CryoFIB milling large tissue samples for cryo-electron tomography
Abstract Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-32716-z |
| _version_ | 1797846052765696000 |
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| author | Sihan Wang Heng Zhou Wei Chen Yifeng Jiang Xuzhen Yan Hong You Xueming Li |
| author_facet | Sihan Wang Heng Zhou Wei Chen Yifeng Jiang Xuzhen Yan Hong You Xueming Li |
| author_sort | Sihan Wang |
| collection | DOAJ |
| description | Abstract Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still difficult steps. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer-thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and localization of the object of interest using cellular secondary electron imaging (CSEI). The milling strategy consists of two steps, a coarse milling step to improve the milling efficiency, followed by a fine milling step. The two-step milling creates a furrow–ridge structure with an additional conductive Pt layer to reduce the beam-induced charging issue. CSEI is highlighted in the workflow, which provides on-the-fly localization during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method. |
| first_indexed | 2024-04-09T17:48:48Z |
| format | Article |
| id | doaj.art-f41a74972a724e1fa77de4bc567b3ecd |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-09T17:48:48Z |
| publishDate | 2023-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-f41a74972a724e1fa77de4bc567b3ecd2023-04-16T11:12:25ZengNature PortfolioScientific Reports2045-23222023-04-0113111210.1038/s41598-023-32716-zCryoFIB milling large tissue samples for cryo-electron tomographySihan Wang0Heng Zhou1Wei Chen2Yifeng Jiang3Xuzhen Yan4Hong You5Xueming Li6Key Laboratory for Protein Sciences of Ministry of Education, School of Life Sciences, Tsinghua UniversityKey Laboratory for Protein Sciences of Ministry of Education, School of Life Sciences, Tsinghua UniversityLiver Research Center, Beijing Friendship Hospital, Capital Medical UniversityZEISS Microscopy Customer Center, Beijing LaboratoryLiver Research Center, Beijing Friendship Hospital, Capital Medical UniversityLiver Research Center, Beijing Friendship Hospital, Capital Medical UniversityKey Laboratory for Protein Sciences of Ministry of Education, School of Life Sciences, Tsinghua UniversityAbstract Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still difficult steps. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer-thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and localization of the object of interest using cellular secondary electron imaging (CSEI). The milling strategy consists of two steps, a coarse milling step to improve the milling efficiency, followed by a fine milling step. The two-step milling creates a furrow–ridge structure with an additional conductive Pt layer to reduce the beam-induced charging issue. CSEI is highlighted in the workflow, which provides on-the-fly localization during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method.https://doi.org/10.1038/s41598-023-32716-z |
| spellingShingle | Sihan Wang Heng Zhou Wei Chen Yifeng Jiang Xuzhen Yan Hong You Xueming Li CryoFIB milling large tissue samples for cryo-electron tomography Scientific Reports |
| title | CryoFIB milling large tissue samples for cryo-electron tomography |
| title_full | CryoFIB milling large tissue samples for cryo-electron tomography |
| title_fullStr | CryoFIB milling large tissue samples for cryo-electron tomography |
| title_full_unstemmed | CryoFIB milling large tissue samples for cryo-electron tomography |
| title_short | CryoFIB milling large tissue samples for cryo-electron tomography |
| title_sort | cryofib milling large tissue samples for cryo electron tomography |
| url | https://doi.org/10.1038/s41598-023-32716-z |
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