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Direct optical nanoscopy with axially localized detection

Nature Photonics volume 9, pages 587–593 (2015)Cite this article

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An Addendum to this article was published on 29 October 2015

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Abstract

Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of ∼150 nm above the coverslip.

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Figure 1: Far- and near-field emission components.
Figure 2: Direct optical nanoscopy with axially localized detection.
Figure 3: DONALD theory.
Figure 4: dSTORM imaging of F-actin in CHO cells immersed in a thiol + oxygen scavenger buffer using DONALD.
Figure 5: dSTORM imaging of microtubules immersed in a thiol + oxygen scavenger-based buffer using DONALD.
Figure 6: SMLM imaging of plasma membrane immersed in a thiol + oxygen scavenger-based buffer using DONALD.

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  • 15 October 2015

    The authors wish to acknowledge a highly relevant manuscript that was published during the reviewing process of this Article, which should have been cited: Deschamps, J., Mund, M., & Ries, J. 3D superresolution microscopy by supercritical angle detection. Opt. Express 22, 29081–29091 (2014). The manuscript reports interesting use of 3D DNA-PAINT origami as a ruler for super-resolution imaging.

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Acknowledgements

The authors thank J. Dompierre for help with immunofluorescence and P. Adenot for providing CellMask Deep Red stain. The authors acknowledge financial support from the AXA Research Fund, Labex WIFI, the French National Research Agency (project SMARTVIEW) and DIM Nano-K (Project NanoSAF).

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Authors and Affiliations

  1. Institut des Sciences Moléculaires d'Orsay (ISMO), Université Paris-Sud, CNRS UMR 8214, Orsay Cedex, F91405, France

    N. Bourg & S. Lévêque-Fort

  2. Université Paris-Sud, Centre de Photonique BioMédicale (CPBM), Fédération LUMAT, CNRS FR 2764, Orsay Cedex, F91405, France

    N. Bourg, C. Mayet, G. Dupuis, S. Lécart & S. Lévêque-Fort

  3. Institut Langevin, ESPCI ParisTech, CNRS, PSL Research University, 1 rue Jussieu, Paris, F-75005, France

    T. Barroca, P. Bon & E. Fort

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Contributions

N.B., G.D., E.F. and S.L.F. conceived and designed the project. N.B. performed the experiments, simulations and analysis. C.M. and N.B. developed the photoswitching buffer. C.M., N.B. and S.L. optimized the immunofluorescence protocol. T.B. and P.B. helped with the simulation and the DONALD module. All authors contributed to writing the manuscript.

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Correspondence to S. Lévêque-Fort.

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The authors declare no competing financial interests.

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Bourg, N., Mayet, C., Dupuis, G. et al. Direct optical nanoscopy with axially localized detection. Nature Photon 9, 587–593 (2015). https://doi.org/10.1038/nphoton.2015.132

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