Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

High-resolution whole-brain staining for electron microscopic circuit reconstruction

Nature Methods volume 12, pages 541–546 (2015)Cite this article

Subjects

Abstract

Currently only electron microscopy provides the resolution necessary to reconstruct neuronal circuits completely and with single-synapse resolution. Because almost all behaviors rely on neural computations widely distributed throughout the brain, a reconstruction of brain-wide circuits—and, ultimately, the entire brain—is highly desirable. However, these reconstructions require the undivided brain to be prepared for electron microscopic observation. Here we describe a preparation, BROPA (brain-wide reduced-osmium staining with pyrogallol-mediated amplification), that results in the preservation and staining of ultrastructural details throughout the brain at a resolution necessary for tracing neuronal processes and identifying synaptic contacts between them. Using serial block-face electron microscopy (SBEM), we tested human annotator ability to follow neural ‘wires’ reliably and over long distances as well as the ability to detect synaptic contacts. Our results suggest that the BROPA method can produce a preparation suitable for the reconstruction of neural circuits spanning an entire mouse brain.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: BROPA overcomes stain-penetration limitations for reduced-osmium staining.
Figure 2: Tissue ultrastructure is preserved well throughout BROPA-prepared brains.
Figure 3: BROPA-prepared brains allow reliable synapse identification and neurite tracing.
Figure 4: Staining density and sample integrity can be assessed using X-ray microCT.

Similar content being viewed by others

References

  1. Denk, W., Briggman, K.L. & Helmstaedter, M. Structural neurobiology: missing link to a mechanistic understanding of neural computation. Nat. Rev. Neurosci. 13, 351–358 (2012).

    Article  CAS  Google Scholar 

  2. Lichtman, J.W. & Denk, W. The big and the small: challenges of imaging the brain's circuits. Science 334, 618–623 (2011).

    Article  CAS  Google Scholar 

  3. Jarrell, T.A. et al. The connectome of a decision-making neural network. Science 337, 437–444 (2012).

    Article  CAS  Google Scholar 

  4. Briggman, K.L., Helmstaedter, M. & Denk, W. Wiring specificity in the direction-selectivity circuit of the retina. Nature 471, 183–188 (2011).

    Article  CAS  Google Scholar 

  5. Bock, D.D. et al. Network anatomy and in vivo physiology of visual cortical neurons. Nature 471, 177–182 (2011).

    Article  CAS  Google Scholar 

  6. Knott, G., Marchman, H., Wall, D. & Lich, B. Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J. Neurosci. 28, 2959–2964 (2008).

    Article  CAS  Google Scholar 

  7. Mikula, S., Binding, J. & Denk, W. Staining and embedding the whole mouse brain for electron microscopy. Nat. Methods 9, 1198–1201 (2012).

    Article  CAS  Google Scholar 

  8. Li, A. et al. Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain. Science 330, 1404–1408 (2010).

    Article  CAS  Google Scholar 

  9. Willingham, M.C. & Rutherford, A.V. The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells. J. Histochem. Cytochem. 32, 455–460 (1984).

    Article  CAS  Google Scholar 

  10. Deerinck, T.J. et al. Enhancing serial block-face scanning electron microscopy to enable high resolution 3-D nanohistology of cells and tissues. Microsc. Microanal. 16, 1138–1139 (2010).

    Article  CAS  Google Scholar 

  11. Zweens, J., Frankena, H., Rispens, P. & Zijlstra, W.G. Determination of extracellular fluid volume in the dog with ferrocyanide. Pflugers Arch. 357, 275–290 (1975).

    Article  CAS  Google Scholar 

  12. Van Harreveld, A., Crowell, J. & Malhotra, S.K. A study of extracellular space in central nervous tissue by freeze-substitution. J. Cell Biol. 25, 117–137 (1965).

    Article  CAS  Google Scholar 

  13. Thorne, R.G. & Nicholson, C. In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space. Proc. Natl. Acad. Sci. USA 103, 5567–5572 (2006).

    Article  CAS  Google Scholar 

  14. Van Harreveld, A. in The Structure and Function of Nervous Tissue (ed. Bourne, G.H.) Ch. 10, 447–511 (Academic Press, 1972).

  15. Cragg, B. Preservation of extracellular space during fixation of the brain for electron microscopy. Tissue Cell 12, 63–72 (1980).

    Article  CAS  Google Scholar 

  16. Rivlin, P.K. & Raymond, P.A. Use of osmium tetroxide-potassium ferricyanide in reconstructing cells from serial ultrathin sections. J. Neurosci. Methods 20, 23–33 (1987).

    Article  CAS  Google Scholar 

  17. Karnovsky, M.J. Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. Proc. Am. Soc. J. Cell Biol. 51, 146a (1971).

    Google Scholar 

  18. Alexander, R., Ko, E.C.F., Mac, Y.C. & Parker, A.J. Solvation of ions. XI. Solubility products and instability constants in water methanol, formamide, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, acetonitrile, and hexamethylphosphorotriamide. J. Am. Chem. Soc. 89, 3703–3712 (1967).

