Abstract

We present a real-time fitter for 3D single-molecule localization microscopy using experimental point spread functions (PSFs) that achieves minimal uncertainty in 3D on any microscope and is compatible with any PSF engineering approach. We used this method to image cellular structures and attained unprecedented image quality for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D super-resolution microscopy broadly accessible, even on standard microscopes without dedicated 3D optics.

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Acknowledgements

We thank J. Mehl for helping with the sample preparation and data acquisition, S. van der Linde (University of Glasgow) for help with the TRABI analysis, E. Klotzsch (University of New South Wales) for the Zeiss Elyra bead stacks, M. Lampe (ALMF, EMBL Heidelberg) for help with the acquisition on the Leica GSD, system and D. Drubin (University of California, Berkeley, Berkeley, California, USA) for the gift of SK-MEL-2 hCLTA(EN)/DNM2(EN) cells. This work was supported by the European Research Council (ERC CoG-724489 to M.M. and J.R.), the Deutsche Forschungs Gemeinschaft (DFG RI 2380/2 to J.R. and J.D.), the EMBL Interdisciplinary Postdoc Programme (EIPOD) under Marie Curie Actions COFUND (Y.L.), the 4D Nucleome/4DN NIH Common Fund (U01 EB021223 to J.E. and J.R.), and the European Molecular Biology Laboratory (Y.L., M.M., P.H., J.D., U.M., B.N., V.J.S., J.E., and J.R.).

Author information

Affiliations

  1. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

    • Yiming Li
    • , Markus Mund
    • , Philipp Hoess
    • , Joran Deschamps
    • , Ulf Matti
    • , Bianca Nijmeijer
    • , Vilma Jimenez Sabinina
    • , Jan Ellenberg
    •  & Jonas Ries
  2. Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland.

    • Ingmar Schoen

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Contributions

Y.L. and J.R. conceived the approach, developed the methods, wrote the software, and analyzed the data. M.M. imaged clathrin-coated pits. P.H. imaged nuclear pore complexes. U.M. carried out DNA-PAINT imaging of microtubules. B.N., V.J.S., and J.E. contributed the Nup107 cell line. I.S. contributed the custom-made DNA-PAINT antibodies. J.D. acquired the aberrated PSFs and helped with the data analysis. Y.L., M.M., and J.R. wrote the manuscript with input from all other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jonas Ries.

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    Supplementary Text and Figures

    Supplementary Figures 1–15

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    Supplementary Software 2

    Compiled software

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DOI

https://doi.org/10.1038/nmeth.4661

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