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An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM

文献类型： 外文期刊

作者： Guo, Jiansheng 1 ; Wang, Guan 4 ; Tang, Wen 1 ; Song, Dandan 2 ; Wang, Xinqiu 6 ; Hong, Jian 3 ; Zhang, Zhongkai 5 ;

作者机构： 1.Zhejiang Univ, Sir Run Run Shaw Hosp, Sch Med, Dept Pathol, Hangzhou 310058, Zhejiang, Peoples R China

2.Zhejiang Univ, Ctr Cryoelectron Microscopy, Sch Med, Hangzhou 310058, Zhejiang, Peoples R China

3.Zhejiang Univ, Ctr Anal & Measurement, Hangzhou 310029, Peoples R China

4.Zhejiang Univ, Dept Neurobiol, Sch Med, Hangzhou 310058, Zhejiang, Peoples R China

5.Yunnan Acad Agr Sci, Key Lab Southwestern Crop Gene Resources & Germpl, Biotechnol & Genet Germplasm Resources Res Inst, Minist Agr & Rural Affairs,Key Lab Agr Biotechnol, Kunming 650205, Yunnan, Peoples R China

6.Zhejiang Univ, Inst Insect Sci, Hangzhou 310058, Peoples R China

关键词： 3D analysis; FIB-SEM; Cryofixation; Image contrast; High resolution

期刊名称：JOURNAL OF STRUCTURAL BIOLOGY （ 影响因子：2.867； 五年影响因子：4.474 ）

ISSN： 1047-8477

年卷期： 2020 年 212 卷 1 期

页码：

收录情况： SCI

摘要： Compared with conventional two-dimensional transmission electron microscopy (TEM), focused ion beam scanning electron microscopy (FIB-SEM) can provide more comprehensive 3D information on cell substructures at the nanometer scale. Biological samples prepared by cryofixation using high-pressure freezing demonstrate optimal preservation of the morphology of cellular structures, as these are arrested instantly in their near-native states. However, samples from cryofixation often show a weak back-scatter electron signal and bad image contrast in FIB-SEM imaging. In addition, it is impossible to do large amounts of heavy metal staining. This is commonly achieved via established osmium impregnation (OTO) en bloc staining protocols. Here, we compared the FIB-SEM image quality of brain tissues prepared using several common freeze-substitution media, and we developed an approach that overcomes these limitations through a combination of osmium tetroxide, uranyl acetate, tannic acid, and potassium permanganate at proper concentrations, respectively. Using this optimized sample preparation protocol for high-pressure freezing and freeze-substitution, perfect smooth membrane morphology, even of the lipid bilayers of the cell membrane, was readily obtained using FIB-SEM. In addition, our protocol is broadly applicable and we demonstrated successful application to brain tissues, plant tissues, Caenorhabditis elegans, Candida albicans, and chlorella. This approach combines the potential of cryofixation for 3D large volume analysis of subcellular structures with the high-resolution capabilities of FIB-SEM.

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