@article{e628e8eb8e3043b28a1678e0c1f36f07,
title = "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",
author = "Neha Karlupia and Schalek, {Richard L.} and Yuelong Wu and Yaron Meirovitch and Donglai Wei and Charney, {Alexander W.} and Kopell, {Brian H.} and Lichtman, {Jeff W.}",
note = "Funding Information: This work was funded by the National Institutes of Health (Grant Nos. UG3-MH123386 [to JWL] and U19-NS104653 [to JWL]) and by the National Institute of Mental Health (Grant No. P50MH094271 [to JWL]). Funding Information: This work was funded by the National Institutes of Health (Grant Nos. UG3-MH123386 [to JWL] and U19-NS104653 [to JWL]) and by the National Institute of Mental Health (Grant No. P50MH094271 [to JWL]). NK and JWL conceived the study. NK designed and performed experiments and collected data. NK and JWL analyzed data. JWL assessed the quality of data. AWC facilitated collection of human cortical biopsies. BHK harvested human cortical biopsies. NK and AWC collected the human biopsies. NK and RLS performed section cutting, screening, and multibeam imaging. YW aligned the human and mouse datasets. NK did manual segmentation in both human and mouse datasets. YM did automatic segmentation of the mouse dataset and computed the largest objects in the volume. NK and YM generated ground truth for automatic segmentation of mouse dataset. DW generated meshes of automatically segmented objects and assessed largest objects. YW wrote the MATLAB script for assessment of extracellular space and for statistical assessment of omegas. NK and JWL wrote the manuscript. We thank Emily Moya, Lillian Wilkins, You Jeong Park, and Alexander Shapson-Coe for help with human biopsy collections. We thank Olga Morozova for help with animal euthanasia and transcardiac perfusion. We thank Savita Karlupia and Rupali Karlupia for manual segmentation of axons in the human dataset. We thank Sarah Hill for help with counting of fused vesicles and Daniel Berger for technical support with the Volume Annotation and Segmentation Tool. The results of this study were partially presented at Society for Neuroscience 2021, 2021-S-3404-SfN: Immersion fixation and staining of large volume of human brain tissue for large scale connectomics. The authors report no biomedical financial interests or potential conflicts of interest. Publisher Copyright: {\textcopyright} 2023 Society of Biological Psychiatry",
year = "2023",
doi = "10.1016/j.biopsych.2023.01.025",
language = "English",
journal = "Biological Psychiatry",
issn = "0006-3223",
publisher = "Elsevier USA",
}