Immersion Fixation and Staining of Multicubic Millimeter Volumes for Electron Microscopy-Based Connectomics of Human Brain Biopsies

Abstract

Connectomics allows mapping of cells and their circuits at the nanometer scale in volumes of approximately 1 mm³. Given that the human cerebral cortex can be 3 mm in thickness, larger volumes are required. Larger-volume circuit reconstructions of human brain are limited by 1) the availability of fresh biopsies; 2) the need for excellent preservation of ultrastructure, including extracellular space; and 3) the requirement of uniform staining throughout the sample, among other technical challenges. Cerebral cortical samples from neurosurgical patients are available owing to lead placement for deep brain stimulation. Described here is an immersion fixation, heavy metal staining, and tissue processing method that consistently provides excellent ultrastructure throughout human and rodent surgical brain samples of volumes 2 × 2 × 2 mm³ and up to 37 mm³ with one dimension ≤2 mm. This method should allow synapse-level circuit analysis in samples from patients with psychiatric and neurologic disorders.

Keywords: Brain biopsy; Connectomics; Electron microscopy; Human cortex; Immersion fixation; Osmium staining.

Figure 1:. Immersion fixation and heavy metal staining of human and mouse cortical biopsies.

A, Shown, are the steps for immersion fixation and heavy metal staining of large brain biopsy tissue blocks. B, Electron micrograph from a human brain biopsy (7.25 × 3 × 1.2 mm³) taken at a z depth of ~300 μm. The arrows point to synapses, the arrowheads show spine apparatus, “M” are mitochondria, and the asterisk shows myelin C, Electron micrograph from mouse cortical block (2 × 2 × 2 mm³) taken at a z depth of 1mm. The arrows, etc. are as in panel B. D, Electron micrograph from the same human cortical biopsy as shown in panel (B). The ECS is shown in red and is 14.5%. E, Electron micrograph from a different human cortical biopsy (8.25 × 2.5 × 1.8 mm³) at center of x-y and a z depth of ~1mm with 12.2% ECS highlighted in red. F, Electron micrograph of cortex from immersion fixed mouse biopsy (shown in C) with 13.4% ECS shown in red. G, Electron micrograph from mouse biopsy (2 × 2 × 2 mm³) that was obtained after trans-cardiac perfusion. ECS is diminished (4.9%). H, Electron micrograph from immersion fixed mouse cortex showing a spinule at a cortical synapse (arrow). I, Electron micrograph from human cortex showing an omega figure at a synapse (arrow). J, Graph showing a significant increase in omega figures in immersion fixed versus trans-cardiac perfused brain samples (*** p<0.0001, Chi-square test; error bars show SEM). CaCl₂, calcium chloride; ECS, extracellular space; GA, glutaraldehyde; IF, immersion fixed; NaC, sodium cacodylate; PF, perfusion fixed; PFA, paraformaldehyde; TCH, thiocarbohydrazide.

Figure 2:. Serial section volume collection, imaging, alignment and segmentation from human and mouse cortical biopsy (Bx) blocks.

A, An immersion fixed human brain biopsy (7.25 × 3 × 1.2 mm³). B, The biopsy shown in A, processed with the heavy metal staining (2 × 2 × 2 mm³ protocol). The red and blue arrow heads in A and B show the same regions. Dotted white lines show sites of trimming before resin embedding. C, An X-ray tomogram of the same stained sample, z-depth ~300 μm. Dotted white lines as in B. The blue box highlights the region from where a series of 1175, 30nm sections were collected. D, A low-resolution electron micrograph (3 × 2.5 mm²) taken from region highlighted in blue box in C. Blue arrowheads are same as in A-C. The black rectangle shows the region of volume imaged with multibeam scanning electron microscope. E, Low resolution 750 × 250 μm² electron micrograph of highlighted rectangle in D. F, An aligned stack of 160 × 160 × 35 μm³ from the region highlighted with yellow box in E. G, An electron micrograph with cluster of axons traced manually from volume in F. H, Rendering of the manually segmented axons from G; all of them could be traced to two edges of the volume. I, Rendering of a spiny dendrite traced from volume in F. J, Inset region from I, (changed orientation) showing presynaptic axons making synapses on spines of the blue-colored dendrite in I. K, Electron micrograph of region in white square in J, showing a synapse from the red-colored axon onto a dendritic spine. L, Rendering of a manually segmented astrocytic process traced to astrocyte soma. M, A magnified view of an astrocytic process (yellow) and glycogen granules (magenta) from area within white box in L. Arrow in M points to a thin process of astrocyte. N, A low-resolution electron micrograph (shown with arrow) superimposed on x-ray tomogram (shown with asterisk) at z-depth of 1 mm from a 2 × 2 × 2 mm³ mouse brain biopsy. The yellow box shows 300 × 300 μm² region from where series of ~100, 30 nm sections were aligned and automatically segmented. Automatically segmented region from the blue box is shown in O. P, An electron micrograph with automatic 2D segmentation shown in Q. R, Rendering of two automatically segmented spiny dendrites (green and yellow as seen in Q). S, Cluster of automatically segmented axons from yellow boxed area in N. T, Three-dimensional rendering of the axons shown in S. U, A rendering of the 8000 largest objects segmented automatically from region highlighted with yellow box in N.