3D Imaging Without Light-Sheet: An Accessible Tissue-Clearing and Confocal Workflow for Human Cortical and Retinal Organoids
TL;DR
This study provides a practical strategy for three-dimensional imaging of intact human neural organoids, facilitating spatial analysis of developmental organization and disease-relevant phenotypes in laboratories without dedicated light-sheet microscopy infrastructure.
Abstract
Human induced pluripotent stem cell (iPSC)-derived neural organoids have emerged as valuable models for investigating human neurodevelopment and neurological disorders. However, their complex three-dimensional architecture poses significant challenges for conventional histological approaches, which rely on physical sectioning and inevitably disrupt spatial relationships within the tissue. Volumetric imaging of intact organoids typically requires light-sheet fluorescence microscopy, a technology not widely accessible to standard cell biology laboratories. Here, we show that solvent-based tissue clearing, using the iDISCO+ and Visikol® HISTO protocols, combined with conventional laser-scanning and spinning-disk confocal microscopy, platforms already available in most imaging facilities, is sufficient to resolve neuroepithelial rosette-like structures, neuronal networks, and astroglial components within intact human cortical and retinal organoids, while preserving immunofluorescent labeling and tissue architecture. The workflow was also compatible with commonly used immunofluorescence markers. Although light-sheet fluorescence microscopy remains advantageous for large-scale whole-sample imaging, our results show that cleared human organoids within the size range analyzed here can be effectively visualized using accessible confocal systems. This study provides a practical strategy for three-dimensional imaging of intact human neural organoids, facilitating spatial analysis of developmental organization and disease-relevant phenotypes in laboratories without dedicated light-sheet microscopy infrastructure.