Clarke Nicholas I, Royle Stephen J
Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry, United Kingdom.
Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry, United Kingdom.
Methods Cell Biol. 2018;145:29-43. doi: 10.1016/bs.mcb.2018.03.010.
The mitotic spindle is a complex structure that coordinates the accurate segregation of chromosomes during cell division. To understand how the mitotic spindle operates at the molecular level, high resolution imaging is needed. Serial block face-scanning electron microscopy (SBF-SEM) is a technique that can be used to visualize the ultrastructure of entire cells, including components of the mitotic spindle such as microtubules, kinetochores, centrosomes, and chromosomes. Although transmission electron microscopy (TEM) has higher resolution, the reconstruction of large volumes using TEM and tomography is labor intensive, whereas SBF-SEM takes only days to process, image, and segment samples. SBF-SEM fills the resolution gap between light microscopy (LM) and TEM. When combined with LM, SBF-SEM provides a platform where dynamic cellular events can be selected and imaged at high resolution. Here we outline methods to use correlation and SBF-SEM to study mitotic spindle architecture in 3D with high resolution.
有丝分裂纺锤体是一种复杂的结构，在细胞分裂过程中协调染色体的精确分离。为了了解有丝分裂纺锤体在分子水平上是如何运作的，需要高分辨率成像。连续块面扫描电子显微镜（SBF-SEM）是一种可用于可视化整个细胞超微结构的技术，包括有丝分裂纺锤体的组成部分，如微管、动粒、中心体和染色体。尽管透射电子显微镜（TEM）具有更高的分辨率，但使用TEM和断层扫描重建大体积样本工作量很大，而SBF-SEM处理、成像和分割样本只需几天时间。SBF-SEM填补了光学显微镜（LM）和TEM之间的分辨率差距。当与LM结合使用时，SBF-SEM提供了一个平台，在这个平台上可以选择动态细胞事件并进行高分辨率成像。在这里，我们概述了使用相关性和SBF-SEM在三维空间中高分辨率研究有丝分裂纺锤体结构的方法。
