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技术文章首页 > 技术文章 > cryo-FIB lift-out 新技术-- Kleindiek
cryo-FIB lift-out 新技术-- Kleindiek
点击次数:142 发布时间:2020-03-25

低温纤维提离技术可以在秀丽隐杆线虫组织中实现分子级别分辨率的低温电子断层扫描,这一成果由马克斯·普朗克生物化学研究所Juergen M. Plitzko和Miroslava Schaffer牵头的联合团队取得。相关论文发表在2019年8月2日出版的《自然—方法学》上。

课题组人员描述了一种制备方法,该方法基于使用低温夹具工具从高压冷冻大块样品中有针对性地提取材料。这种提离技术使低温电子断层扫描可用于多细胞生物和组织,扩大了原位结构生物学的应用范围。研究组开展了对秀丽隐杆线虫(Caenorhabditis elegans)蠕虫中细胞溶质80S核糖体的结构研究,从而证明了该提离技术的潜力。制备质量允许亚图分析,具有足够的分辨率来区分单个核糖体易位状态,并显示核糖体结构中细胞间的显著变化。

据了解,低温聚焦离子束铣削冷冻水化细胞技术,最近为细胞内部空间的研究提供了视角。结合低温电子层析技术,这种方法可以进入细胞内部天然结构,从而实现对大分子的原位结构研究。然而,这种方法主要局限于可以被完全玻璃化冷冻保存的单个细胞。

 

附:英文原文

Title: A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue

Author: Miroslava Schaffer, Stefan Pfeffer, Julia Mahamid, Stephan Kleindiek, Tim Laugks, Sahradha Albert, Benjamin D. Engel, Andreas Rummel, Andrew J. Smith, Wolfgang Baumeister, Juergen M. Plitzko

Issue&Volume:  Volume 16 Issue 8

Abstract: Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure.

DOI: 10.1038/s41592-019-0497-5

 

 

期刊信息

Nature Methods:《自然—方法学》,创刊于2004年。隶属于施普林格·自然出版集团,IF:28.467
 

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