MULTISCALE 3D IMAGING AND QUANTITATIVE PHENOTYPING OF PATHOLOGICAL HUMAN BRAIN TISSUE

Deciphering the three-dimensional (3D) multiscale organization of neurons and their connections across large volumes of the human brain remains a major challenge due to the complexity of tissue composition, limited sample availability, and variability in preservation conditions. Nevertheless, 3D analysis provides a comprehensive view of tissue spatial organization, revealing structural complexity and enabling structure-function correlations, as well as insights into how these are altered in brain disease. Achieving this requires high-resolution volumetric imaging techniques, such as light-sheet fluorescence microscopy (LSFM) and two-photon fluorescence microscopy (TPFM), combined with specialized optical clearing and labeling strategies. However, the spatial resolution of these modalities is typically limited to ≈ 250 nm by optical diffraction, making it necessary to use super-resolution techniques or tissue expansion methods to visualize nanometer-sized structures.
Here, we present specialized tissue deparaffinization, clearing, and labeling strategies, together with an optimized protocol for isotropic brain tissue expansion, named SHORT-Ex.
These approaches enabled multiscale 3D imaging of the cytoarchitectural and myeloarchitectural organization of large (centimeter-sized) human brain specimens and were integrated with machine learning–based methods for automated 3D quantitative cellular phenotyping. We applied these methodologies to formalin-fixed and deparaffinized specimens from diverse sources, including postmortem human brainstem, cortical tissue from patients with Alzheimer’s disease, and postsurgical brain specimens from pediatric patients with malformations of cortical development (MCDs).
Collectively, this framework provides comprehensive 3D structural analysis across the spatial hierarchy of the human brain, enabling new insights into the fundamental principles of brain function and facilitating the identification of brain disease-specific cytoarchitectural alterations.