Parallel cryo electron tomography on in situ lamellae
Parallel cryo electron tomography on in situ lamellae
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DOI：
10.1038/s41592-022-01690-1
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发表时间：
2022-12-01
期刊：
影响因子：
48
通讯作者：
Danev, Radostin
中科院分区：
文献类型：
--
作者：
Eisenstein, Fabian;Yanagisawa, Haruaki;Danev, Radostin
低温聚焦离子束研磨样品的原位低温电子层析成像技术近年来已成为研究天然细胞环境中大分子复合物结构的一种强有力的技术。然而，以高通量方式记录层析倾斜系列的可能性受到限制，部分原因是薄片状样本。在这里，我们利用一个几何样本模型和光学图像偏移记录几十个倾斜系列并行，从而节省时间，并获得传统上用于跟踪标本运动的样本区域。并行低温电子断层扫描（PACE-tomo）方法实现了每个倾斜系列的吞吐量快于5分钟，并允许收集以前无法到达的样品区域，从而最大限度地提高每个薄片的数据量。在最先进的显微镜和通用显微镜上进行的体外和原位核糖体性能测试证明了PACE-tomo的高通量和高质量。
In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. However, the possibilities for recording tomographic tilt series in a high-throughput manner are limited, in part by the lamella-shaped samples. Here we utilize a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining access to sample areas conventionally used for tracking specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows for the collection of sample areas that were previously unreachable, thus maximizing the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high throughput and quality of PACE-tomo.