期刊
ELIFE
卷 10, 期 -, 页码 -出版社
eLIFE SCIENCES PUBL LTD
DOI: 10.7554/eLife.70506
关键词
cryo-electron tomography; correlative light and electron microscopy; cryo-FIB-SEM; automation; in situ structural biology; electron microscopy; cryo-lift-out; Chlamydomonas reinhardtii; Drosophila melanogaster; Human; S; cerevisiae; Emiliania huxleyi
类别
资金
- European Research Council [760067, 871037]
- European Molecular Biology Laboratory [664726]
- Max Planck Institute Magdeburg
- Marie Sklodowska-Curie Actions [664726]
- European Research Council (ERC) [760067] Funding Source: European Research Council (ERC)
- Marie Curie Actions (MSCA) [664726] Funding Source: Marie Curie Actions (MSCA)
Lamella micromachining by cryo-FIB at low temperature has become a widely used method for cellular cryo-electron tomography. The improved stability of new-generation cryo-FIB instruments now allows for automated operations. The SerialFIB software tool enables the creation of automated and customizable cryo-FIB preparation protocols, laying the foundation for further development of advanced cryogenic imaging and sample preparation protocols.
Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new-generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with three-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.
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