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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.

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

References

  1. Hell, S. W. Microscopy and its focal switch. Nat. Meth. 6, 24–32 (2009).

    Article  CAS  Google Scholar 

  2. Huang, B., Babcock, H. & Zhuang, X. Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143, 1047–1058 (2010).

    Article  CAS  Google Scholar 

  3. Klein, T., Proppert, S. & Sauer, M. Eight years of single-molecule localization microscopy. Histochem Cell Bio. 141, 561–575 (2014).

    Article  CAS  Google Scholar 

  4. Ries, J., Kaplan, C., Platonova, V., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Meth. 9, 582–587 (2012).

    Article  CAS  Google Scholar 

  5. Raulf, A. et al. Click chemistry facilitates direct labeling and super-resolution imaging of nucleic acids and proteins. RSC Adv. 4, 30462–30466 (2014).

    Article  CAS  Google Scholar 

  6. Li, H. & Vaughan, J. C. Switchable fluorophores for single-molecule localization microscopy. Chem. Rev. 118, 9412–9454 (2018).

    Article  CAS  Google Scholar 

  7. Strauss, S. et al. Modified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imaging. Nat. Meth. 15, 685–688 (2018).

    Article  CAS  Google Scholar 

  8. Heydarian, H. et al. Template-free 2D particle fusion in localization microscopy. Nat. Meth. 15, 781–784 (2018).

    Article  CAS  Google Scholar 

  9. Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Meth. 12, 244–250 (2015).

    Article  CAS  Google Scholar 

  10. Kaufmann, R. et al. Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions. Nano Lett. 14, 4171–4175 (2014).

    Article  CAS  Google Scholar 

  11. Weisenburger, S. et al. Cryogenic optical localization provides 3D protein structure data with Angstrom resolution. Nat. Meth. 14, 141–144 (2017).

    Article  CAS  Google Scholar 

  12. Hulleman, C. H., Li, W., Gregor, I., Rieger, B. & Enderlein, J. Photon yield enhancement of red fluorophores at cryogenic temperatures. Chem. Phys. Chem. 19, 1774–1780 (2018).

    Article  CAS  Google Scholar 

  13. Rieger, B. & Stallinga, S. The lateral and axial localization uncertainty in super-resolution light microscopy. Chem. Phys. Chem. 15, 664–670 (2014).

    Article  CAS  Google Scholar 

  14. Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2016).

    Article  Google Scholar 

  15. Heintzmann, R. & Huser, T. Super-resolution structured illumination microscopy. Chem. Rev. 117, 13890–13908 (2017).

    Article  CAS  Google Scholar 

  16. Busoni, L., Dornier, A., Viovy, J.-L., Prost, J. & Cappello, G. Fast subnanometer particle localization by traveling-wave tracking. J. Appl. Phys. 98, 064302 (2005).

    Article  Google Scholar 

  17. Wicker, K. Non-iterative determination of pattern phase in structured illumination microscopy using autocorrelations in Fourier space. Opt. Exp. 21, 24692–24701 (2013).

    Article  Google Scholar 

  18. Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Prot. 12, 1198–1228 (2017).

    Article  CAS  Google Scholar 

  19. Nieuwenhuizen, R. P. J. et al. Measuring image resolution in optical nanoscopy. Nat. Meth. 10, 557–562 (2013).

    Article  CAS  Google Scholar 

  20. Li, Y. et al. Real-time 3D single molecule localization using experimental point spread functions. Nat. Meth. 15, 367–369 (2018).

    Article  CAS  Google Scholar 

  21. Gu, L. et al. Molecular resolution imaging by repetitive optical selective exposure. Nat. Meth., https://doi.org/10.1038/s41592-019-0544-2 (2019).

    Article  CAS  Google Scholar 

  22. Chmyrov, A. et al. Nanoscopy with more than 100,000 ‘doughnuts’. Nat. Meth. 10, 737–740 (2013).

    Article  CAS  Google Scholar 

  23. Stallinga, S. & Rieger, B. Accuracy of the Gaussian point spread function model in 2D localization microscopy. Opt. Exp. 18, 24461–24476 (2010).

    Article  CAS  Google Scholar 

  24. Thorsen, R. Ø. et al. Impact of optical aberrations on axial position determination by photometry. Nat. Meth. 15, 989–993 (2018).

    Article  CAS  Google Scholar 

  25. Mulliken, J. C. et al. Methods for CCD camera characterization. In Proc. SPIE 2173, Image Acquisition and Scientific Imaging Systems (eds Titus, H. C. and Waks, A.) 73–84 (SPIE, 1994).

  26. Heintzmann, R., Relich, P. K., Nieuwenhuizen, R. P. J., Lidke, K. A. & Rieger, B. Calibrating photon counts from a single image. Preprint at https://arxiv.org/abs/1611.05654 (2019).

  27. Huang, F., Schwartz, S. L., Byars, J. M. & Lidke, K. A. Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomed. Opt. Exp. 2, 1377–1393 (2011).

    Article  Google Scholar 

  28. Smith, C. S. et al. Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking. J. Cell. Biol. 209, 609–619 (2015).

    Article  CAS  Google Scholar 

  29. Smith, C. S., Joseph, N., Rieger, B. & Lidke, K. A. Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat. Meth. 7, 373–375 (2010).

    Article  CAS  Google Scholar 

  30. Huang, F. et al. Video-rate nanoscopy using sCMOS-camera specific single-molecule localization algorithms. Nat. Meth. 10, 653–658 (2013).

    Article  CAS  Google Scholar 

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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.

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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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