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Engineering bright sub-10-nm upconverting nanocrystals for single-molecule imaging

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Abstract

Imaging at the single-molecule level reveals heterogeneities that are lost in ensemble imaging experiments, but an ongoing challenge is the development of luminescent probes with the photostability, brightness and continuous emission necessary for single-molecule microscopy1,2,3,4,5,6. Lanthanide-doped upconverting nanoparticles overcome problems of photostability and continuous emission7,8,9,10,11,12 and their upconverted emission can be excited with near-infrared light at powers orders of magnitude lower than those required for conventional multiphoton probes13,14. However, the brightness of upconverting nanoparticles has been limited by open questions about energy transfer and relaxation within individual nanocrystals and unavoidable tradeoffs between brightness and size15,16,17,18. Here, we develop upconverting nanoparticles under 10 nm in diameter that are over an order of magnitude brighter under single-particle imaging conditions than existing compositions, allowing us to visualize single upconverting nanoparticles as small (d = 4.8 nm) as fluorescent proteins. We use advanced single-particle characterization and theoretical modelling to find that surface effects become critical at diameters under 20 nm and that the fluences used in single-molecule imaging change the dominant determinants of nanocrystal brightness. These results demonstrate that factors known to increase brightness in bulk experiments lose importance at higher excitation powers and that, paradoxically, the brightest probes under single-molecule excitation are barely luminescent at the ensemble level.

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Figure 1: Luminescence of UCNPs.
Figure 2: UCNP size-dependent luminescence intensity and heterogeneity.
Figure 3: Single UCNP luminescence lifetime as a function of particle size and excitation power.
Figure 4: Simulated UCNP emission intensity.
Figure 5: Luminescence intensity of single UCNPs as a function of Er3+ and Yb3+ doping.

Change history

  • 24 March 2014

    In the version of this Letter originally published online, in Fig. 2b caption, the last two sentences were missing. This error has now been corrected in all versions of the Letter.

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Acknowledgements

The authors thank M. Salmeron and R. Johns for discussions and comments on the manuscript, as well as A. Nievergelt and A. Mueller for assistance with data processing and visualization. P.J.S. acknowledges Bica for support. A.D.O. was supported by a fellowship from the National Institute of Biomedical Imaging and Bioengineering, under NIH Award F32EB014680. Work at the Molecular Foundry was supported by the Director, Office of Science, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, of the US Department of Energy (contract no. DE-AC02-05CH11231).

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Contributions

P.J.S., B.E.C., D.J.M., D.J.G., E.M.C. and A.D.O. developed the idea for the project. A.D.O., E.M.C. and B.E.C. synthesized the nanocrystals. E.M.C. performed the kinetic modelling and simulations. D.J.G. performed the single-UCNP optical imaging, lifetime and spectroscopy measurements, as well as single-UCNP scanning TEM imaging. S.A. and M.V.P.A. provided TEM and analysis. J.J.U. provided in-depth discussions. E.S.B. and B.S. provided in-depth discussions and performed data analysis. D.J.G., E.M.C., B.E.C. and P.J.S. wrote the paper. All authors reviewed the paper.

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Correspondence to Bruce E. Cohen or P. James Schuck.

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Gargas, D., Chan, E., Ostrowski, A. et al. Engineering bright sub-10-nm upconverting nanocrystals for single-molecule imaging. Nature Nanotech 9, 300–305 (2014). https://doi.org/10.1038/nnano.2014.29

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