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Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)

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Abstract

We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

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Figure 1: STORM with photo-switchable fluorophores.
Figure 2: The high localization accuracy of individual switches during each switching cycle defines the intrinsic resolution of STORM.
Figure 3: STORM can resolve structures with sub-diffraction-limit resolution.

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Acknowledgements

This work is supported by in part by the US National Institutes of Health, the Defense Advance Research Projects Agency and a Packard Science and Engineering Fellowship (to X.Z.). X.Z. is a Howard Hughes Medical Institute Investigator.

Author information

Author notes

  1. Michael J Rust and Mark Bates: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physics, Harvard University, 12 Oxford Street, Cambridge, 02138, Massachusetts, USA

    Michael J Rust & Xiaowei Zhuang

  2. Division of Engineering and Applied Sciences, Harvard University, 12 Oxford Street, Cambridge, 02138, Massachusetts, USA

    Mark Bates

  3. Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, 02138, Massachusetts, USA

    Xiaowei Zhuang

  4. Howard Hughes Medical Institute, Harvard University, 12 Oxford Street, Cambridge, 02138, Massachusetts, USA

    Xiaowei Zhuang

Authors
  1. Michael J Rust
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  2. Mark Bates
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  3. Xiaowei Zhuang
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Contributions

M.J.R. and M.B. conceived the STORM imaging concept. M.J.R., DNA imaging and data analysis; M.B., RecA imaging and data acquisition. X.Z. supervised the project.

Corresponding author

Correspondence to Xiaowei Zhuang.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Switching of Cy3-Cy5–labeled antibody. (PDF 45 kb)

Supplementary Fig. 2

Localization accuracy of single switches before and after drift correction. (PDF 49 kb)

Supplementary Fig. 3

Fluorescence time trace of DNA labeled with multiple switches over many STORM imaging cycles. (PDF 78 kb)

Supplementary Methods (PDF 50 kb)

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Rust, M., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3, 793–796 (2006). https://doi.org/10.1038/nmeth929

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  • DOI: https://doi.org/10.1038/nmeth929

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