Cryo-plasma FIB/SEM volume imaging of biological specimens
Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in-situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.
Raw data, along with segmentation and associated TEM overviews (if relevant) are deposited on the EMPIAR data repository: EMPIAR-11414, EMPIAR-11415, EMPIAR-11416, EMPIAR-11417, EMPIAR-11418, EMPIAR-11419, EMPIAR-11420, EMPIAR-11421. Code can be found on the Rosalind Franklin Institute GitHub (https://github.com/rosalindfranklininstitute/) and the serialFIB GitHub (https://github.com/sklumpe/SerialFIB/).
Article and author information
Wellcome Trust (220526/Z/20/Z)
- James H Naismith
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All of the animals were handled according to approved, reviewed institutional animal care procedures. Mice were euthanised in a schedule 1 procedure via intraperitoneal injection of sodium pentobarbital followed by decapitation following licensed procedures approved by the Mary Lyon Centre and the Home Office UK. All operating procedures were designed to minimise any suffering for the animals involved in the study.
- Giulia Zanetti, Birkbeck, University of London, United Kingdom
- Preprint posted: September 21, 2022 (view preprint)
- Received: September 21, 2022
- Accepted: February 20, 2023
- Accepted Manuscript published: February 21, 2023 (version 1)
- Version of Record published: March 8, 2023 (version 2)
© 2023, Dumoux 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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