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Cryo-plasma FIB/SEM volume imaging of biological specimens
- Source :
- eLife. 12
- Publication Year :
- 2023
- Publisher :
- eLife Sciences Publications, Ltd, 2023.
-
Abstract
- 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.
Details
- ISSN :
- 2050084X
- Volume :
- 12
- Database :
- OpenAIRE
- Journal :
- eLife
- Accession number :
- edsair.doi...........e4272a59f81d81df6a84ef46fe9de7dc
- Full Text :
- https://doi.org/10.7554/elife.83623