Volume EM connectomics

Microscopy & ex vivo · reads

Volume electron microscopy is electron imaging of fixed, heavy-metal-stained tissue, serial section by serial section, stitched into a three-dimensional volume; the short wavelength of electrons resolves synapses. What it reads is structure: electron-image contrast from the stain, from which cell shapes, synapses and the wiring between cells are reconstructed; it records no voltage or spikes. In a cubic millimetre of human temporal cortex resected during epilepsy surgery, 5019 sections about 34 nm thick were imaged at 4 nm pixels, and a classifier detected about 150 million synapses.

Spatial resolution

2.7–3.5 nm

Temporal resolution

n/a

Field of view

1.05 mm³ (acquired volume)

Neurons covered

82247 neurons (acquired volume)

Reads

heavy-metal-stained electron-image contrast; ultrastructure; 3D cellular geometry and segmentation; automatically detected synapses classified excitatory or inhibitory; inferred synaptic connectivity between pre- and postsynaptic partners

Writes

n/a

Destructive?

terminal

Prerequisites

surgery: Excess anterior middle temporal gyrus tissue from human surgical resection performed as treatment for drug-resistant epilepsy; other: Rapid immersion fixation of the resected human cortical tissue; other: Ex vivo staining and further preservation with osmium and other heavy metals using a ROTO staining protocol; other: Resin embedding and trimming of the stained sample; other: Serial sectioning with an automatic tape-collecting ultramicrotome (ATUM): 5019 sections at an average thickness of 33.9 nm, 92.5% between 30 and 33 nm, for a total sample thickness of 0.170 mm

Theoretical temporal resolution

n/a

Description

Volume electron microscopy resolves dense neural tissue at the synaptic level because the short wavelength of electrons gives extremely high spatial resolution. This makes reconstruction of every cellular element and synapse possible in dense central nervous system networks. In the scoped human example, excess anterior middle temporal gyrus tissue resected during treatment for drug-resistant epilepsy undergoes rapid immersion fixation, which preserves ultrastructure. It is then further preserved and stained with osmium and other heavy metals under a ROTO staining protocol. The stained sample is embedded in resin, trimmed, and cut into thin serial sections that an automatic tape-collecting ultramicrotome collects. A multibeam scanning electron microscope images each section as tiles, and a workflow manager assesses each tile for quality during acquisition and flags problems. Cutting compresses sections in the cutting direction, and a pixel-size correction adjusts for this compression. Stitching and coarse alignment of tiles use microscope stage coordinates, semi-automated feature correspondences, and image patch cross-correlations that relax an elastic triangular mesh of each tile and each section. A fine-scale alignment based on optical flow between neighboring sections removes remaining drift and jitter. Tiles containing cortex combine into a unified image volume of human cerebral cortex.

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