从玻璃化细胞中光光光光光光光光光光光,电子和离子束显微镜的光光光光光光的光光磨工作流程
Elise G Perton1,2, Daan B Boltje1,3, Arjen J Jakobi2
1Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
Bio-protocol
|July 31, 2025
概括
通过使用一种新的光导向的聚焦离子束扫描电子显微镜 (FIB-SEM) 方法,提高了冷电子断层扫描 (cryo-ET) 样品的准备. 这种技术可以确保目标分子位于薄膜内,从而实现高质量的细胞成像.
科学领域:
- 细胞和分子成像技术
- 结构生物学 结构生物学
- 低温电子显微镜 低温电子显微镜
背景情况:
- 低温电子断层扫描 (cryo-ET) 需要与完整细胞不相容的薄样本 (<200 nm).
- 聚焦离子束扫描电子显微镜 (FIB-SEM) 用于片制备缺乏对目标结构和精确厚度控制的指导.
- 这些局限性阻碍了冷ET的成功率和工作流量吞吐量.
研究的目的:
- 介绍一项用于光引导聚焦离子束 (FIB) 磨削的协议,使用一致的三束冷显微镜.
- 改进用于冷电子断层扫描的玻璃化细胞薄片的制备.
- 提高冷-ET工作流程的成功率和吞吐量.
主要方法:
- 以光显微镜,冷式微冷器和压制阶段为FIB-SEM进行改装.
- 在FIB削过程中对光标记分子进行实时监控.
- 采用反射光显微镜来检测片的厚度和质量控制.
主要成果:
- 通过光指导成功制备含有特定细胞结构的薄片.
- 精确控制叶片的厚度和均性.
- 缓解与盲加工和低于最佳的板块质量相关的问题.
结论:
- 巧合的三束冷显微镜使得精确的,光引导的FIB磨粉用于冷ET样品的准备.
- 这一协议显著提高了向叶片内的特定细胞结构的能力.
- 该方法提高了用于结构生物学的冷ET工作流程的可靠性和效率.
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