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Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton


By combining astigmatism imaging with a dual-objective scheme, we improved the image resolution of stochastic optical reconstruction microscopy (STORM) and obtained <10-nm lateral resolution and <20-nm axial resolution when imaging biological specimens. Using this approach, we resolved individual actin filaments in cells and revealed three-dimensional ultrastructure of the actin cytoskeleton. We observed two vertically separated layers of actin networks with distinct structural organizations in sheet-like cell protrusions.

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Figure 1: Experimental setup and spatial resolution of dual-objective 3D STORM.
Figure 2: Dual-objective 3D STORM resolves individual actin filaments in cells.
Figure 3: Sheet-like cell protrusion comprises two layers of actin networks with distinct structures.


  1. Hell, S.W. Science 316, 1153–1158 (2007).

    Article  CAS  Google Scholar 

  2. Huang, B., Babcock, H. & Zhuang, X.W. Cell 143, 1047–1058 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Gould, T.J. et al. Nat. Methods 5, 1027–1030 (2008).

    Article  CAS  Google Scholar 

  5. Heilemann, M. et al. Angew. Chem. Int. Ed. 47, 6172–6176 (2008).

    Article  CAS  Google Scholar 

  6. Vogelsang, J., Cordes, T., Forthmann, C., Steinhauer, C. & Tinnefeld, P. Proc. Natl. Acad. Sci. USA 106, 8107–8112 (2009).

    Article  CAS  Google Scholar 

  7. Chhabra, E.S. & Higgs, H.N. Nat. Cell Biol. 9, 1110–1121 (2007).

    Article  CAS  Google Scholar 

  8. Pollard, T.D. & Borisy, G.G. Cell 112, 453–465 (2003).

    Article  CAS  Google Scholar 

  9. Svitkina, T. Methods Cell Biol. 79, 295–319 (2007).

    Article  CAS  Google Scholar 

  10. Urban, E., Jacob, S., Nemethova, M., Resch, G.P. & Small, J.V. Nat. Cell Biol. 12, 429–435 (2010).

    Article  CAS  Google Scholar 

  11. Rust, M.J., Bates, M. & Zhuang, X.W. Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Aquino, D. et al. Nat. Methods 8, 353–359 (2011).

    Article  CAS  Google Scholar 

  15. Zhuang, X.W. Nat. Photonics 3, 365–367 (2009).

    Article  CAS  Google Scholar 

  16. Pellegrin, S. & Mellor, H. J. Cell Sci. 120, 3491–3499 (2007).

    Article  CAS  Google Scholar 

  17. Geiger, B., Spatz, J.P. & Bershadsky, A.D. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009).

    Article  CAS  Google Scholar 

  18. Svitkina, T.M. & Borisy, G.G. J. Cell Biol. 145, 1009–1026 (1999).

    Article  CAS  Google Scholar 

  19. Giannone, G. et al. Cell 128, 561–575 (2007).

    Article  CAS  Google Scholar 

  20. Small, J.V., Rottner, K., Hahne, P. & Anderson, K.I. Microsc. Res. Tech. 47, 3–17 (1999).

    Article  CAS  Google Scholar 

  21. Koestler, S.A., Auinger, S., Vinzenz, M., Rottner, K. & Small, J.V. Nat. Cell Biol. 10, 306–313 (2008).

    Article  CAS  Google Scholar 

  22. Auinger, S. & Small, J.V. Methods Cell Biol. 88, 257–272 (2008).

    Article  CAS  Google Scholar 

  23. Dempsey, G.T. et al. J. Am. Chem. Soc. 131, 18192–18193 (2009).

    Article  CAS  Google Scholar 

  24. Goshtasby, A. 2-D and 3-D Image Registration for Medical, Remote Sensing, and Industrial Applications (Wiley, 2005).

Download references


We thank G. Danuser for helpful discussion. This work was supported in part by the US National Institutes of Health and a Collaborative Innovation Award (43667) from Howard Hughes Medical Institute and Gatsby Charitable Foundation (to X.Z.). X.Z. is funded by the Howard Hughes Medical Institute.

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Authors and Affiliations



K.X., H.P.B. and X.Z. designed research. K.X. did experiments and data analysis. H.P.B. assisted with the optical setup. K.X. and X.Z. prepared the manuscript. X.Z. supervised the project.

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Correspondence to Xiaowei Zhuang.

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The authors declare no competing financial interests.

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Supplementary Figures 1–7, Supplementary Results, Supplementary Discussion and Supplementary Protocols 1–2 (PDF 23681 kb)

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Xu, K., Babcock, H. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat Methods 9, 185–188 (2012).

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