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

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

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Authors

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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