Bright sub-20-nm cathodoluminescent nanoprobes for electron microscopy

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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.

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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.

Data availability

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

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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.

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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.

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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

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