Using X-Ray Microscopy to Visualize 3D Architecture of Native Spheroid and Organoid Samples

Introduction Organoid and spheroid cell cultures have revolutionized in-vitro research allowing for the creation of three-dimensional cell constructs that have the potential of closely mimicking conditions in-vivo. However, three-dimensional cell constructs cannot be visualized sufficiently by histology as only two-dimensional slices can be observed and further sample deterioration may occur in preparation. A novel approach to this issue is using X-ray tomography allowing for three-dimensional visualization and further segmentation of the sample without sample destruction.

Material & Methods Extrahepatic biliary organoids as well as hepatic spheroids were cultivated, and paraffin embedded. 5mm cylinders were cut and mounted for measurement using the ZEISS VersaXRM-500 Xradia micro-CT. Measurement consisted of an overview image of the sample allowing for a spatial identification of samples within the cylinder. Based on the overview, high resolution images were obtained. High resolution images were then reconstructed, and three-dimensional volumes of samples were obtained. Those were further segmented using a region-growing segmentation algorithm to obtain quantitative information about the sample volume.

Results Tissue slices obtained using X-ray tomography highly correlated with histological slides. Three-dimensional reconstruction allowed for the appreciation of morphological features of both organoids and spheroids, giving a clear indication of sample structure and spatial configuration.

Conclusion X-ray microscopy offers a novel approach to analyzing three-dimensional cell culture samples. Using unstained samples, it allows for high spatial resolution allowing for the appreciation of three-dimensional features without the need for the destruction of the samples which can still be used for histological analysis.

Publication History

Article published online:
20 January 2025