State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China; Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou 310058, China.
Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou 310058, China.
Micron. 2023 Mar;166:103413. doi: 10.1016/j.micron.2023.103413. Epub 2023 Jan 13.
The quantitative study of plasmodesmata (PD) frequency is routine in plant science for providing information on the potential of intercellular transportation. Here, we report quantification of plasmodesmatal frequency in virus-infected tobacco vascular tissues using serial sectioning and image analysis. The image datasets were collected by focused ion beam-scanning electron microscopy (FIB-SEM), and the measurements of plasmodesmatal frequency were performed after image analysis with commercial computational programs. With a 5-nm step size (less than half the diameter of PD) during FIB sectioning, exhaustive PD sampling was performed in regions of interest. Segmentation of cell wall (CW) and PD from the background densities was performed manually, and PD were assigned automatically to individual CW interfaces by image analysis and then quantified. The PD quantification results were used to compare the plamodesmatal frequencies among different CW interfaces of individual cells and the average frequencies among different cell types were calculated. CWs lacking PD distribution were found in several cellular types, and the PD frequency were used to determine the possible pathways of PD-based symplasmic transportation. The method enables imaging of samples of several cells containing multiple CW interfaces and minimizes PD omission during sectioning and imaging.
质体连丝（PD）频率的定量研究是植物科学中的常规操作，可为细胞间运输的潜力提供信息。在这里，我们报告了使用连续切片和图像分析来量化病毒感染的烟草血管组织中 PD 频率的方法。图像数据集是通过聚焦离子束扫描电子显微镜（FIB-SEM）收集的，并且在使用商业计算程序进行图像分析后，对 PD 频率进行了测量。在 FIB 切片过程中，使用 5nm 的步长（小于 PD 直径的一半），在感兴趣的区域进行了详尽的 PD 采样。通过手动对细胞壁（CW）和 PD 从背景密度中进行分割，并通过图像分析将 PD 自动分配给各个 CW 界面，然后对其进行定量。使用 PD 定量结果来比较个体细胞不同 CW 界面之间的 PD 频率，并计算不同细胞类型之间的平均频率。在几种细胞类型中发现了缺乏 PD 分布的 CW，并且 PD 频率可用于确定基于 PD 的胞质运输的可能途径。该方法能够对包含多个 CW 界面的多个细胞的样本进行成像，并最大限度地减少切片和成像过程中的 PD 遗漏。
