Article | Published:

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

Nature Methods volume 8, pages 499505 (2011) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

  2. 2.

    , & Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143, 1047–1058 (2010).

  3. 3.

    & Subdiffraction resolution in far-field fluorescence microscopy. Opt. Lett. 24, 954–956 (1999).

  4. 4.

    , & Saturated patterned excitation microscopy—a concept for optical resolution improvement. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 19, 1599–1609 (2002).

  5. 5.

    Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA 102, 13081–13086 (2005).

  6. 6.

    , & Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

  7. 7.

    et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

  8. 8.

    , & Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

  9. 9.

    , & Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

  10. 10.

    et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).

  11. 11.

    et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008).

  12. 12.

    , & Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proc. Natl. Acad. Sci. USA 105, 14271–14276 (2008).

  13. 13.

    , , , & Super-resolution video microscopy of live cells by structured illumination. Nat. Methods 6, 339–342 (2009).

  14. 14.

    , , & Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat. Methods 5, 417–423 (2008).

  15. 15.

    et al. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc. Natl. Acad. Sci. USA 104, 17370–17375 (2007).

  16. 16.

    et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).

  17. 17.

    et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat. Methods 6, 153–159 (2009).

  18. 18.

    et al. Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat. Methods 5, 947–949 (2008).

  19. 19.

    , , & Super-resolution imaging with small organic fluorophores. Angew. Chem. Int. Edn. Engl. 48, 6903–6908 (2009).

  20. 20.

    et al. Live-cell super-resolution imaging with trimethoprim conjugates. Nat. Methods 7, 717–719 (2010).

  21. 21.

    et al. Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys. J. 99, 2686–2694 (2010).

  22. 22.

    et al. Live-cell dSTORM with SNAP-tag fusion proteins. Nat. Methods 8, 7–9 (2011).

  23. 23.

    , , & Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).

  24. 24.

    , , & Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Methods 5, 1047–1052 (2008).

  25. 25.

    Nano-imaging with Storm. Nat. Photonics 3, 365–367 (2009).

  26. 26.

    , , & Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

  27. 27.

    et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

  28. 28.

    et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl. Acad. Sci. USA 106, 2995–2999 (2009).

  29. 29.

    et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. USA 106, 3125–3130 (2009).

  30. 30.

    , , , & Near-isotropic 3D optical nanoscopy with photon-limited chromophores. Proc. Natl. Acad. Sci. USA 107, 10068–10073 (2010).

  31. 31.

    et al. Photoswitching mechanism of cyanine dyes. J. Am. Chem. Soc. 131, 18192–18193 (2009).

  32. 32.

    et al. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285–3290 (2007).

  33. 33.

    & Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether. J. Cell Biol. 129, 1559–1574 (1995).

  34. 34.

    , , & Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat. Struct. Mol. Biol. 11, 567–573 (2004).

  35. 35.

    et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

  36. 36.

    , , & Spatial control of coated-pit dynamics in living cells. Nat. Cell Biol. 1, 1–7 (1999).

  37. 37.

    & Glass beads load macromolecules into living cells. J. Cell Sci. 88, 669–678 (1987).

  38. 38.

    , , , & A bright and photostable photoconvertible fluorescent protein. Nat. Methods 6, 131–133 (2009).

  39. 39.

    et al. EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc. Natl. Acad. Sci. USA 101, 15905–15910 (2004).

  40. 40.

    & Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 9, 929–943 (2008).

  41. 41.

    , & Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell 124, 997–1009 (2006).

  42. 42.

    & Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection. Opt. Lett. 29, 2611–2613 (2004).

Download references

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.

Author information

Author notes

    • Sara A Jones
    •  & Sang-Hee Shim

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Sara A Jones
    • , Sang-Hee Shim
    •  & Xiaowei Zhuang
  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Jiang He
  3. Department of Physics, Harvard University, Cambridge, Massachusetts, USA.

    • Xiaowei Zhuang
  4. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

    • Xiaowei Zhuang

Authors

  1. Search for Sara A Jones in:

  2. Search for Sang-Hee Shim in:

  3. Search for Jiang He in:

  4. Search for Xiaowei Zhuang in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Sang-Hee Shim or Xiaowei Zhuang.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10, Supplementary Table 1

Videos

  1. 1.

    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.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.1605

Further reading