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

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Abstract

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.

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Acknowledgements

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

  1. Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Ke Xu & Xiaowei Zhuang

  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Hazen P Babcock

  3. Department of Physics, Harvard University, Cambridge, Massachusetts, USA

    Xiaowei Zhuang

Authors
  1. Ke Xu
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  2. Hazen P Babcock
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  3. Xiaowei Zhuang
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Contributions

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.

Corresponding author

Correspondence to Xiaowei Zhuang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

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

Supplementary Software

Analysis software (ZIP 4 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). https://doi.org/10.1038/nmeth.1841

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