| A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy
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Author:
Date:
2021-04-05
[Abstract] The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum ...
[摘要] [摘要]自由生活的线虫秀丽隐杆线虫是研究发育生物学的流行模型系统。在这里，我们描述了详细的协议，以高压冷冻线虫的胚胎（解剖后离体，或完整的蠕虫内），然后快速冷冻替代。经过处理的样品适合通过常规电子显微镜（EM）或更新的体积EM（vEM）方法（如Focuse d离子束扫描电子显微镜（FIB-SEM））进行超微结构分析。的细胞特征，例如超微结构的NUCL耳信封，染色体，内质网和线粒体保存良好这些实验程序后，并产生精确的三维模型用于可视化和分析（张等人，2020）。在秀丽隐杆线虫合子的前核相遇后，该方案被用于膜和染色体的3D重建（Rahman等，2020）。
[背景技术]线虫是自由生活线虫具有许多特性，使其适合于科学的研究：（1）将蠕虫是〜1毫米长; ...
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| Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography
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Author:
Date:
2015-09-05
[Abstract] Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the ...
[摘要] 低电子层析成像（CET）是用于在亚纳米分辨率下成像细胞和分子结构的成熟技术。 由于该方法仅限于比500nm薄的样品，因此需要合适的样品制备以从较大的细胞体积获得CET数据。 最近，冷冻生物材料的低温聚焦离子束（cryo-FIB）研磨已被证明可再现地产生大的，均匀的，无失真的玻璃体横截面用于最先进的CET。 可以使用cryo-FIB技术稀释所有可以浸入冷冻的真核细胞和原核细胞。 连同低剂量显微镜的进展，这已经转移了原位结构生物学的前沿。 在本协议中，我们描述了冷冻FIB技术的典型步骤，从完全生长的细胞培养开始。 最近研究的三个生物样品作为例子。
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