Plant Tissues in 3D via X-Ray Tomography: Simple Contrasting Methods Allow High Resolution Imaging

Computed tomography remains strongly underused in plant sciences despite its high potential in delivering detailed 3D phenotypical information because of the low X-ray absorption of most plant tissues. Existing protocols to study soft tissues display poor performance, especially when compared to those used on animals. More efficient protocols to study plant material are therefore needed. Flowers of Arabidopsis thaliana and Marcgravia caudata were immersed in a selection of contrasting agents used to treat samples for transmission electron microscopy. Grayscale values for floral tissues and background were measured as a function of time. Contrast was quantified via a contrast index. The thick buds of Marcgravia were scanned to determine which contrasting agents best penetrate thick tissues. The highest contrast increase with cytoplasm-rich tissues was obtained with phosphotungstate, whereas osmium tetroxide and bismuth tatrate displayed the highest contrast increase with vacuolated tissues. Phosphotungstate also displayed the best sample penetration. Furthermore, infiltration with phosphotungstate allowed imaging of all plants parts at a high resolution of 3 µm, which approaches the maximum resolution of our equipment: 1.5 µm. The high affinity of phosphotungstate for vasculature, cytoplasm-rich tissue, and pollen causes these tissues to absorb more X-rays than the surrounding tissues, which, in turn, makes these tissues appear brighter on the scan data. Tissues with different brightness can then be virtually dissected from each other by selecting the bracket of grayscale to be visualized. Promising directions for the future include in silico phenotyping and developmental studies of plant inner parts (e.g., ovules, vasculature, pollen, and cell nuclei) via virtual dissection as well as correlations of quantitative phenotypes with omics datasets. Therefore, this work represents a crucial improvement of previous methods, allowing new directions of research to be undertaken in areas ranging from morphology to systems biology.尽管计算机断层扫描（computed tomography）在获取精细三维表型信息方面具备极高潜力，但由于多数植物组织的X射线吸收率较低，其在植物科学领域的应用仍严重不足。现有用于植物软组织研究的实验方案性能欠佳，相较于动物组织研究方案尤为突出。因此，亟需开发更为高效的植物材料研究方案。本研究将拟南芥（Arabidopsis thaliana）与Marcgravia caudata的花浸泡于一系列用于透射电子显微镜（transmission electron microscopy）样品处理的对比剂中，随后随时间变化测量花组织与背景的灰度值，并通过对比度指数量化成像对比度。针对Marcgravia caudata的粗壮花蕾开展扫描实验，以筛选出最易穿透厚组织的对比剂。结果表明，富细胞质组织的对比度提升效果最优者为磷钨酸盐；而液泡化组织的对比度提升效果最佳者则为四氧化锇与酒石酸铋。此外，磷钨酸盐同样展现出最优的样品穿透能力。采用磷钨酸盐浸润处理后，本研究可实现3 μm分辨率下所有植物部位的成像，该分辨率接近本实验设备的最大分辨率1.5 μm。磷钨酸盐对维管组织、富细胞质组织及花粉具有高亲和力，可使这些组织相较于周围组织吸收更多X射线，进而在扫描数据中呈现出更高的亮度。通过选取特定灰度区间进行可视化，可对不同亮度的组织实现虚拟解剖分离。未来的研究方向包括通过虚拟解剖开展植物内部结构（如胚珠、维管组织、花粉及细胞核）的计算机模拟表型分析与发育研究，以及定量表型与组学数据集的关联分析。综上，本研究相较此前的方法实现了关键性改进，为从形态学到系统生物学等多个领域的全新研究方向开辟了可能。