Cryo-plasma FIB/SEM volume imaging of biological specimens.

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular 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. 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 20-50 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. © 2023, Dumoux et al.

Citation

Maud Dumoux, Thomas Glen, Jake L R Smith, Elaine M L Ho, Luis M A Perdigão, Avery Pennington, Sven Klumpe, Neville B Y Yee, David Andrew Farmer, Pui Y A Lai, William Bowles, Ron Kelley, Jürgen M Plitzko, Liang Wu, Mark Basham, Daniel K Clare, C Alistair Siebert, Michele C Darrow, James H Naismith, Michael Grange. Cryo-plasma FIB/SEM volume imaging of biological specimens. eLife. 2023 Feb 21;12

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PMID: 36805107

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