Abstract
Human cerebral organoids serve as a quintessential model for deciphering the complexities of brain development in a three-dimensional milieu. However, imaging these organoids, particularly when they exceed several millimeters in size, has been curtailed by the technical impediments such as phototoxicity, slow imaging speeds, and inadequate resolution and imaging depth. Addressing these pivotal challenges, our study has pioneered a high-speed scanning microscope, synergistically coupled with advanced computational image processing. This ensemble has empowered us to monitor the intricate dynamics of neuron and oligodendrocyte development within cerebral organoids across a trajectory of approximately two months. Line-shaped illumination mitigates photodamage and, alongside refined spatial gating, maximizes signal collection through integrating with computational processing. The integration of deconvolution and compressive sensing has improved image contrast by 6-fold, elucidating fine features of the neurites. Thus, noninvasive imaging enabled us to perform long-term tracking of neural and oligodendroglial development in the large-scale human cerebral organoid. Furthermore, our sophisticated volumetric segmentation algorithm has yielded a robust four-dimensional quantitative analysis, encapsulating both neuronal and oligodendroglial maturation. Collectively, these advances mark a significant advancement in the field of neurodevelopment, providing a powerful tool for in-depth study of complex brain organoid systems.