015).

    Article  CAS  Google Scholar 

  38. Kasthuri, N. et al. Saturated reconstruction of a volume of neocortex. Cell 162, 648–661 (2015).

    Article  CAS  Google Scholar 

  39. Stephenson, R. S. et al. High resolution 3-dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling. Sci. Rep. 7, 7188 (2017).

    Article  Google Scholar 

  40. Angelo, M. et al. Multiplexed ion beam imaging of human breast tumors. Nat. Med. 20, 436–442 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Gordon and Betty Moore Foundation (grant no. 4309), the National Institutes of Health (1R01GM128089-01A1) and the National Cancer Institute CCNE-TD at Stanford University (U54CA199075). Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract no. DE-AC02–05CH11231. Part of this work was performed at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-1542152. Access to the JEOL TEM 1400 was provided through the Stanford Microscopy Facility, NIH grant SIG no. 1S10RR02678001. M.B.P. was supported by the Helen Hay Whitney Foundation Postdoctoral Fellowship, and P.C.M. was supported through the Stanford Neuroscience Interdisciplinary Award. M.D.W. and J.A.D. acknowledge financial support provided as part of the DOE ‘Light–Material Interactions in Energy Conversion’ Energy Frontier Research Center under grant no. DE-SC0001293, as well as funding provided by the Global Climate and Energy Project at Stanford University. The authors thank N. Ginsberg and C. Aiello for providing software used in time-gated CL imaging experiments and for discussions. The authors are also grateful to J.J. Perrino and D.H. Burns for providing expertise in electron microscopy and for discussions, and R. Walsworth, D. Glenn, H. Zhang, T.C. Sudhof, J. Trotter, J. Collins and X. Zheng for discussions.

Author information

Author notes

  1. Nian Liu

    Present address: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA

  2. Guosheng Song

    Present address: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, China

  3. Lydia-Marie Joubert

    Present address: EM Unit, Central Analytical Facilities, Stellenbosch University, Stellenbosch, South Africa

  4. These authors contributed equally: Maxim B. Prigozhin and Peter C. Maurer.

Authors and Affiliations

  1. Department of Physics, Stanford University, Stanford, CA, USA

    Maxim B. Prigozhin, Peter C. Maurer & Steven Chu

  2. Department of Chemistry, University of California at Berkeley, Berkeley, CA, USA

    Alexandra M. Courtis & A. Paul Alivisatos

  3. Department of Materials Science and Engineering, Stanford University, Stanford, CA, USA

    Nian Liu, Michael D. Wisser, Chris Siefe, Stefan Fischer, Yi Cui & Jennifer A. Dionne

  4. Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Bining Tian, Emory Chan, Shaul Aloni, D. Frank Ogletree, Edward S. Barnard & Bruce E. Cohen

  5. Department of Radiology, Stanford University, Stanford, CA, USA

    Guosheng Song & Jianghong Rao

  6. CSIF Beckman Center, Stanford University, Stanford, CA, USA

    Lydia-Marie Joubert

  7. Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    A. Paul Alivisatos

  8. Department of Materials Science and Engineering, University of California, Berkeley, CA, USA

    A. Paul Alivisatos

  9. Kavli Energy NanoScience Institute, Berkeley, CA, USA

    A. Paul Alivisatos

  10. IBM Research – Almaden, San Jose, CA, USA

    Roger M. Macfarlane

  11. Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA

    Steven Chu

Authors
  1. Maxim B. Prigozhin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter C. Maurer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexandra M. Courtis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nian Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael D. Wisser
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chris Siefe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bining Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Emory Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Guosheng Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Stefan Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Shaul Aloni
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. D. Frank Ogletree
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Edward S. Barnard
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Lydia-Marie Joubert
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Jianghong Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. A. Paul Alivisatos
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Roger M. Macfarlane
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Bruce E. Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Yi Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Jennifer A. Dionne
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Steven Chu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.B.P., P.C.M. and S.C. conceived the project, designed experiments, analysed the data and interpreted the results. M.B.P. and P.C.M. conducted CL imaging experiments and wrote software for data analysis. M.B.P., P.C.M., A.M.C., N.L., M.D.W., C.S., B.T., G.S. and S.F. synthesized and characterized rare-earth nanoparticles. E.C. provided software for the simulation of the nanoparticle spectra. S.A., D.F.O., E.S.B. and L.-M.J. provided assistance and expertise in electron microscopy hardware and sample preparation. D.F.O. and S.A. developed the CL optics, and E.S.B. and D.F.O. developed the CL software. S.C. supervised the research. J.R., A.P.A., R.M.M., B.E.C., Y.C. and J.A.D. supervised the relevant portions of the research such as sample preparation and training M.B.P. and P.C.M. on the nanoparticle synthesis. M.B.P., P.C.M. and S.C. wrote the manuscript.

Corresponding author

Correspondence to Steven Chu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Text and Supplementary Figures 1–30

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Prigozhin, M.B., Maurer, P.C., Courtis, A.M. et al. Bright sub-20-nm cathodoluminescent nanoprobes for electron microscopy. Nat. Nanotechnol. 14, 420–425 (2019). https://doi.org/10.1038/s41565-019-0395-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-019-0395-0

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing