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Localization microscopy at doubled precision with patterned illumination

Nature Methods volume 17, pages 59–63 (2020)Cite this article

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Abstract

MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of view. Here we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micrometer-sized field of view. We show a near two-fold improvement in precision over standard localization with the same photon count on DNA-origami nanostructures and tubulin in cells, using DNA-PAINT and STORM imaging.

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Fig. 1: Principle of SIMFLUX.
Fig. 2: Demonstration of SIMFLUX on DNA-origami nanostructures.
Fig. 3: Demonstration of SIMFLUX on cellular tubulin with DNA-PAINT and (d)STORM.

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Data availability

Raw image data and processed conventional SMLM and SIMFLUX localization data is available at https://doi.org/10.4121/uuid:b1078e64-48d5-4f42-a1a8-3386ed14d4c7

Code availability

Software for processing SIMFLUX datasets is available as Supplementary Software. Updates will be made available at https://www.github.com/qnano/simflux

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Acknowledgements

J.C. and C.S.S. were supported by the Netherlands Organisation for Scientific Research (NWO), under NWO START-UP project no. 740.018.015 and NWO Veni project no. 16761. T.H., R.Ø.T. and B.R. acknowledge National Institutes of Health (grant no. U01EB021238). F.S. and R.J. acknowledge support by an ERC Starting Grant (MolMap, grant agreement no. 680241). C.S.S. acknowledges a research fellowship through Merton College, Oxford, UK. B.R. acknowledges an ERC Consolidator Grant (Nano@cryo, grant agreement no. 648580). We thank F. Balzarotti for advice on theoretical analysis of localization precision, and D. Jurriens and L. Kapitein for help with imaging cellular samples.

Author information

Author notes

  1. These authors contributed equally: Jelmer Cnossen, Taylor Hinsdale.

  2. These authors jointly supervised this work: Carlas S. Smith, Bernd Rieger, Sjoerd Stallinga.

Authors and Affiliations

  1. Department of Imaging Physics, Delft University of Technology, Delft, the Netherlands

    Jelmer Cnossen, Taylor Hinsdale, Rasmus Ø. Thorsen, Carlas S. Smith, Bernd Rieger & Sjoerd Stallinga

  2. Delft Center for Systems and Control, Delft University of Technology, Delft, the Netherlands

    Jelmer Cnossen & Carlas S. Smith

  3. Department of Biology, University of Utrecht, Utrecht, the Netherlands

    Marijn Siemons

  4. Department of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany

    Florian Schueder & Ralf Jungmann

  5. Max Planck Institute of Biochemistry, Martinsried, Germany

    Florian Schueder & Ralf Jungmann

  6. Department of Engineering Science, University of Oxford, Oxford, UK

    Carlas S. Smith

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  1. Jelmer Cnossen
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Contributions

Imaging experiments were done by T.H., M.S. and F.S. Data analyses were done by J.C. and T.H. Simulations were done by R.Ø.T. and J.C. M.S., F.S. and R.J. provided samples. C.S.S., B.R. and S.S. devised key concepts and supervised the study. T.H., C.S.S., B.R. and S.S. wrote the paper. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Carlas S. Smith, Bernd Rieger or Sjoerd Stallinga.

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Competing interests

The authors declare no competing interests.

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Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–21, Supplementary Table and Supplementary Notes 1–7.

Reporting Summary

Supplementary Software

simflux-publication-release.zip contains the code developed for processing SIMFLUX datasets (source code and binaries and example dataset).

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Cnossen, J., Hinsdale, T., Thorsen, R.Ø. et al. Localization microscopy at doubled precision with patterned illumination. Nat Methods 17, 59–63 (2020). https://doi.org/10.1038/s41592-019-0657-7

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