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.

中文翻译：

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通过基于实验室的 X 射线显微镜和相位对比计算机断层扫描，对发芽时小活种子的内部 3D 结构进行可视化。

背景 在植物研究中，需要以微米分辨率显示组织内部 3D 结构而不被对比试剂染色，这可以通过具有相位检索技术的 X 射线计算机断层扫描 (CT) 来实现。最近，一种基于实验室的 X 射线显微镜采用相衬 CT 被开发为观察弱吸收生物样品的有力工具。在这里，我们报告了使用基于实验室的 X 射线相位对比 CT 对未染色三色堇种子的观察。结果 将 2 毫米大小的活三色堇种子简单地安装在塑料管内，并通过内部 X 射线照射，以使用基于实验室的 X 射线显微镜收集投影图像。应用相位检索技术来增强投影图像的对比度。除了干燥的种子，试验了对比较差的湿种子发芽。来自干种子和湿种子的相位检索断层扫描显示细胞水平的空间分辨率足以分辨种子中的细胞，并提供足够的对比度来手动描绘胚胎的边界。手动分割允许在萌发的三个不同阶段对胚胎进行 3D 渲染，从而可视化萌发时胚胎的整体形态变化以及胚胎内细胞的空间排列。结论 我们的结果证实了基于实验室的 X 射线相位对比 CT 可用于小种子发育的 3D 结构研究。