A modular platform for automated cryo-FIB workflows
- Max Planck Institute of Biochemistry, Germany
- European Molecular Biology Laboratory, Germany
- Max Planck Institute of Biophysics, Germany
Abstract
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 3-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.
Data availability
All code developed in this work is available on GitHub. SerialFIB and a written tutorial can be obtained on: https://github.com/sklumpe/SerialFIB. A comprehensive video tutorial session is uploaded to YouTube: https://www.youtube.com/watch?v=QR7ngJ0apBk. The documentation is available on GitHub (https://github.com/sklumpe/SerialFIB/blob/main/documentation/SFIB.pdf). A Python script for post-processing of cryo-FIB-SEM volume imaging data as described in the method section is available in the same repository. The Python 3-ported 3DCT is available on: https://github.com/hermankhfung/3dct. New 3DCT functions described in this work, scripts for cryo-FLM virtual slice series creation, 3D point-based registration and transformation of cryo-FLM data with respect to cryo-FIB-SEM data, and bUnwarpJ-based analysis of FIB images before and after milling are available on: https://github.com/hermankhfung/tools3dct. All tomograms and subtomogram averages depicted in the figures are deposited to EMDB under accession codes EMD-13832, EMD-13833, EMD-13836, EMD-13837, EMD-13838, EMD-13834, EMD-13835. FIB-SEM volume imaging data for the Sum159 cells and Chlamydomonas reinhardtii are deposited in EMPIAR under accession codes EMPIAR-10847, EMPIAR-10870. FIB, SEM, TEM and fluorescence images used for 3D correlation are available on the BioImage Archive (S-BSST730, S-BSST729). Numerical data for lamella widths and thicknesses presented in Figure 2-figure supplement 4 are tabulated in Figure 2-figure supplement 4-Source Data 1 and 2.
Article and author information
Author details
Christoph W Müller
Funding
European research council (760067)
- Julia Mahamid
European research council (871037)
- Julia Mahamid
EMBL Interdisciplinary Postdoctoral Program under Marie Curie Actions COFUND (664726)
- Herman K H Fung
Max Planck Institute for Biochrmistry
- Sven Klumpe
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Klumpe et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A modular platform for automated cryo-FIB workflows
eLife 10:e70506.
https://doi.org/10.7554/eLife.70506
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