    Article  CAS  Google Scholar 

  19. Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004).

    Article  Google Scholar 

  20. Bahr, G.F. Osmium tetroxide and ruthenium tetroxide and their reactions with biologically important substances: electron stains III. Exp. Cell Res. 7, 457–479 (1954).

    Article  CAS  Google Scholar 

  21. Hayat, M.A. Principles and Techniques of Electron Microscopy: Biological Applications 4th edn. (Cambridge Univ. Press, 2000).

  22. Greedan, J.E., Willson, D.B. & Haas, T.E. Metallic nature of osmium dioxide. Inorg. Chem. 7, 2461–2463 (1968).

    Article  CAS  Google Scholar 

  23. Eberle, A.L. et al. High-resolution, high-throughput imaging with a multibeam scanning electron microscope. J. Microsc. 10.1111/jmi.12224 (27 January 2015).

  24. Titze, B. & Denk, W. Automated in-chamber specimen coating for serial block-face electron microscopy. J. Microsc. 250, 101–110 (2013).

    Article  CAS  Google Scholar 

  25. Pfenninger, K.H. The cytochemistry of synaptic densities. I. An analysis of the bismuth iodide impregnation method. J. Ultrastruct. Res. 34, 103–122 (1971).

    Article  CAS  Google Scholar 

  26. Tapia, J.C. et al. High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy. Nat. Protoc. 7, 193–206 (2012).

    Article  CAS  Google Scholar 

  27. Helmstaedter, M., Briggman, K.L. & Denk, W. High-accuracy neurite reconstruction for high-throughput neuroanatomy. Nat. Neurosci. 14, 1081–1088 (2011).

    Article  CAS  Google Scholar 

  28. Tomassy, G.S. et al. Distinct profiles of myelin distribution along single axons of pyramidal neurons in the neocortex. Science 344, 319–324 (2014).

    Article  CAS  Google Scholar 

  29. Hayworth, K.J. et al. Ultrastructurally smooth thick partitioning and volume stitching for large-scale connectomics. Nat. Methods 10.1038/nmeth.3292 (16 February 2015).

  30. Hayworth, K.J., Kasthuri, N., Schalek, R. & Lichtman, J.W. Automating the collection of ultrathin serial sections for large volume TEM reconstructions. Microsc. Microanal. 12, 86–87 (2006).

    Article  Google Scholar 

  31. Siegel, S.M. & Siegel, B.Z. Autoxidation of pyrogallol: general characteristics and inhibition by catalase. Nature 181, 1153–1154 (1958).

    Article  CAS  Google Scholar 

  32. Spurr, A.R. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31–43 (1969).

    Article  CAS  Google Scholar 

  33. Binding, J., Mikula, S. & Denk, W. Low-dosage maximum-a-posteriori focusing and stigmation (MAPFoSt). Microsc. Microanal. 19, 38–55 (2013).

    Article  CAS  Google Scholar 

  34. Gundersen, H.J.G. et al. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 96, 857–881 (1988).

    Article  CAS  Google Scholar 

  35. Fiala, J.C. & Harris, K.M. Extending unbiased stereology of brain ultrastructure to three-dimensional volumes. J. Am. Med. Inform. Assoc. 8, 1–16 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K.L. Briggman, K. Hayworth and S.K. Mikula for discussions; J. Bollmann, J. Kornfeld and S.K. Mikula for comments on the manuscript; A. Scherbarth, S.K. Mikula, M. Mueller, C. Roome, R. Shoeman, B. Titze and J. Tritthardt for technical support, D. Zeidler for providing the multi-beam images, J. Kornfeld, I. Sonntag, and F. Svara for help with traceability analysis and tracer organization; and D. Bornhorst, A. Greiss, U. Häusler, J. Hügle, A. Ivanova, F. Kaufhold, C. Kehrel, P. Kroemer, J. Loeffler, L. Muenster, S. Oberrauch, J. Phillip, N. Reisert, N. Scherer, F. Scheu and M. Webeler for tracing neurites. This work was supported by the Max Planck Society.

Author information

Author notes

  1. Shawn Mikula & Winfried Denk

    Present address: Present address: Electrons – Photons – Neurons, Max Planck Institute for Neurobiology, Martinsried, Germany.,

Authors and Affiliations

  1. Department of Biomedical Optics, Max Planck Institute for Medical Research, Heidelberg, Germany

    Shawn Mikula & Winfried Denk

Authors
  1. Shawn Mikula
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Winfried Denk
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.M. and W.D. conceived of the project and wrote the paper. S.M. designed the study, carried out the experiments and analyzed the data.

Corresponding author

Correspondence to Shawn Mikula.

