对光遗传定位细胞器进行精确的原位冷冻相关光镜和电子显微镜观察。

IF 4.3 3区 材料科学 Q1 ENGINEERING, ELECTRICAL & ELECTRONIC
ACS Applied Electronic Materials Pub Date : 2024-10-15 Epub Date: 2024-10-30 DOI:10.1242/jcs.262163
Vikas A Tillu, Gregory M I Redpath, James Rae, Juanfang Ruan, Yin Yao, Maria L Cagigas, Renee Whan, Edna C Hardeman, Peter W Gunning, Vaishnavi Ananthanarayanan, Robert G Parton, Nicholas Ariotti
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引用次数: 0

摘要

在细胞质异质、致密和致密的环境中进行原位冷冻电镜观察时,准确定位细胞结构仍然是一项挑战。在这里,我们开发了一种低温关联光电子显微镜(cryo-CLEM)工作流程,它将生长在机械定义基底上的薄细胞结合起来,通过低温电子断层扫描(cryo-ET)快速分析细胞器和大分子复合物。我们将这些先进技术与光遗传学结合起来,将核周围定位的细胞器重新分配到细胞外围,这样就能对细胞器进行可视化,否则细胞区域太厚就无法进行低温电子断层扫描。这种可靠而稳健的工作流程可实现快速原位分析,而无需冷冻聚焦离子束铣削。使用该方案,可在一天内完成细胞冷冻、冷冻荧光显微镜成像和批量冷冻电子显微镜分析。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Precision in situ cryogenic correlative light and electron microscopy of optogenetically positioned organelles.

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense and compacted environment of the cytoplasm remains challenging. Here, we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow that utilizes thin cells grown on a mechanically defined substratum for rapid analysis of organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles that would otherwise be positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.

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来源期刊
CiteScore
7.20
自引率
4.30%
发文量
567
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