使用 SEM、离子铣削、TEM 和连续切片对植物叶片细胞进行体积 3D 重建。
Volumetric 3D reconstruction of plant leaf cells using SEM, ion milling, TEM, and serial sectioning.
机构信息
Center for Microscopy and Imaging, Baylor University, One Bear Place #97046, Waco, TX, 76798, USA.
Institute of Biology, Plant Sciences, University of Graz, NAWI Graz, Schubertstrasse 51, 8010, Graz, Austria.
出版信息
Planta. 2022 May 6;255(6):118. doi: 10.1007/s00425-022-03905-3.
Focused ion beam scanning electron microscopy is well suited for volumetric extractions and 3D reconstructions of plant cells and its organelles. The three-dimensional (3D) reconstruction of individual plant cells is an important tool to extract volumetric data of organelles and is necessary to fully understand ultrastructural changes and adaptations of plants to their environment. Methods such as the 3D reconstruction of cells based on light microscopical images often lack the resolution necessary to clearly reconstruct all cell compartments within a cell. The 3D reconstruction of cells through serial sectioning transmission electron microscopy (ssTEM) and focused ion beam scanning electron microscopy (FIB-SEM) are powerful alternatives but not widely used in plant sciences. Here, we present a method for the 3D reconstruction and volumetric extraction of plant cells based on FIB milling and compare the results with 3D reconstructions obtained with ssTEM. When compared to 3D reconstruction based on ssTEM, FIB-SEM delivered similar results. The data extracted in this study demonstrated that tobacco cells were larger (31410 µm) than pumpkin cells (20697 µm) and contained more chloroplasts (175 vs. 124), mitochondria (1317 vs. 291) and peroxisomes (745 vs. 79). While individual chloroplasts, mitochondria, peroxisomes were larger in pumpkin plants (25, 53, and 50%, respectively) they covered more total volume in tobacco plants (5390, 395, 374 µm, respectively) due to their higher number per cell when compared to pumpkin plants (4762, 134, 59 µm, respectively). While image acquisition with FIB-SEM was automated, software controlled, and less difficult than ssTEM, FIB milling was slower and sections could not be revised or re-imaged as they were destroyed by the ion beam. Nevertheless, the results in this study demonstrated that both, FIB-SEM and ssTEM, are powerful tools for the 3D reconstruction of and volumetric extraction from plant cells and that there were large differences in size, number, and organelle composition between pumpkin and tobacco cells.
聚焦离子束扫描电子显微镜非常适合于植物细胞及其细胞器的体积提取和 3D 重建。单个植物细胞的 3D 重建是提取细胞器体积数据的重要工具，对于充分了解植物的超微结构变化和对其环境的适应至关重要。基于光学显微镜图像的细胞 3D 重建等方法往往缺乏清晰重建细胞内所有细胞区室所需的分辨率。通过连续切片透射电子显微镜（ssTEM）和聚焦离子束扫描电子显微镜（FIB-SEM）进行细胞的 3D 重建是强大的替代方法，但在植物科学中并未广泛使用。在这里，我们提出了一种基于 FIB 铣削的植物细胞 3D 重建和体积提取方法，并将结果与通过 ssTEM 获得的 3D 重建进行了比较。与基于 ssTEM 的 3D 重建相比，FIB-SEM 提供了相似的结果。本研究中提取的数据表明，烟草细胞（31410 µm）比南瓜细胞（20697 µm）更大，并且含有更多的叶绿体（175 个比 124 个）、线粒体（1317 个比 291 个）和过氧化物酶体（745 个比 79 个）。虽然单个叶绿体、线粒体、过氧化物酶体在南瓜植物中更大（分别为 25%、53%和 50%），但由于与南瓜植物相比，每个细胞的数量更多，它们在烟草植物中覆盖的总体积更大（分别为 5390 µm、395 µm 和 374 µm）（分别为 4762 µm、134 µm 和 59 µm）。虽然 FIB-SEM 的图像采集是自动化的、软件控制的，并且比 ssTEM 更容易，但 FIB 铣削速度较慢，并且由于离子束的破坏，无法对切片进行修订或重新成像。尽管如此，本研究的结果表明，FIB-SEM 和 ssTEM 都是植物细胞 3D 重建和体积提取的有力工具，并且南瓜和烟草细胞在大小、数量和细胞器组成方面存在很大差异。
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