MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1.

    Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

  2. 2.

    Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).

  3. 3.

    Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

  4. 4.

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

  5. 5.

    Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017).

  6. 6.

    Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

  7. 7.

    Eilers, Y., Ta, H., Gwosch, K. C., Balzarotti, F. & Hell, S. W. MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution. Proc. Natl Acad. Sci. USA 115, 6117–6122 (2018).

  8. 8.

    Backlund, M. P., Shechtman, Y. & Walsworth, R. L. Fundamental precision bounds for three-dimensional optical localization microscopy with Poisson statistics. Phys. Rev. Lett. 121, 023904 (2018).

  9. 9.

    Thevathasan, J. V. et al. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nat. Methods 16, 1045–1053 (2019).

  10. 10.

    von Appen, A. et al. In situ structural analysis of the human nuclear pore complex. Nature 526, 140–143 (2015).

  11. 11.

    Banterle, N., Bui, K. H., Lemke, E. A. & Beck, M. Fourier ring correlation as a resolution criterion for super-resolution microscopy. J. Struct. Biol. 183, 363–367 (2013).

  12. 12.

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

  13. 13.

    Hell, S., Reiner, G., Cremer, C. & Stelzer, E. H. K. Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. J. Microsc. 169, 391–405 (1993).

  14. 14.

    Masch, J.-M. et al. Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling. Proc. Natl Acad. Sci. USA 115, E8047–E8056 (2018).

  15. 15.

    MacGillavry, H. D., Song, Y., Raghavachari, S. & Blanpied, T. A. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615–622 (2013).

  16. 16.

    Fukata, Y. et al. Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J. Cell Biol. 202, 145–161 (2013).

  17. 17.

    MacGillavry, H. D. & Hoogenraad, C. C. The internal architecture of dendritic spines revealed by super-resolution imaging: what did we learn so far? Exp. Cell Res. 335, 180–186 (2015).

  18. 18.

    Zhang, Z., Kenny, S. J., Hauser, M., Li, W. & Xu, K. Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nat. Methods 12, 935–938 (2015).

  19. 19.

    Dai, M., Jungmann, R. & Yin, P. Optical imaging of individual biomolecules in densely packed clusters. Nat. Nanotechnol. 11, 798–807 (2016).

  20. 20.

    Li, B. & Kohler, J. J. Glycosylation of the nuclear pore. Traffic 15, 347–361 (2014).

  21. 21.

    Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).

  22. 22.

    Jungmann, R. et al. Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett. 10, 4756–4761 (2010).

  23. 23.

    Gao, P., Prunsche, B., Zhou, L., Nienhaus, K. & Nienhaus, G. U. Background suppression in fluorescence nanoscopy with stimulated emission double depletion. Nat. Photonics 11, 163–169 (2017).

  24. 24.

    Leutenegger, M., Rao, R., Leitgeb, R. A. & Lasser, T. Fast focus field calculations. Opt. Express 14, 11277–11291 (2006).

  25. 25.

    Pham, T.-A., Soubies, E., Sage, D. & Unser, M. Closed-form expression of the Fourier ring-correlation for single-molecule localization microscopy. https://doi.org/10.1109/ISBI.2019.8759279. In IEEE International Symposium on Biomedical Imaging (IEEE, 2019).

  26. 26.

    Ong, W. Q., Citron, Y. R., Schnitzbauer, J., Kamiyama, D. & Huang, B. Heavy water: a simple solution to increasing the brightness of fluorescent proteins in super-resolution imaging. Chem. Commun. 51, 13451–13453 (2015).

  27. 27.

    D’Este, E., Kamin, D., Göttfert, F., El-Hady, A. & Hell, S. W. STED nanoscopy reveals the ubiquity of subcortial cytoskeleton periodicity in living neurons. Cell Rep. 10, 1246–1251 (2015).

Download references


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

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.

Correspondence to Stefan W. Hell.

Ethics declarations

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.

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–9, 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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation