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MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells

Nature Methods volume 17, pages 217–224 (2020)Cite this article

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Matters Arising to this article was published on 15 December 2022

Abstract

The ultimate goal of biological super-resolution fluorescence microscopy is to provide three-dimensional resolution at the size scale of a fluorescent marker. Here we show that by localizing individual switchable fluorophores with a probing donut-shaped excitation beam, MINFLUX nanoscopy can provide resolutions in the range of 1 to 3 nm for structures in fixed and living cells. This progress has been facilitated by approaching each fluorophore iteratively with the probing-donut minimum, making the resolution essentially uniform and isotropic over scalable fields of view. MINFLUX imaging of nuclear pore complexes of a mammalian cell shows that this true nanometer-scale resolution is obtained in three dimensions and in two color channels. Relying on fewer detected photons than standard camera-based localization, MINFLUX nanoscopy is poised to open a new chapter in the imaging of protein complexes and distributions in fixed and living cells.

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Fig. 1: Iterative MINFLUX setup and localization.
Fig. 2: MINFLUX nanoscopy in fixed and living cells.
Fig. 3: Iterative 3D MINFLUX localization yields isotropic nanometer precision.
Fig. 4: MINFLUX imaging of the post-synaptic protein PSD-95 with 3D resolution of 2–3 nm s.d.
Fig. 5: Two-color MINFLUX nanoscopy in 2D and 3D.

Data availability

The data that support the findings of this study are available from the corresponding author S.W.H. upon reasonable request.

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Acknowledgements

We thank the following MPI colleagues: E. D’Este for support with biological sample optimization; M. Bates for discussions about single-molecule techniques; H. Ta for help with DNA origami; D. Kamin and I. Herfort for support with cultured neurons; the Facility for Synthetic Chemistry for the Alexa Fluor 647-HaloTag ligand. We acknowledge S. Grant (University of Edinburgh) for providing the PSD-95–Halo mouse line. J. Thevathasan, B. Nijmeijer, M. Kueblbeck and U. Matti (all EMBL) made the Nup96 cell lines. P.H. and J.R. acknowledge the European Research Council (ERC grant CoG-724489) and the Human Frontier Science Program (grant RGY0065/2017 to J.R.). S.W.H. acknowledges support from DFG grant SFB1286/A7.

Author information

Author notes

  1. These authors contributed equally: Klaus C. Gwosch, Jasmin K. Pape, Francisco Balzarotti.

Authors and Affiliations

  1. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Klaus C. Gwosch, Jasmin K. Pape, Francisco Balzarotti & Stefan W. Hell

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

    Philipp Hoess, Jan Ellenberg & Jonas Ries

  3. Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany

    Stefan W. Hell

Authors
  1. Klaus C. Gwosch
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  2. Jasmin K. Pape
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  3. Francisco Balzarotti
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  4. Philipp Hoess
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  5. Jan Ellenberg
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  7. Stefan W. Hell
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Contributions

K.C.G., J.K.P. and F.B. designed and built hardware, programmed software and performed experiments and data analysis with input from S.W.H. K.C.G., J.K.P. and P.H. prepared samples. P.H. and J.R. proposed the Nup96 cell line as a resolution test sample. J.E. made the Nup96 cell line. F.B. carried out theoretical analysis of iterative MINFLUX with K.C.G. and co-supervised the project. S.W.H. supervised the project and was responsible for its conception. K.C.G., J.K.P., F.B. and S.W.H. wrote the manuscript.

Corresponding author

Correspondence to Stefan W. Hell.

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Competing interests

S.W.H. is a co-founder of the company Abberior Instruments, which commercializes super-resolution microscopy systems, including MINFLUX. S.W.H., K.C.G. and F.B. hold patents on the principles, embodiments and procedures of MINFLUX.

Additional information

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. 1–9, Supplementary Tables 1–3 and Supplementary Notes 1–4

Reporting Summary

Supplementary Video 1

3D MINFLUX nanoscopy of NUP96 in a mammalian cell. A U-2 OS cell expressing NUP96-SNAP labeled with Alexa Fluor 647 after fixation as displayed in Fig. 3. The color indicates the z position of the localization.

Supplementary Video 2

3D MINFLUX nanoscopy of the synaptic protein PSD-95. Primary hippocampal neurons from transgenic mice expressing PSD-95–Halo conjugated to Alexa Fluor 647 after fixation as displayed in Fig. 4. The color indicates the 3D localization density. PSD-95 appears in clusters distributed on a curved surface (gray surface) displayed also as a contour line projection to the xy-bounding plane.

Supplementary Video 3

3D two-color MINFLUX nanoscopy of the nuclear pore complex in a mammalian cell. A U-2 OS cell expressing NUP96–SNAP labeled with Alexa Fluor 647 (green) and WGA conjugated to CF680 (magenta) after fixation as displayed in Fig. 5. The colors indicate the molecular species assigned in the two-color classification.

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Gwosch, K.C., Pape, J.K., Balzarotti, F. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat Methods 17, 217–224 (2020). https://doi.org/10.1038/s41592-019-0688-0

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