Ethics declarations

Competing interests

W.D. receives license income for SBEM technology (Gatan, 3View).

Integrated supplementary information

Supplementary Figure 1 ROTO staining and ultrastructural preservation over different depths.

SEM images for ROTO-prepared samples without, (a-e), and with, (f-i), extracellular space (ECS) preservation. Locations for high-resolution images indicated in the corresponding low-resolution scans. Imaging parameters are 440 nm pixel size, 2.8 kV, 100 pA, and 0.0064 e-/nm2 in a and f, 5 nm pixel size, 2.8 kV, 300 pA, and 600 e-/nm2 in b-e, and 10 nm pixel size, 2.8 kV, 100 pA, and 100 e-/nm2 in g-i. Scales bars are 100 μm in a and f, 250 nm in b-e and 500 nm in g-i.

Supplementary Figure 2 Mechanical disruption in large ROTO-prepared samples.

SEM block-face image (220 nm pixel size, 2.8 kV, 100 pA, and 0.0256 e-/nm2) taken from a ROTO sample (with 10% formamide added to the reduced osmium step to increase stain penetration) prepared using an ECS-preserving perfusion medium. The scale bar corresponds to 100 μm.

Supplementary Figure 3 Multibeam SEM imaging of a BROPA-prepared sample.

(a) Schematic of the prototype multi-beam microscope. (b) Image (3.8 nm pixel size, 430 pA, 100 ns pixel dwell time, 470 MPixel/s effective scan rate, 18.6 e-/nm2) of a block-face coated with a thin film of palladium to avoid charging. Each tile corresponds to an image taken by one of the 61 beams that scan the sample simultaneously. (c) Subregions as indicated in b. (d) Subregion from multi-beam SEM image acquired at high current (4.6 nm pixel size, 3 nA, 50 ns pixel dwell time, 755 MPixel/s effective scan rate, 44.2 e-/nm2). The sample is the same as in b. Asymmetric synapse onto a spine head (arrowhead). Scale bars are 10 μm in b and 1 μm in c. Scale bar in c also applies to d.

Supplementary Figure 4 Inter-areal SBEM and neurite traceability.

(a) Complete block-face SEM image (top, 440 nm pixel size, 4 kV, 150 pA, 0.0097 e-/nm2) of a BROPA brain and a higher-magnification view of the region of interest (bottom). The red rectangle shows the approximate extent of a continuous SBEM stack. The normal of the stack images is along the long axis of the rectangle. (b) From the left: surface view of aligned stack; manually identified neuronal (blue) and glial (red) nuclei; neurites emerging from 381 randomly-selected nuclei. 6 of 381 neurons (3 each in cortex and striatum) together with 100 randomly selected external-capsule axons. Note that one of them veers into cortex. (c) Single cortical cell with dendrites (thin blue lines), the initial segment and an ascending collateral (thick red, both unmyelinated), and the descending axon (thin blue line) with nodes of Ranvier (red). Scale bars are 1 mm in a (top), 500 µm in a (bottom), and 40 µm in c.

Supplementary Figure 5 Crack in BROPA sample.

(a) Cracks, generally devoid of epoxy, are occasionally observed in epoxy-embedded BROPA samples (see Supplementary Video 4). (b) High-magnification image of the red asterisk in a. Imaging parameters are 220 nm pixel size, 3.0 kV and 0.29 e-/nm2 in a and 10 nm pixel size, 3.0 kV and 140 e-/nm2 in b. Scale bars are 100 μm in a and 5 μm in b.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 1366 kb)

Supplementary Video 1

Striatum SBEM stack High-magnification striatum SBEM stack (cropped to 6.4 × 5.1 × 15.8 micron) showing the dendrite and synapse segmentation from Fig. 3a overlaid onto the original data. (MOV 11250 kb)

Supplementary Video 2

Somatosensory cortex SBEM stack High-magnification somatosensory cortex SBEM stack (cropped to 6.4 × 5.1 × 15.8 micron). (MOV 17593 kb)

Supplementary Video 3

External capsule SBEM stack High-magnification external capsule SBEM stack (cropped to 6.4 × 5.1 × 15.8 micron). (MOV 17648 kb)

Supplementary Video 4

X-ray microCT fly-through X-ray microCT fly-through of whole-brain in Fig. 4a. (MOV 4674 kb)

Supplementary Data

Multi-page tiff stacks of synapses corresponding to Fig. 3b,c and the inset of c. (ZIP 9112 kb)

Supplementary Software

Matlab code used for the neurite traceability analysis (RESCOP) provided as a compressed folder. See “readme” file in folder. (ZIP 171 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mikula, S., Denk, W. High-resolution whole-brain staining for electron microscopic circuit reconstruction. Nat Methods 12, 541–546 (2015). https://doi.org/10.1038/nmeth.3361

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.3361

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing