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Updated: Jul 24, 2025
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Visualization of Organelles In Situ by Cryo-STEM Tomography
Published on: June 23, 2023
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通过冷-STEM断层扫描在现场可视化器官细胞
Peter Kirchweger1, Debakshi Mullick1, Sharon Grayer Wolf2
1Department of Chemical and Biological Physics, Weizmann Institute of Sciences.
Journal of visualized experiments : JoVE
|July 10, 2023
概括
使用扫描传输电子显微镜 (STEM) 的冷电子断层扫描 (cryo-ET) 克服了样品厚度的限制. 这种方法可以在高分辨率下揭示细胞结构,如线粒体和有机体,而无需样本削.
科学领域:
- 结构生物学 结构生物学
- 细胞生物学 细胞生物学
- 显微镜的使用方法
背景情况:
- 低温电子显微镜 (cryo-EM) 确定生物大分子的近原子分辨率结构.
- 传统的冷电磁断层扫描受到样品厚度的限制,需要样品削,这会破坏3D背景.
- 扫描传输电子显微镜 (STEM) 提供了高分辨率,但需要小心处理敏感的生物样本.
研究的目的:
- 提出使用STEM的冷断层扫描协议,以克服冷EM的厚度限制.
- 描述显微镜配置和自动化用于冷-STEM断层扫描.
- 展示冷-STEM断层扫描用于成像细胞结构的应用.
主要方法:
- 开发了一种使用扫描传输电子显微镜 (STEM) 的冷断层扫描装置.
- 描述了显微镜配置 (两和三电容系统) 和使用SerialEM软件的自动化.
- 实施了批量采集和与光显微镜相关对齐的改进.
主要成果:
- 成功重建了线粒体,可视化了其膜,颗粒和周围的细胞质成分.
- 证明了在细胞质和附着细胞的核外围内的细胞器的成像能力.
- 在不需要聚焦离子束削的情况下实现了高分辨率成像.
结论:
- 化STEM断层扫描绕过了传统化EM的厚度限制.
- 这种技术为细胞器官和宏分子复合体的空间组织提供了详细的见解.
- 低温STEM断层扫描是一种强大的工具,用于在现场研究细胞结构.
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