Visualization of internal 3D structure of small live seed on germination by laboratory-based X-ray microscopy with phase contrast computed tomography

BACKGROUND: The visualization of internal 3D-structure of tissues at micron resolutions without staining by contrast reagents is desirable in plant researches, and it can be achieved by an X-ray computed tomography (CT) with a phase-retrieval technique. Recently, a laboratory-based X-ray microscope adopting the phase contrast CT was developed as a powerful tool for the observation of weakly absorbing biological samples. Here we report the observation of unstained pansy seeds using the laboratory-based X-ray phase-contrast CT. RESULTS: A live pansy seed within 2 mm in size was simply mounted inside a plastic tube and irradiated by in-house X-rays to collect projection images using a laboratory-based X-ray microscope. The phase-retrieval technique was applied to enhance contrasts in the projection images. In addition to a dry seed, wet seeds on germination with the poorer contrasts were tried. The phase-retrieved tomograms from both the dry and the wet seeds revealed a cellular level of spatial resolutions that were enough to resolve cells in the seeds, and provided enough contrasts to delineate the boundary of embryos manually. The manual segmentation allowed a 3D rendering of embryos at three different stages in the germination, which visualized an overall morphological change of the embryo upon germination as well as a spatial arrangement of cells inside the embryo. CONCLUSIONS: Our results confirmed an availability of the laboratory-based X-ray phase-contrast CT for a 3D-structural study on the development of small seeds. The present method may provide a unique way to observe live plant tissues at micron resolutions without structural perturbations due to the sample preparation.