Extracting tissue and cell outlines of Arabidopsis seeds using refraction contrast X-ray CT at the SPring-8 facility

How biological form is determined is one of the important questions in developmental biology. Physical forces are thought to be the primary determinants of the biological forms, and several theories for this were proposed nearly a century ago. To evaluate how physical forces can influence biological forms, precise determination of cell and tissue shapes and their geometries is necessary. Computed tomography (CT) is useful for visualizing three-dimensional structures without destroying a sample. Because recent progress in micro-CT has enabled visualizing cells and tissues at the sub-micron level, we investigated if we could extract cell and tissue outlines of seeds using refraction contrast X-ray CT available at the SPring-8 synchrotron radiation facility. We used Arabidopsis seeds because Arabidopsis is a well-known model plant and its seed size is small enough to obtain whole images using the X-ray CT experimental system. We could trace the outlines of tissues in dry seeds using beamline BL20B2 (10 keV, 2.4μm·pixel^-1). Although we could also detect the outlines of some cell types, the image resolution was not adequate to extract whole cell edges. To detect the edges of cells in the epidermis and cortex, we obtained CT images using beamline BL20XU (8 keV, 0.5 μm·pixel^-1). With these CT images, we could extract the facets and edges of each cell and determine cell vertices. This method enabled us to compare the numbers of cell facets among various cell types. We could also describe cell geometry as a set of points that showed these cell vertices.