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低温ICLEM:低温相关光和电子显微镜,具有沉浸目标

Niko Faul¹, Shih-Ya Chen¹, Christian Lamberz¹

¹Technische Universität Darmstadt, Department of Electrical Engineering and Information Technology, Merckstrasse 25, 64283 Darmstadt, Germany.

Journal of structural biology

|February 19, 2025

概括

我们开发了一种用于关联式冷浸泡光显微镜和电子冷显微镜 (cryo-iCLEM) 的新工作流. 这种方法保留了样品的完整性和超结构,使得在低温温度下进行详细的细胞检测.

科学领域:

细胞和分子成像技术
冷电子显微镜的使用方法
先进的光显微镜技术技术

背景情况:

相对光电子显微镜 (CLEM) 对于分子层次的细胞研究至关重要.
电子显微镜从冷温度中获益,但光显微镜往往缺乏与冷相容的沉浸目标.
现有的冷浸泡光显微镜 (cryo-iLM) 技术尚未被整合到相关的工作流程中.

研究的目的:

介绍一种新的工作流程,用于相关的冷浸泡光显微镜和电子冷显微镜 (cryo-iCLEM).
为了证明冷-iLM与冷电子断层扫描 (cryo-ET) 和聚焦离子束 (FIB) 研磨的兼容性.
为了展示cryo-iCLEM在低温下高分辨率细胞成像的优势.

主要方法:

开发一个cryo-iCLEM工作流程,包括在低温温度下将样品嵌入粘性浸泡介质中.
使用定制的冷浸泡镜头对冷LM进行样本的检查.
在cryo-iLM后,浸泡介质溶解在液体乙中,从而使其能够进行随后的冷电子显微镜 (cryo-EM) 成像.
在cryo-iLM之前和之后应用冷电子断层扫描 (cryo-ET) 来评估样品完整性和超结构.

主要成果:

Cryo-iCLEM保持了超薄,电子透明的样品机械完好无损,而不会降低超结构性保存从结.
工作流程成功成像了FIB磨砂的片,显示机械敏感的样品保持无损.
嵌入浸泡液减少污染,允许扩展数据采集 (小时) 与低光体漂白率.

结论:

Cryo-iCLEM提供了一种强大的方法,可以将高分辨率光显微镜与冷电子显微镜相结合.
这种技术保护了样品的完整性,并提高了在低温温度下成像的能力.
预计开发的工作流程将在冷-CLEM应用中推进各种光显微镜技术.

关键词:

这就是Clem Clem.相对显微镜是相对显微镜.在冷凍-FIB.低温光显微镜的显微镜.低温浸泡显微镜的使用方法样品的准备样品的准备

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