Cryo-plasma FIB/SEM volume imaging of biological specimens
Maud Dumoux, Thomas Glen, Jake L. R. Smith, Elaine ML Ho, Luı́s M. A. Perdigão, Avery Pennington, Sven Klumpe, Neville B.-y. Yee, David A. Farmer, Pui YA Lai, W Bowles, Ron Kelley, Jürgen M. Plitzko, Liang Wu, Mark Basham, Daniel K. Clare, C. Alistair Siebert, Michele C. Darrow, James H. Naismith, Michael Grange
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.