Brain specimens
Hemispheres from five individuals (including half of the cerebrum, cerebellum and brainstem), were used in this study, following informed consent to use the tissue for research and the ethical approval for research by the National Research Ethics Service Committee London - Central. All hemispheres were fixed in 10% neutral buffered formalin (Fig. 1a). The laterality and demographics are summarized in Supplementary Table 1; the donors were neurologically normal, but one case had an undiagnosed, asymptomatic tumour (diameter roughly 10 mm) in the white matter, adjacent to the pars opercularis. This tumour did not pose issues in any of the processing steps described below.
Data acquisition
Our data acquisition pipeline largely leverages our previous work26. We summarize it here for completeness; the reader is referred to the corresponding publication for further details.
MRI scanning
Before dissection, the hemispheres were scanned on a 3-T Siemens MAGNETOM Prisma scanner. The specimens were placed in a container filled with Fluorinert (perfluorocarbon), a proton-free fluid with no MRI signal that yields excellent ex vivo MRI contrast and does not affect downstream histological analysis58. The MRI scans were acquired with a T2-weighted sequence (optimized long echo train 3D fast spin echo59) with the following parameters: TR = 500 ms, TEeff = 69 ms, BW = 558 hertz per pixel, echo spacing = 4.96 ms, echo train length = 58, 10 averages, with 400-μm isotropic resolution, acquisition time for each average = 547 s, total scanning time = 91 min. These scans were processed with a combination of SAMSEG35 and the FreeSurfer 7.0 cortical stream51 to bias-field-correct the images, generate rough subcortical segmentations and obtain white matter and pial surfaces with corresponding parcellations according to the Desikan–Killiany atlas29 (Fig. 1b).
Dissection
After MRI scanning, each hemisphere is dissected to fit into standard 74 mm × 52 mm cassettes. First, each hemisphere was split into cerebrum, cerebellum and brainstem. Using a metal frame as a guide, these were subsequently cut into 10-mm-thick slices in coronal, sagittal and axial orientation, respectively. These slices were photographed inside a rectangular frame of known dimensions for pixel size and perspective correction; we refer to these images as ‘whole slice photographs’. Although the brainstem and cerebellum slices all fit into the cassettes, the cerebrum slices were further cut into as many blocks as needed. ‘Blocked slice photographs’ were also taken for these blocks (Fig. 1c, left).
Tissue processing and sectioning
After standard tissue processing steps, each tissue block was embedded in paraffin wax and sectioned with a sledge microtome at 25-μm thickness. Before each cut, a photograph was taken with a 24 MPx Nikon D5100 camera (ISO = 100, aperture = f/20, shutter speed = automatic) mounted right above the microtome, pointed perpendicularly to the sectioning plane. These photographs (henceforth ‘blockface photographs’) were corrected for pixel size and perspective using fiducial markers. The blockface photographs have poor contrast between grey and white matter (Fig. 1c, right) but also negligible nonlinear geometric distortion, so they can be readily stacked into 3D volumes. A two-dimensional convolutional neural network (CNN) pretrained on the ImageNet dataset60 and fine-tuned on 50 manually labelled examples was used to automatically produce binary tissue masks for the blockface images.
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