Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Photometry unlocks 3D information from 2D localization microscopy data

Nature Methods volume 14, pages 41–44 (2017)Cite this article

Subjects

Abstract

We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Photometry-based 3D super-resolution imaging.
Figure 2: Virtual 3D imaging obtained by TRABI from 2D dSTORM data on different structures.
Figure 3: 3D super-resolution imaging of microtubules in U2OS cells, stained with AF647-labeled antibodies.

References

  1. Betzig, E. et al. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  2. van de Linde, S. et al. Nat. Protoc. 6, 991–1009 (2011).

    Article  CAS  Google Scholar 

  3. Huang, B., Wang, W., Bates, M. & Zhuang, X. Science 319, 810–813 (2008).

    Article  CAS  Google Scholar 

  4. Ram, S., Prabhat, P., Chao, J., Ward, E.S. & Ober, R.J. Biophys. J. 95, 6025–6043 (2008).

    Article  CAS  Google Scholar 

  5. Juette, M.F. et al. Nat. Methods 5, 527–529 (2008).

    Article  CAS  Google Scholar 

  6. Pavani, S.R. et al. Proc. Natl. Acad. Sci. USA 106, 2995–2999 (2009).

    Article  CAS  Google Scholar 

  7. Shtengel, G. et al. Proc. Natl. Acad. Sci. USA 106, 3125–3130 (2009).

    Article  CAS  Google Scholar 

  8. Xu, K., Babcock, H.P. & Zhuang, X. Nat. Methods 9, 185–188 (2012).

    Article  CAS  Google Scholar 

  9. Bourg, N. et al. Nat. Photonics 9, 587–593 (2015).

    Article  CAS  Google Scholar 

  10. Deschamps, J., Mund, M. & Ries, J. Opt. Express 22, 29081–29091 (2014).

    Article  Google Scholar 

  11. Howell, S.B. Publ. Astron. Soc. Pac. 101, 616–622 (1989).

    Article  Google Scholar 

  12. Holden, S.J. et al. Biophys. J. 99, 3102–3111 (2010).

    Article  CAS  Google Scholar 

  13. Wolter, S. et al. Nat. Methods 9, 1040–1041 (2012).

    Article  CAS  Google Scholar 

  14. Gibson, S.F. & Lanni, F. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 9, 154–166 (1992).

    Article  CAS  Google Scholar 

  15. Schücker, K., Holm, T., Franke, C., Sauer, M. & Benavente, R. Proc. Natl. Acad. Sci. USA 112, 2029–2033 (2015).

    Article  Google Scholar 

  16. Ehmann, N. et al. Nat. Commun. 5, 4650 (2014).

    Article  CAS  Google Scholar 

  17. Grimm, J.B. et al. Angew. Chem. Int. Edn Engl. 55, 1723–1727 (2016).

    Article  CAS  Google Scholar 

  18. Huang, B., Jones, S.A., Brandenburg, B. & Zhuang, X. Nat. Methods 5, 1047–1052 (2008).

    Article  CAS  Google Scholar 

  19. Heilemann, M., Kasper, R., Tinnefeld, P. & Sauer, M. J. Am. Chem. Soc. 128, 16864–16875 (2006).

    Article  CAS  Google Scholar 

  20. Löschberger, A. et al. J. Cell Sci. 125, 570–575 (2012).

    Article  Google Scholar 

  21. Schäfer, P., van de Linde, S., Lehmann, J., Sauer, M. & Doose, S. Anal. Chem. 85, 3393–3400 (2013).

    Article  Google Scholar 

  22. Mehlitz, A. et al. Cell. Microbiol. 16, 1224–1243 (2014).

    Article  CAS  Google Scholar 

  23. Ober, R.J., Ram, S. & Ward, E.S. Biophys. J. 86, 1185–1200 (2004).

    Article  CAS  Google Scholar 

  24. Small, A. & Stahlheber, S. Nat. Methods 11, 267–279 (2014).

    Article  CAS  Google Scholar 

  25. Parthasarathy, R. Nat. Methods 9, 724–726 (2012).

    Article  CAS  Google Scholar 

  26. Kirshner, H., Aguet, F., Sage, D. & Unser, M. J. Microsc. 249, 13–25 (2013).

    Article  CAS  Google Scholar 

  27. Sage, D. et al. Nat. Methods 12, 717–724 (2015).

    Article  CAS  Google Scholar 

  28. Ovesný, M., Křížek, P., Borkovec, J., Svindrych, Z. & Hagen, G.M. Bioinformatics 30, 2389–2390 (2014).

    Article  Google Scholar 

  29. Herbert, A. GDSC SMLM ImageJ Plugins http://www.sussex.ac.uk/gdsc/intranet/microscopy/imagej/smlm_plugins (2015).

