Evaluation of the validity of large-scale serial sectioning TEM for three-dimensional reconstruction of rice mesophyll cells and chloroplasts.
Evaluation of the validity of large-scale serial sectioning TEM for three-dimensional reconstruction of rice mesophyll cells and chloroplasts.
复制标题
大规模连续切片 TEM 三维重建水稻叶肉细胞和叶绿体的有效性评估。
DOI：
10.1007/s00709-021-01728-9
复制
发表时间：
2022
期刊：
影响因子：
2.9
通讯作者：
Taniguchi M.
中科院分区：
文献类型：
--
作者：
Yamane K;Oi T;Taniguchi M.
连续切片透射电子显微镜是一种利用超微切割机获得的连续切片进行三维重建的经典方法。然而，生产出均匀的长丝带是困难的。这里，在制作了15-30个连续切片(切割间隔为100纳米)的色带后，暂停了超微切割机的运动，然后将色带安装在单独的单槽网格上。然而，由于这种单层扫描电子显微镜方法可能包含影响因素，如切片厚度的不正确间距和切片的扭曲，这是在透射电子显微镜观察下用金刚石刀和光束相互作用产生的，因此对水稻叶肉细胞和叶绿体的定性和定量数据与聚焦离子束扫描电子显微镜(FIB-SEM)(切割间隔为50 nm)得到的数据进行了比较。在3D模型中没有观察到结构变形。此外，两种方法在体积和表面积上也没有显著差异。截面厚度的增加对表面积/体积比有显著影响，但不同方法的差异不显著。我们的方法有助于观察大量的植物细胞和细胞器，从而识别不同大小和类型的叶绿体。在中等生长条件下生长的水稻叶片中，用单层透射电子显微镜证实了叶绿体口袋的形成，这是围绕着其他细胞内隔室的结构。由于90个叶绿体中只有4个形成了口袋结构，所以在适度的生长条件下，这种形成被认为是罕见的。
Serial sectioning transmission electron microscopy (ssTEM) is a classical method of 3D reconstruction using serial sections obtained with an ultramicrotome. However, producing a long ribbon with homogeneity is difficult. Here, ultramicrotome movement was suspended after producing a ribbon of 15–30 serial sections (cutting intervals, 100 nm), and then, the ribbon was mounted on an individual one-slot grid. However, as this ssTEM method may include influencing factors such as incorrect intervals of section thickness and distortion of sections, which is produced by cutting sections using a diamond knife and beam interaction under TEM observation, qualitative and quantitative data on rice mesophyll cells and chloroplasts were compared with those obtained from a focused ion beam scanning electron microscopy (FIB-SEM) (cutting intervals, 50 nm). No structural distortion in 3D models was observed. In addition, no significant differences in the volume and surface area were observed between the two methods. The surface to volume ratio was significantly affected by the increase in section thickness, but not the difference of methodologies. Our method was useful for observing large volumes of plant cells and organelles, leading to the identification of various sizes and types of chloroplasts. The formation of a chloroplast pocket, which is a structure surrounding other intracellular compartments, was confirmed in rice leaves grown under moderate growth conditions using the ssTEM method. As only four out of 90 chloroplasts formed pocket structures, the formation was considered to be rare under the applied moderate growth conditions.