Brief Communication

Staining and embedding the whole mouse brain for electron microscopy

Received:
Accepted:
Published online:

Abstract

The development of methods for imaging large contiguous volumes with the electron microscope could allow the complete mapping of a whole mouse brain at the single-axon level. We developed a method based on prolonged immersion that enables staining and embedding of the entire mouse brain with uniform myelin staining and a moderate preservation of the tissue's ultrastructure. We tested the ability to follow myelinated axons using serial block-face electron microscopy.

  • Subscribe to Nature Methods for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    & Nat. Methods 4, 307–308 (2007).

  2. 2.

    et al. PLoS Comput. Biol. 5, e1000334 (2009).

  3. 3.

    et al. J. Neurosci. 29, 14553–14570 (2009).

  4. 4.

    , & J. Cell Biol. 17, 19–58 (1963).

  5. 5.

    , , & J. Natl. Cancer Inst. 11, 439–461 (1950).

  6. 6.

    , , & J. Cell Biol. 12, 385–410 (1962).

  7. 7.

    , & J. Cell Biol. 30, 424–432 (1966).

  8. 8.

    et al. Science 330, 1404–1408 (2010).

  9. 9.

    et al. Front. Neuroinform. 5, 29 (2011).

  10. 10.

    Am. J. Anat. 107, 59–72 (1960).

  11. 11.

    & Histochem. Cell Biol. 2, 1–4 (1960).

  12. 12.

    & Electron Microscopy: Principles and Techniques for Biologists. (Jones & Bartlett Learning, 1999).

  13. 13.

    Principles and Techniques of Electron Microscopy: Biological Applications, 4th edn. (Cambridge University Press, 2000).

  14. 14.

    J. Phys. Chem. 63, 1574–1577 (1959).

  15. 15.

    Exp. Cell Res. 7, 457–479 (1954).

  16. 16.

    & J. Chem. Soc. Trans. 125, 1215–1218 (1924).

  17. 17.

    in Proc. 4th Eur. Reg. Conf. Electron Microsc. (ed. Bocciarelli, D.S.) 11–65 (Rome: Tipografia Polyglotta Vaticana, 1968).

  18. 18.

    in Proc. 11th Meet. Am. Soc. Cell Biol. 146 (1971).

  19. 19.

    , , , & J. Histochem. Cytochem. 13, 629–639 (1965).

  20. 20.

    , & in Basic Neurochemistry: Molecular, Cellular, and Medical Aspects, 7th edn. (eds. Siegel, G., Albers, R.W., Brady, S. & Price, D.) Ch. 4, 51–72 (Academic, 2006).

  21. 21.

    J. Ultrastruct. Res. 26, 31–43 (1969).

  22. 22.

    J. Electron Microsc. (Tokyo) 23, 197 (1974).

  23. 23.

    , & J. Neurocytol. 32, 1165–1179 (2003).

  24. 24.

    Neuropathol. Appl. Neurobiol. 6, 415–420 (1980).

  25. 25.

    & PLoS Biol. 2, e329 (2004).

  26. 26.

    J. Ultrastruct. Res. 22, 168–184 (1968).

  27. 27.

    J. Histochem. Cytochem. 27, 1337–1342 (1979).

  28. 28.

    & J. Histochem. Cytochem. 32, 455–460 (1984).

  29. 29.

    et al. Microsc. Microanal. 16 (suppl. 2), 1138–1139 (2010).

  30. 30.

    Anat. Rec. 133, 305 (1959).

  31. 31.

    , & Microsc. Microanal. (in the press).

  32. 32.

    & Commun. Stat. Theory Methods 6, 813–827 (1977).

  33. 33.

    et al. in Proc. 6th PIMS Ind. Problem Solving Workshop (ed. Macki, J.) Ch. 1, 1–25 (PIMS, 2002).

  34. 34.

    , , & Neuroimage 35, 9–15 (2007).

  35. 35.

    J. Cell Biol. 17, 208–212 (1963).

  36. 36.

    & The Mouse Brain in Stereotaxic Coordinates 3rd edn. (Academic, 2008).

Download references

Acknowledgements

We thank I. Sonntag and M. Helmstaedter for help with traceability analysis, S. Hillmer and U. Mersdorf for help with transmission electron microscopy, K. Briggman, S.K. Mikula and A. Scherbarth for help with staining procedures, B. Titze for help with conductive coating, J. Tritthardt for developing electronic circuits and M. Mueller for help with scanning electron microscopy–related software. We also thank the following student tracers: M. Diemer, C. Domnick, J. Hanne, P. Hofmann, A. Ivanova, H. Jakobi, A. Klein, J. Löffler, J. Nassel, J. Trendel and P. Weber. This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG).

Author information

Affiliations

  1. Max-Planck Institute for Medical Research, Heidelberg, Germany.

    • Shawn Mikula
    • , Jonas Binding
    •  & Winfried Denk

Authors

  1. Search for Shawn Mikula in:

  2. Search for Jonas Binding in:

  3. Search for Winfried Denk in:

Contributions

S.M. and W.D. designed the study and devised the analysis; S.M. carried out the experiments; J.B. and W.D. devised the aberration correction algorithm; S.M. analyzed the data; and S.M. and W.D. wrote the paper.

Competing interests

W.D. receives license income for 3View serial block-face electron microscopy technology from Gatan.

Corresponding authors

Correspondence to Shawn Mikula or Winfried Denk.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figure 1, Supplementary Tables 1–3 and Supplementary Protocol

Videos

  1. 1.

    Fly-through of a subregion from the internal capsule region of interest.

    Voxel size is 40 nm isotropic

  2. 2.

    Rotation animation of axon tracings from the ventroposterolateral nucleus of the dorsal thalamus.

    Nodes of Ranvier are indicated by small gray spheres