扫描传输电子显微镜断层扫描在病毒学中:高压冷,冷替代样本的3D成像
Johannes Georg Wieland1, Julia La Roche2, Tim Bergner2
1Boehringer Ingelheim Pharma GmbH & Co. KG; Central Facility for Electron Microscopy, Ulm University; johannes.wieland@boehringer-ingelheim.com.
Journal of visualized experiments : JoVE
|August 25, 2025
概括
扫描传输电子显微镜 (STEM) 断层扫描可提供高分辨率的3D成像,用于高达1μm厚的生物样本. 本协议详细说明了用于STEM断层扫描的样本准备,使得详细的结构病毒学和相关显微镜研究成为可能.
科学领域:
- 结构生物学
- 显微镜技术
- 病毒学
背景情况:
- 三维 (3D) 电子显微镜 (EM) 对于理解病毒诱导的细胞变化至关重要.
- 传输电子显微镜 (TEM) 断层扫描等常规技术受到样品厚度 (≤200 nm) 的限制.
- 基于扫描电子显微镜 (SEM) 的方法提供更大的体积,但通常具有较低的分辨率或Z轴限制.
研究的目的:
- 提出用于扫描传输电子显微镜 (STEM) 断层扫描的病毒学样本的准备方案.
- 为了实现高分辨率的生物超结构3D成像,直至1μm厚度.
- 证明STEM断层扫描在结构病毒学和相关显微镜中的实用性.
主要方法:
- 高压冷,冷替代和树脂嵌入病毒学标本.
- 扫描传输电子显微镜 (STEM) 断层扫描用于3D超结构分析.
- 相对光电子显微镜 (CLEM) 集成福斯特共振能量转移 (FRET) 成像与STEM断层扫描 (FRET-3D-CLEM).
主要成果:
- 一个强大的准备近原生,室温STEM断层扫描样本的协议.
- 复合性囊泡性口腔炎病毒 (VSV) 病毒形态和芽的详细3D可视化.
- 使用FRET-3D-CLEM对增强感染的纳米纤维的吸收和拆卸的洞察.
结论:
- STEM断层扫描是一种高分辨率3D生物样本结构分析的强大技术,克服了其他EM方法的局限性.
- 本方案有助于在病毒学和纳米技术方面进行先进的研究.
- 与STEM断层扫描相结合的FRET-3D-CLEM提供了前所未有的关于动态细胞过程的见解.
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