集成光显微镜 (iFLM) 用于冷-FIB-研磨和现场冷-ET
Jae Yang1,2, Veronika Vrbovská3, Tilman Franke3
1Department of Biochemistry, University of Wisconsin, Madison, WI USA.
bioRxiv : the preprint server for biology
|July 28, 2023
概括
我们为冷-FIB-SEM开发了一种集成光光显微镜 (iFLM),可实现精确的3D相关工作流程. 这种自动化方法有效地准备了冷保存的细胞层,用于冷电子断层扫描 (cryo-ET) 分析.
科学领域:
- 显微镜和成像技术
- 结构生物学 结构生物学
- 生物技术是生物技术.
背景情况:
- 相对的冷-光光显微镜聚焦离子束 (cryo-FLM-FIB) 研磨对于在位冷电子断层扫描 (cryo-ET) 是至关重要的.
- 对于冷ET样本准备的相关工作流程中精确的定位仍然是一个重大挑战.
研究的目的:
- 开发和验证一个集成的光光显微镜 (iFLM) 模块用于冷-FIB-SEM.
- 建立一个以坐标为基础的,两点的3D相关工作流程,以实现高效的叶片制备.
主要方法:
- 将iFLM模块集成到冷-FIB-SEM系统中.
- 由iFLM引导的自动化FIB削,用于精确准感兴趣的地区.
- 电子断层扫描 (cryo-ET) 从已制备的片收集数据.
主要成果:
- 在24小时内有效制备9-12片片 (厚度≤200纳米).
- 从FIB磨砂的HeLa细胞板块成功获取了冷ET数据.
- 对冷保存样品进行飞行准和自动研磨的演示.
结论:
- 开发的iFLM集成的冷-FIB-SEM工作流显著提高了冷-ET样本的准备.
- 这种方法促进了高效和精确的叶片磨削,用于详细的细胞超结构分析.
- 该工作流适用于各种冷保存的生物标本,包括细胞,细菌和组织.
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