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Fast, three-dimensional super-resolution imaging of live cells

Abstract

We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolutions of 25 nm and temporal resolutions as fast as 0.5 s. We also demonstrated live-cell 3D super-resolution imaging. We obtained 3D spatial resolution of 30 nm in the lateral direction and 50 nm in the axial direction at time resolutions as fast as 1–2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.

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Figure 1: STORM images of transferrin in live cells.
Figure 2: 3D STORM images of CCPs labeled with photoswitchable cyanine dyes via a SNAP tag in live cells.
Figure 3: Two-color 3D STORM images of CCPs and transferrin in live cells.
Figure 4: Comparison of CCPs labeled with various probes.

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Acknowledgements

We thank M. Davidson (Florida State University) and L. Looger (Janelia Farm) for Eos fluorescent protein plasmids. This work is supported in part by the US National Institutes of Health (to X.Z.) and a Collaborative Innovation Award (43667) from Howard Hughes Medical Institute. S.-H.S. is in part supported by the Mary Fieser fellowship. X.Z. receives support from the Howard Hughes Medical Institute.

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Contributions

X.Z. conceived of the project. S.A.J., S.-H.S. and X.Z. designed the experiments. S.A.J. and S.-H.S. performed all experiments and analysis. J.H. assisted with bead-loading experiments. S.A.J., S.-H.S. and X.Z. wrote the manuscript.

Corresponding authors

Correspondence to Sang-Hee Shim or Xiaowei Zhuang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Table 1 (PDF 2966 kb)

Supplementary Movie 1

A differential interference contrast movie of a cell under the STORM imaging conditions. A BS-C-1 cell was placed in imaging buffer and irradiated with a 657-nm laser at 15 kW cm−2 (the maximum laser excitation intensity used in this work). The recording of the movie started immediately after the laser illumination was turned on. The red area corresponds to the illuminated region, which is equivalent to the typical beam size used in STORM experiments. The intracellular vesicles continue to move and cell edge probes its environment throughout the imaging time under this condition. Scale bar, 10 μm. (MOV 5637 kb)

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Jones, S., Shim, SH., He, J. et al. Fast, three-dimensional super-resolution imaging of live cells. Nat Methods 8, 499–505 (2011). https://doi.org/10.1038/nmeth.1605

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