Japan Textile Products Quality and Technology Center, Kobe, Hyogo, Japan.
Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto, Japan.
J Microsc. 2020 Apr;278(1):42-48. doi: 10.1111/jmi.12885. Epub 2020 Mar 18.
Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra-high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three-dimensional array of microtubules in a 0.7-µm-thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7-µm-thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial-section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely-distributed cellular organelles such as microtubules for neurite outgrowth. LAY DESCRIPTION: An optimal 3D visualisation of microtubule cytoskeleton using ultra-high voltage electron microscopy tomography The ultra-high voltage electron microscope (UHVEM) is able to visualise a micrometre-thick specimen at nanoscale spatial resolution because of the high-energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7-µm-thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network.
电子断层扫描方法结合传统透射电子显微镜已广泛应用于研究各种细胞结构的三维分布模式，包括神经突中的微管。由于电子的穿透能力取决于切片厚度和加速电压，因此最高加速电压可达 200kV 的传统 TEM 仅限于厚度为 0.2μm 或更薄的样品。在本文中，我们表明，加速电压高于 1000kV（1MV）的超高电压电子显微镜（UHVEM）可实现 0.7μm 厚神经突切片中微管三维排列的清晰重建。与 2MV 下 1.0μm 切片相比，在 1MV 加速电压下，可更清晰地重建 0.7μm 厚切片中的微管详细结构。此外，通过 UHVEM 连续切片断层扫描可以重建神经突中每个微管的整个分布。优化的 UHVEM 断层扫描的应用将提供新的见解，弥合广泛分布的细胞细胞器（如微管）的结构和功能之间的差距，以促进神经突生长。
中文描述：利用超高电压电子显微镜断层扫描术对微管细胞骨架进行最佳 3D 可视化 超高电压电子显微镜（UHVEM）由于高能电子束能够穿透如此厚的标本，因此能够在纳米级空间分辨率下对微米厚的标本进行可视化。在这项研究中，我们结合使用 UHVEM 和电子断层扫描方法，确定了在 0.7μm 厚切片中对微管细胞骨架成像所需的最佳条件。我们的方法提供了有关组成微管网络的各个微管纤维复杂排列的优异 3D 信息。
