Article

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

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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.

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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

    • Shawn Mikula
    •  & Winfried Denk

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

Affiliations

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

    • Shawn Mikula
    •  & Winfried Denk

Authors

  1. Search for Shawn Mikula in:

  2. Search for Winfried Denk in:

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.

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

Videos

  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.

  2. 2.

    Supplementary Video 2

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

  3. 3.

    Supplementary Video 3

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

  4. 4.

    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.