A modular platform for automated cryo-FIB workflows
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
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.
Reviewing Editor
- Axel T Brunger, Stanford University School of Medicine, Howard Hughes Medical Institute, United States
Version history
- Received: May 18, 2021
- Preprint posted: May 20, 2021 (view preprint)
- Accepted: December 23, 2021
- Accepted Manuscript published: December 24, 2021 (version 1)
- Version of Record published: January 17, 2022 (version 2)
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.
Metrics
-
- 5,263
- views
-
- 775
- downloads
-
- 75
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.
-
- Developmental Biology
- Structural Biology and Molecular Biophysics
The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.