3D histology of human heart-forming organoids by X-ray phase-contrast tomography.

Three-dimensional (3D) imaging is crucial for elucidating the complex structure of organoid models which involve complex spatial cellular and tissue organization in 3D. While a variety of volume imaging methods, including novel light microscopy tools, are now well established to probe the cellular complexity of organoids in 3D, the gold standard for obtaining a precise morphological picture is histology, a traditionally 2D imaging technique that relies on slicing the specimen and therefore has severe limitations in scalability and volumetric imaging. X-ray phase-contrast tomography (XPCT) has emerged as an imaging modality capable of extending conventional histology into the third dimension. While it has been applied to various types of animal and human tissues, its applicability to organoid systems, however, is yet in its infancy. Here, we use XPCT for 3D histology of unstained and formalin-fixed paraffin-embedded human heart-forming organoids (HFOs) at multiple scales and with isotropic resolution. Derived from human pluripotent stem cells, HFOs are a complex and highly structured in vitro model of early heart, foregut and vasculature development, resembling the early human heart-forming region. Using highly coherent synchrotron radiation, we show that HFOs and their different tissue elements can be visualized in their full three-dimensionality and at subcellular scale.