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Nature Methods | Brief Communication

Staining and embedding the whole mouse brain for electron microscopy

Journal name:
Nature Methods
Volume:
9,
Pages:
1198–1201
Year published:
DOI:
doi:10.1038/nmeth.2213
Received
Accepted
Published online

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.

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Figures

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  1. Whole mouse brain stained with wbPATCO and embedded with Quetol.
    Figure 1: Whole mouse brain stained with wbPATCO and embedded with Quetol.

    (a) Block-face image of a whole-brain cross-section cut at the level of bregma −1.26, coated with 5 nm platinum-carbon and imaged with scanning electron microscopy (SEM) using secondary-electron detection. Inset, horizontal view of the entire mouse brain after embedding; block dimensions are 6 × 8.5 × 14 mm3. AP, anterior-posterior. (b) Single image tile (green box in a). Inset, magnified subregion indicated by the blue box. (c,d) High-magnification SEM images from labeled asterisks of the block face in a, demonstrating ultrastructural preservation of deep (c) and superficial (d) regions. Note membrane discontinuities (asterisks) and postsynaptic densities (arrowheads). (e) High-magnification transmission electron microscopy (TEM) image of a 70-nm section taken from a region in the dorsolateral striatum (labeled asterisk in a) in a different sample. Note the intra-period and major dense lines in the inset in e. mit, mitochondrion; Ax, axon; m, microtubules.

  2. Traceability analysis of eight regions of interest (ROIs) in the mouse brain.
    Figure 2: Traceability analysis of eight regions of interest (ROIs) in the mouse brain.

    (a) Parasagittal view showing projections of ROIs. (b) Horizontal view of ROIs through a transparent brain. (c) Serial block-face electron microscopy stack from the corpus callosum, cut down the middle, with 50 traced axons emerging, randomly colored. (d) Single xy image from the stack. (e) yz reslice from the center of a cube (same as in c) showing locations of the 50 seed tracings (cyan points) for the axons shown in c. Amyg, amygdala; CC, corpus callosum; DHC, dorsal hippocampal commissure; DpMe, deep mesencephalic nucleus; EC, external capsule; IC, internal capsule; V1, primary visual cortex (superficial layers); VPL, ventroposterolateral nucleus of the dorsal thalamus. Color coding in a and b denotes major brain subdivisions (cerebral cortex, olfactory bulb, cerebellum, brainstem, superior colliculus and so on).

  3. Analysis of axon morphological diversity and tracing error rate.
    Figure 3: Analysis of axon morphological diversity and tracing error rate.

    (a–c) Axons traced in the ventroposterolateral nucleus (VPL) of the dorsal thalamus (a) with corresponding volumetric representation (b), and axons traced in the internal capsule (c). Fifty axons are displayed per region, with five axons highlighted in different colors to emphasize individual axon morphologies in relation to axon bundles and groupings (a,c). Nodes of Ranvier are indicated by small gray spheres. (d) Quantification of node-of-Ranvier length versus axon caliber in all eight regions of interest for axons containing a node of Ranvier with matching paranodes contained in the serial block-face electron microscopy cube. Ellipses indicate the s.d. of the data (n = 339 axons) for white- and gray-matter regions. (e,f) White-matter (e) and gray-matter (f) tracing error rates for myelinated axons of different axon-diameter bands and data display resolutions (n = 200 axons for each plot).

Videos

  1. Fly-through of a subregion from the internal capsule region of interest.
    Video 1: Fly-through of a subregion from the internal capsule region of interest.
    Voxel size is 40 nm isotropic
  2. Rotation animation of axon tracings from the ventroposterolateral nucleus of the dorsal thalamus.
    Video 2: Rotation animation of axon tracings from the ventroposterolateral nucleus of the dorsal thalamus.
    Nodes of Ranvier are indicated by small gray spheres

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Author information

Affiliations

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

    • Shawn Mikula,
    • Jonas Binding &
    • Winfried Denk

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 financial interests

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

Corresponding authors

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Supplementary information

Video

  1. Video 1: Fly-through of a subregion from the internal capsule region of interest. (24,048 KB, Download)
    Voxel size is 40 nm isotropic
  2. Video 2: Rotation animation of axon tracings from the ventroposterolateral nucleus of the dorsal thalamus. (17,599 KB, Download)
    Nodes of Ranvier are indicated by small gray spheres

PDF files

  1. Supplementary Text and Figures (680 KB)

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

Additional data