Brief Communication | Published:

Correlative super-resolution fluorescence and metal-replica transmission electron microscopy

Nature Methods volume 11, pages 305308 (2014) | Download Citation

Abstract

We combine super-resolution localization fluorescence microscopy with transmission electron microscopy of metal replicas to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. We validate robust correlation on the scale of 20 nm by imaging endogenous clathrin (in two and three dimensions) and apply the method to find the previously unknown three-dimensional position of the endocytic protein epsin on clathrin-coated structures at the plasma membrane.

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.

    et al. Science 313, 1642–1645 (2006).

  2. 2.

    et al. Angew. Chem. Int. Ed. Engl. 47, 6172–6176 (2008).

  3. 3.

    et al. Mol. Cell. Biol. 31, 4994–5010 (2011).

  4. 4.

    , , & Science 317, 1749–1753 (2007).

  5. 5.

    , , , & PLoS ONE 8, e77209 (2013).

  6. 6.

    et al. Proc. Natl. Acad. Sci. USA 106, 3125–3130 (2009).

  7. 7.

    , , & Curr. Biol. 21, 1167–1175 (2011).

  8. 8.

    et al. J. Cell Biol. 154, 1209–1223 (2001).

  9. 9.

    et al. eLife 2, e01149 (2013).

  10. 10.

    et al. Nat. Methods 8, 80–84 (2011).

  11. 11.

    J. Cell Biol. 84, 560–583 (1980).

  12. 12.

    et al. Nature 419, 361–366 (2002).

  13. 13.

    et al. Cell 149, 124–136 (2012).

  14. 14.

    et al. Traffic 7, 262–281 (2006).

  15. 15.

    , , , & Int. J. Biochem. Cell Biol. 39, 1765–1770 (2007).

  16. 16.

    , & PLoS Biol. 7, e1000191 (2009).

  17. 17.

    J. Struct. Biol. 152, 36–51 (2005).

  18. 18.

    , & J. Struct. Biol. 116, 71–76 (1996).

  19. 19.

    et al. Nature Methods 9, 727–729 (2012).

  20. 20.

    , , & Nat. Cell Biol. 1, 1–7 (1999).

  21. 21.

    et al. Nature 468, 580–584 (2010).

  22. 22.

    et al. Nature Commun. 3, 1154 (2012).

  23. 23.

    , & Nat. Methods 9, 671–675 (2012).

Download references

Acknowledgements

We thank M. Daniels and the US National Heart, Lung, and Blood Institute (NHLBI) electron microscopy core for help with EM; H. Shroff, J. Shaw and K. Neuman for critical reading of the manuscript; W. Li and Y. Wang (Janelia Farm) for EM grid preparation; L. Greene (NHLBI) for antibodies; S. Yu (NHLBI) for plasmid preparation; A. Nagy for acquiring atomic force microscopy (AFM) images; and E. Tyler and A. Hoofring of NIH Medical Arts for design work on Figure 1. J.W.T. is supported by the Intramural Research Program of the NHLBI, NIH. G.S. and H.F.H. are supported by the Howard Hughes Medical Institute.

Author information

Author notes

    • Kem A Sochacki
    •  & Gleb Shtengel

    These authors contributed equally to this work.

Affiliations

  1. National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, Maryland, USA.

    • Kem A Sochacki
    •  & Justin W Taraska
  2. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.

    • Gleb Shtengel
    •  & Harald F Hess
  3. National Institute of Child Health and Human Development, US National Institutes of Health, Bethesda, Maryland, USA.

    • Schuyler B van Engelenburg

Authors

  1. Search for Kem A Sochacki in:

  2. Search for Gleb Shtengel in:

  3. Search for Schuyler B van Engelenburg in:

  4. Search for Harald F Hess in:

  5. Search for Justin W Taraska in:

Contributions

K.A.S., G.S. and J.W.T. designed the experiments. K.A.S. and G.S. performed the experiments. G.S. and K.A.S. processed data. K.A.S. analyzed the results. K.A.S. and J.W.T. wrote the manuscript. S.B.v.E. designed plasmids. J.W.T. and H.F.H. oversaw the project. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Harald F Hess or Justin W Taraska.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8

Videos

  1. 1.

    Correlated clathrin-AF647 tomograms viewed in XY

    Tomograms of six clathrin structures correlated with clathrin-AF647 (clathrin-AF647 is magenta, myristoylated psCFP2 is cyan). The tomograms are viewed in the XY plane and scan the Z dimension over time. Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the Z-dimension. Each tomogram is 459 nm wide and tall.

  2. 2.

    Correlated clathrin-AF647 tomograms viewed in YZ

    The tomograms from Supplementary Video 1 are viewed in the YZ plane and scan the X dimension over time (clathrin-AF647 is magenta, myristoylated psCFP2 is cyan). Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the X-dimension. Each tomogram is 459 nm wide in the Y dimension.

  3. 3.

    Membrane iPALM/EM alignment validation

    A tomogram of clathrin data is viewed in the YZ plane and scans the X dimension over time (clathrin-AF647 is magenta, myristoylated psCFP2 is cyan). An example of membrane misalignment between fluorescence and EM is shown in frames 19-106 on the right hand side. Regions with membrane misalignment were not used for analysis. Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the X-dimension. The tomogram is 215.7 nm high in the Z dimension.

  4. 4.

    Correlated epsin-AF647 tomograms viewed in XY

    Tomograms of six clathrin structures correlated with epsin-AF647 (epsin-AF647 is magenta, myristoylated psCFP2 is cyan). The tomograms are viewed in the XY plane and scan the Z dimension over time. Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the Z-dimension. Each tomogram is 459 nm wide and tall.

  5. 5.

    Correlated epsin-AF647 tomograms viewed in YZ

    The tomograms from Video 4 are viewed in the YZ plane and scan the X dimension over time (epsin-AF647 is magenta, myristoylated psCFP2 is cyan). Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the X-dimension. Each tomogram is 459 nm wide in the Y dimension.

  6. 6.

    Isosurface models of epsin-AF647 tomograms

    TEM tomograms (grey) and their corresponding epsin-AF647 fluorescence (magenta) are represented as isosurface models (Fig. 3f–h). Two models are shown, including three CCSs. Each model is 459 nm in the X and Y dimension and is rotated around the X-axis from 0-90°.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.2816

Further reading