A modular platform for automated cryo-FIB workflows

eLife

Philipp S Erdmann¹  Sven Klumpe¹  Jürgen M Plitzko¹  Xiaojie Zhang²  Janina Baumbach²  Christoph W Müller²  Herman KH Fung²  Bernhard Hampoelz²  Ievgeniia Zagoriy²  Julia Mahamid²  Martin Beck³  Sara K Goetz⁴ 

[1] Department Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany;Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany;Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany;Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany;Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany;Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences, Heidelberg, Germany;

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;   

DOI  :  10.7554/eLife.70506

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY