Optimizing Tissue Clearing Protocols for Whole-Organoid Imaging of Human Cortical and Retinal iPSC-Derived Models

Background: Three-dimensional brain organoids derived from human induced pluripotent stem cells provide valuable in vitro systems for studying human brain development and neurological disorders. However, their dense and heterogeneous structure presents significant challenges for high-resolution imaging, particularly when using conventional histological sectioning, which disrupts native cellular architecture. Improved methodologies are needed to preserve the three-dimensional integrity of organoids while enabling detailed visualization of internal structures and cell types. Results: We present a combined methodological approach that integrates solvent-based tissue clearing protocols with high-resolution volumetric imaging using commonly accessible microscopy platforms. Specifically, we applied two clearing techniques—iDISCO plus and Visikol HISTO—to cortical and retinal organoids, followed by imaging with spinning disk confocal microscopy, laser scanning confocal microscopy, and structured illumination microscopy. This strategy enabled deep tissue penetration and preservation of structural features, such as neural rosettes, cortical plate-like zones, and astrocytic networks. Immunostaining with markers for radial glial progenitors, adherent junctions, and mature neuronal and glial elements revealed fine morphological details and spatial organization without the need for sectioning. Minimal necrosis was observed in central regions of organoids, supporting the effectiveness of the clearing protocols in preserving tissue quality. Importantly, our results demonstrate that conventional imaging platforms can achieve sufficient resolution and depth for comprehensive three-dimensional analysis when paired with optimized clearing methods. Conclusions: Our combined protocol for tissue clearing and volumetric imaging offers an accessible, scalable, and effective approach for three-dimensional visualization of brain organoids. By avoiding the use of more complex light-sheet microscopy, this methodology broadens access to high-content imaging of organoids in standard laboratory settings. This platform can facilitate detailed investigation of neurodevelopmental processes, disease phenotypes, and therapeutic responses in organoid models, supporting a wide range of applications in developmental biology and translational research.