Sato Ethel Mendocilla, Baroux Célia
Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland.
Bio Protoc. 2017 Jun 20;7(12):e2355. doi: 10.21769/BioProtoc.2355.
The advent of non-invasive, high-resolution microscopy imaging techniques and computational pipelines for high-throughput image processing has contributed to gain insights in plant organ morphogenesis at the cellular level. Confocal scanning laser microscopy (CSLM) allows the generation of three dimensional images constituted of serial optical sections reporting on stained subcellular structures. Fluorescent labels of cell walls or cell membranes, either chemically or through reporter proteins, are particularly useful for the analyses of tissue organization and cellular shapes in 3D. Image segmentation based on cell boundary signals is used as an input to generate 3D-segments representing cells. These digitalized, 3D objects provide quantitative data on cell shape, size, geometry, position or on (intercellular) intensity signals if additional reporters are used. Herein, we report a detailed, annotated workflow for image segmentation using microscopic data. We used it in the context of a study of tissue patterning during ovule primordium development in Whole carpels are stained for cell boundaries using a modified pseudo-Schiff propidium iodide (mPS-PI) protocol, 3D images are acquired at high resolution by CSLM, segmented and annotated for individual cell types using ImarisCell. This allows for quantitative analyses of cell shape and cell number that are relevant for tissue morphodynamic studies.
非侵入性、高分辨率显微镜成像技术以及用于高通量图像处理的计算流程的出现，有助于在细胞水平上深入了解植物器官形态发生。共聚焦扫描激光显微镜（CSLM）能够生成由一系列光学切片构成的三维图像，这些切片反映了经染色的亚细胞结构。通过化学方法或借助报告蛋白对细胞壁或细胞膜进行荧光标记，对于三维组织结构和细胞形状的分析尤为有用。基于细胞边界信号的图像分割被用作生成代表细胞的三维片段的输入。如果使用额外的报告基因，这些数字化的三维物体可提供有关细胞形状、大小、几何形状、位置或（细胞间）强度信号的定量数据。在此，我们报告了一种使用显微镜数据进行图像分割的详细且带注释的工作流程。我们在一项关于胚珠原基发育过程中组织模式形成的研究中使用了该流程。完整的心皮使用改良的假希夫碘化丙啶（mPS-PI）方案对细胞边界进行染色，通过CSLM以高分辨率获取三维图像，使用ImarisCell对单个细胞类型进行分割和注释。这使得对与组织形态动力学研究相关的细胞形状和细胞数量进行定量分析成为可能。
