Time-resolved cryogenic electron tomography for the study of transient cellular processes.

IF 4.3 3区 材料科学 Q1 ENGINEERING, ELECTRICAL & ELECTRONIC ACS Applied Electronic Materials Pub Date : 2024-07-01 Epub Date: 2024-05-08 DOI:10.1091/mbc.E24-01-0042
Joseph Yoniles, Jacob A Summers, Kara A Zielinski, Cali Antolini, Mayura Panjalingam, Stella Lisova, Frank R Moss, Maximus Aldo Di Perna, Christopher Kupitz, Mark S Hunter, Lois Pollack, Soichi Wakatsuki, Peter D Dahlberg
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Abstract

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling subnanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second before freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 s of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a subsecond temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.

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用于研究瞬时细胞过程的时间分辨低温电子断层扫描。
低温电子断层成像技术(cryo-ET)是生命科学领域分辨率最高的成像技术,可对近原生状态下保存的样本进行亚纳米级的观察。用于制备样品的快速冷冻过程适合进行时间分辨研究,几十年来,研究人员一直致力于体外样品的研究。在这里,我们重点开发一种用于原位时间分辨研究的冷冻设备。该装置先将细胞样本与溶液相刺激剂混合,然后直接喷洒到进入低温液态乙烷的电子显微镜网格上。通过改变装置内细胞和刺激剂溶液的流速,我们可以控制反应时间,从几十毫秒到冷冻前的一秒多。在原理验证演示中,我们将冷冻方法应用于与酸性缓冲液混合的模型细菌新月酵母菌。通过低温电子显微镜,我们解析了整个细胞的结构变化,包括表层蛋白质溶解、外膜变形和细胞质重排,所有这些都在 1.5 秒的反应时间内完成。这种名为时间分辨冷冻电子显微镜(TR-cryo-ET)的新方法通过加入亚秒级时间轴增强了冷冻电子显微镜的功能,并使分子、细胞器或细胞水平的诱导结构变化可视化成为可能。[媒体:见正文]。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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CiteScore
7.20
自引率
4.30%
发文量
567
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