  30. Badieirostami, M., Lew, M.D., Thompson, M.A. & Moerner, W.E. Appl. Phys. Lett. 97, 161103 (2010).

    Article  Google Scholar 

  31. Böhmer, M. & Enderlein, J. J. Opt. Soc. Am. B 20, 554–559 (2003).

    Article  Google Scholar 

  32. Patra, D., Gregor, I. & Enderlein, J. J. Phys. Chem. A 108, 6836–6841 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Enderlein (University of Göttingen) for providing us with software for the refractive index mismatch correction and D. Birch for critically reading the manuscript. We are very grateful to T. Klein, as well as to K. Schücker, R. Benavente (University of Würzburg) and L. Lavis (Janelia Research Campus) for the provision of raw data and to L. Pließ for support with cell culture.

Author information

Author notes

  1. Sebastian van de Linde

    Present address: Present address: Department of Physics, University of Strathclyde, Glasgow, UK.,

Authors and Affiliations

  1. Department of Biotechnology and Biophysics, University of Würzburg, Würzburg, Germany

    Christian Franke, Markus Sauer & Sebastian van de Linde

Authors
  1. Christian Franke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Markus Sauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sebastian van de Linde
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.F. and S.v.d.L. designed the TRABI algorithm, developed software, performed experiments and evaluated the data. C.F., M.S. and S.v.d.L. discussed results and commented on the manuscript. S.v.d.L. conceived the project and wrote the manuscript.

Corresponding author

Correspondence to Sebastian van de Linde.

Ethics declarations

Competing interests

C.F., M.S. and S.v.d.L. are in the process of filing a patent application.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–19 and Supplementary Notes 1 and 2. (PDF 5981 kb)

Source data to Supplementary Figure 1

Source data to Supplementary Figure 2

Source data to Supplementary Figure 3

Source data to Supplementary Figure 4

Supplementary Software

TRABI 1.0 Software. (ZIP 161 kb)

Source data

Source data tables for Supplementary Figures 1, 3, 4 and 10 (ZIP 32 kb)

Virtual 3D imaging of synaptonemal complex

Rendered virtual 3D structure of synaptonemal complex of the inset shown in Fig. 2b (ii). Left: x-y projection color-coded in P(z); right: 360° rotational view, scale bar 500 nm. (MOV 1350 kb)

Virtual 3D imaging of active zone protein Bruchpilot in Drosophila

Rendered virtual 3D structure of the presynaptic protein Bruchpilot in Drosophila neuromuscular junction of a detail shown in Fig. 2c. Left: x-y projection color-coded in P(z); right: 360° rotational view, scale bar 200 nm. (MOV 906 kb)

Virtual 3D imaging of F-Actin in COS-7 cells

Virtual 3D image stack of the data shown in Fig. 2d and Supplementary Fig. 15. P(z) interval in 5% steps. (MOV 4415 kb)

3D imaging of microtubules in U2OS cells

Rendered 3D microtubule structure of the inset shown in Fig. 3c. Left: x-y projection, right: 3D TRABI 360° rotational view, x-y-z scale 20 nm/px, Scale bar 250 nm. (MOV 729 kb)

3D imaging of microtubules in U2OS cells

Rendered 3D microtubule structure of the inset shown in Fig. 3d. Left: x-y projection, right: 3D TRABI 360° rotational view, x-y-z scale 20 nm/px, Scale bar 250 nm. (MOV 479 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Franke, C., Sauer, M. & van de Linde, S. Photometry unlocks 3D information from 2D localization microscopy data. Nat Methods 14, 41–44 (2017). https://doi.org/10.1038/nmeth.4073

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.4073

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing