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VOLUME ELECTRON MICROSCOPY OF CORTICAL ORGANOIDS: METHODS FOR REGION IDENTIFICATION, CONNECTOME RECONSTRUCTION AND ORGANELLE SEGMENTATION
Sveva Dallereand 8 co-authors
Neuroscience Institute Cavalieri Ottolenghi, Department of Neuroscience "Rita Levi Montalcini", University of Turin
FENS Forum 2026 (2026)
Barcelona, Spain
Board PS05-09AM-044
Presentation
Date TBA
Board: PS05-09AM-044
Event Information
Poster Board
PS05-09AM-044
Poster
View posterAbstract
Volume electron microscopy (vEM) enables three-dimensional visualization of neural tissue at nanometer resolution, but to date only a limited number of studies have applied vEM techniques to study the cytoarchitecture and ultrastructure of organoids. In this study, we developed an integrated and scalable vEM workflow tailored to human cortical organoids, designed to support precise region identification and high-resolution ultrastructural analysis. We combine correlative light and electron microscopy: toluidine blue-stained semithin sections, large-area SEM mosaic imaging, focused ion beam-scanning electron microscopy (FIB-SEM), and transmission electron microscopy for ultrastructural validation. By directly comparing two widely used resin embedding strategies, we demonstrate that the protocol derived from DeFelipe and Fairén (1993) and Cano-Astorga et al. (2024) provides superior compatibility with light microscopy and semithin sectioning, facilitating reliable identification of regions of interest and downstream synaptic analysis. On the other hand, Deerinck’s protocol (2010) enhances overall membrane contrast but limits postsynaptic density visualization. High-resolution FIB-SEM imaging of neuropil-like peripheral regions of cortical organoids enabled detailed three-dimensional reconstruction of synapses, neurites, and intracellular organelles, allowing quantitative analysis of synaptic contact geometry, neurite organization, and organelle distribution within defined cellular compartments. The proposed workflow expands the applicability of vEM to organoid-based models and lays the groundwork for comparative investigations of human brain development, neurological disease, and therapeutic interventions.
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