尽量减少低温软 X 射线断层扫描和低温电子断层扫描标本制备过程中的冰污染

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Journal of Structural Biology: X Pub Date : 2024-10-18 DOI:10.1016/j.yjsbx.2024.100113
Chia-Chun Hsieh, Zi-Jing Lin, Lee-Jene Lai
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引用次数: 0

摘要

低温软 X 射线断层成像(Cryo-SXT)是一种新开发的技术,用于对接近原生状态的三维全细胞进行成像。Cryo-SXT 用户需要制备大量低温样品网格,以利用分配的光束时间研究各种条件下的细胞现象。因此,必须尽可能高效、仔细地及时制备冷冻样品网格,以尽量减少冰对冷冻样品网格的污染。在这项研究中,我们设计了一种低温多栅格盒存储系统,包括外壳、漏斗支架和多栅格盒容器。我们的系统不仅增加了可临时存储的冷冻样本网格数量,还减少了冷冻网格-盒式容器的转移频率,从而降低了网格上结冰的概率。我们还将这一系统应用于 A549 低温细胞网格制备。冷冻光学显微镜和冷冻-SXT 的相关图像显示,在使用我们的系统进行制备时,网格上形成的冰非常有限。此外,冷冻-SXT 结果还能观察到线粒体的三维图像,其嵴呈片状。我们的低温多网格盒存储系统可用于低温-SXT 和低温电子断层扫描(cryo-ET)应用。
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Minimizing ice contamination during specimen preparation for cryo-soft X-ray tomography and cryo-electron tomography
Cryo-soft X-ray tomography (cryo-SXT) is a newly developed technique for imaging 3D whole cells in nearly native states. Cryo-SXT users require the preparation of numerous cryo-sample grids to use the allocated beamtime to study cellular phenomena under various conditions. Therefore, it is important to promptly prepare cryo-sample grids as efficiently and carefully as possible to minimize ice contamination on the frozen sample grid. In this study, we designed a cryo-multi-grid-box storage system, which includes a shell, funnel holder, and multi-grid-box container. Our system not only increases the number of cryo-sample grids that can be temporarily stored but also reduces the frequency of cryo grid-box container transfers, thus decreasing the probability of forming ice on the grid. We have also applied this system to A549 cryo cell grid preparation. The correlative images from cryo-light microscopy and cryo-SXT showed that limited ice had formed on the grid when preparation was performed using our system. Additionally, 3D images of mitochondria with the lamellar shape of the cristae could be observed in our cryo-SXT results. Our cryo-multi-grid-box storage system can be used for cryo-SXT and cryo-electron tomography (cryo-ET) applications.
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来源期刊
Journal of Structural Biology: X
Journal of Structural Biology: X Biochemistry, Genetics and Molecular Biology-Structural Biology
CiteScore
6.50
自引率
0.00%
发文量
20
审稿时长
62 days
期刊最新文献
Corrigendum to “Minimizing ice contamination during specimen preparation for cryo-soft X-ray tomography and cryo-electron tomography” [J. Struct. Biol.: X 10(2024) 100113] Editorial by Natalie Reznikov [for Buss et al., “Hierarchical organization of bone in three dimensions: A twist of twists” (2022)] Structural analysis of the stable form of fibroblast growth factor 2 – FGF2-STAB Localization of albumin with correlative super resolution light- and electron microscopy in the kidney Minimizing ice contamination during specimen preparation for cryo-soft X-ray tomography and cryo-electron tomography
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