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bioRxiv · 10.64898/2026.03.19.712931

Quantitative 3D cytoarchitecture of human brain organoids using light-sheet microscopy

Vinchure, O. S.·Job, A. V.·Alonso-Olivares, H.·Alkuraya, F. S.·Gabriel, E.·Gopalakrishnan, J.
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Abstract

Human brain organoids are valuable models for studying human brain development and disorders. Although organoids enable multi-omics analyses, understanding developmental mechanisms requires precise three-dimensional mapping of cell types and their dynamics within their cytoarchitecture. Traditional thin-section immunohistochemistry and imaging provide insights but disrupt spatial information. Light-sheet microscopy allows volumetric imaging of intact organoids, but methods and analytical algorithms remain technically challenging. Here, we introduce LUCID-org, an optimized and reproducible technique for Light-sheet imaging of Unsectioned, Cleared, Immunolabeled, and Depth-resolved human brain organoids. Coupled with machine-learning-based quantitative analysis, LUCID-org enables unbiased 3D characterization of cell-type composition and cytoarchitecture. We validated this approach using brain organoids derived from a patient with CENPJ-mutated microcephaly, revealing quantifiable defects in the ventricular zone (VZ), progenitor density, dynamics, cell-type variability, VZ lumen volume, and neuronal distribution compared with healthy controls. The LUCID-org pipeline is straightforward, taking approximately one week, significantly cheaper, non-toxic, and compatible with standard light-sheet microscopes. Therefore, the LUCID-org method could serve as a benchmark for comprehensive 3D analysis of human brain organoids.

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Vinchure, O. S., Job, A. V., Alonso-Olivares, H., Alkuraya, F. S., Gabriel, E., Gopalakrishnan, J.. 2026-03-20. Quantitative 3D cytoarchitecture of human brain organoids using light-sheet microscopy. https://doi.org/10.64898/2026.03.19.712931

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