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

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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.

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  1. 1.

    , & Structural neurobiology: missing link to a mechanistic understanding of neural computation. Nat. Rev. Neurosci. 13, 351–358 (2012).

  2. 2.

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

  3. 3.

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

  4. 4.

    , & Wiring specificity in the direction-selectivity circuit of the retina. Nature 471, 183–188 (2011).

  5. 5.

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

  6. 6.

    , , & Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J. Neurosci. 28, 2959–2964 (2008).

  7. 7.

    , & Staining and embedding the whole mouse brain for electron microscopy. Nat. Methods 9, 1198–1201 (2012).

  8. 8.

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

  9. 9.

    & 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).

  10. 10.

    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).

  11. 11.

    , , & Determination of extracellular fluid volume in the dog with ferrocyanide. Pflugers Arch. 357, 275–290 (1975).

  12. 12.

    , & A study of extracellular space in central nervous tissue by freeze-substitution. J. Cell Biol. 25, 117–137 (1965).

  13. 13.

    & 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).

  14. 14.

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

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

    , , & 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).

  19. 19.

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

  20. 20.

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

  21. 21.

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

  22. 22.

    , & Metallic nature of osmium dioxide. Inorg. Chem. 7, 2461–2463 (1968).

  23. 23.

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

  24. 24.

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

  25. 25.

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

  26. 26.

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

  27. 27.

    , & High-accuracy neurite reconstruction for high-throughput neuroanatomy. Nat. Neurosci. 14, 1081–1088 (2011).

  28. 28.

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

  29. 29.

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

  30. 30.

    , , & Automating the collection of ultrathin serial sections for large volume TEM reconstructions. Microsc. Microanal. 12, 86–87 (2006).

  31. 31.

    & Autoxidation of pyrogallol: general characteristics and inhibition by catalase. Nature 181, 1153–1154 (1958).

  32. 32.

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

  33. 33.

    , & Low-dosage maximum-a-posteriori focusing and stigmation (MAPFoSt). Microsc. Microanal. 19, 38–55 (2013).

  34. 34.

    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).

  35. 35.

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

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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

    • Shawn Mikula
    •  & Winfried Denk

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


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

    • Shawn Mikula
    •  & Winfried Denk


  1. Search for Shawn Mikula in:

  2. Search for Winfried Denk in:


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.

Competing interests

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

Corresponding author

Correspondence to Shawn Mikula.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5


  1. 1.

    Supplementary Video 1

    Striatum SBEM stackHigh-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.

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    Supplementary Video 2

    Somatosensory cortex SBEM stackHigh-magnification somatosensory cortex SBEM stack (cropped to 6.4 × 5.1 × 15.8 micron).

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    Supplementary Video 3

    External capsule SBEM stackHigh-magnification external capsule SBEM stack (cropped to 6.4 × 5.1 × 15.8 micron).

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    Supplementary Video 4

    X-ray microCT fly-throughX-ray microCT fly-through of whole-brain in Fig. 4a.

Zip files

  1. 1.

    Supplementary Data

    Multi-page tiff stacks of synapses corresponding to Fig. 3b,c and the inset of c.

  2. 2.

    Supplementary Software

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