Serial block-face scanning electron microscopy

What is SBF-SEM?

Serial block-face scanning electron microscopy (SBF-SEM) is an imaging technique that generates 3D views of whole cells and tissue regions at higher resolutions than those achieved by light microscopy. It produces images of the internal ultrastructure of cells and tissues similar to those of transmission electron microscopy (albeit at slightly lower resolution), with the advantage of generating 3D datasets by serial imaging of the same area.

The combination of electron microscopy resolution and serial imaging means that SBF-SEM allows us to see small details of the tissue/cell ultrastructure, while at the same time being able to place the ultrastructural detail in the broader 3D context of the cell and tissue architecture.

Given their 3D nature, SBF-SEM datasets can be used to produce 3D models of whole cells, organelles and other cellular structures.

How does SBF-SEM work?

In SBF-SEM, a piece of tissue or cell pellet embedded in plastic resin is placed inside a special imaging chamber called 3View (see video), which is fitted to a high-resolution scanning electron microscope. The face of the block is imaged by SEM, and then an ultrathin slice of the block face is removed while the block remains inside the microscope. The newly exposed block face is then imaged. This process is repeated hundreds of times to produce consecutive images of the sample, which are then combined into an image series referred to as a '3D volume'.

Although samples embedded in resin for transmission electron microscopy (TEM) can often be imaged by SBF-SEM, the best SBF-SEM datasets are produced using samples prepared especially for this technique, with protocols designed to enhance sample contrast before embedding. SBF-SEM is performed in very sensitive and complex equipment that is operated exclusively by highly trained staff and selected users with solid electron microscopy experience.

Publications

Zoltner M, Campagnaro GD, Taleva G, Burrell A, Cerone M, Leung KF, Achcar F, Horn D, Vaughan S, Gadelha C, Zíková A, Barrett MP, de Koning HP, Field MC. (2020). Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes. J Biol Chem. 295(24):8331-8347. doi: 10.1074/jbc.
Boulogne C, Gillet C, Hughes L, LE Bars R, Canette A, Hawes CR, Satiat-Jeunemaitre B. (2020). Functional organisation of the endomembrane network in the digestive gland of the Venus flytrap: revisiting an old story with a new microscopy toolbox. J Microsc. 280(2):86-103. doi: 10.1111/jmi.12957. Epub 2020 Sep 23. PMID: 32844427.
Pain C, Kriechbaumer V, Kittelmann M, Hawes C, Fricker M. (2019). Quantitative analysis of plant ER architecture and dynamics. Nat Commun. 10(1):984. doi: 10.1038/s41467-019-08893-9.
Abeywickrema M, Vachova H, Farr H, Mohr T, Wheeler RJ, Lai DH, Vaughan S, Gull K, Sunter JD, Varga V (2019). Non-equivalence in old- and new-flagellum daughter cells of a proliferative division in Trypanosoma brucei. Mol Microbiol 112(3):1024-1040.
Hoffmann A, Käser S, Jakob M, Amodeo S, Peitsch C, Týč J, Vaughan S, Zuber B, Schneider A, Ochsenreiter T. (2018). Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei. Proc Natl Acad Sci U S 115(8):E1809-E1818. doi: 10.1073/pnas.1716582115.
Bertiaux E, Mallet A, Fort C, Blisnick T, Bonnefoy S, Jung J, Lemos M, Marco S, Vaughan S, Trépout S, Tinevez JY, Bastin P (2018). Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum. J Cell Biol 217(12):4284-4297.
Protein Storage Vacuoles Originate from Remodeled Preexisting Vacuoles in Arabidopsis thaliana. Feeney M, Kittelmann M, Menassa R, Hawes C, Frigerio L. (2018) Plant Physiol. 177(1):241-254. doi: 10.1104/pp.18.00010. Epub 2018 Mar 19.
Hughes L, Borrett S, Towers K, Starborg T, Vaughan S (2017). Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy. J Cell Sci 130(3):637-647.
Käser S, Oeljeklaus S, Týč J, Vaughan S, Warscheid B, Schneider A (2016). Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria. Proc Natl Acad Sci U S A 113(31):E4467-75.
Scheuring D, Löfke C, Krüger F, Kittelmann M, Eisa A, Hughes L, Smith RS, Hawes C, Schumacher K, Kleine-Vehn J. (2016) Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression. Proc Natl Acad Sci U S A. 113(2):452-7. doi: 10.1073/pnas.1517445113.
Sanchez MA, Tran KD, Valli J, Hobbs S, Johnson E, Gluenz E, Landfear SM. (2016). KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes. J Biol Chem. 291(38):19760-73. doi: 10.1074/jbc.M116.739235.
Hughes L, Towers K, Starborg T, Gull K, Vaughan S. (2013). A cell-body groove housing the new flagellum tip suggests an adaptation of cellular morphogenesis for parasitism in the bloodstream form of Trypanosoma brucei. J Cell Sci 126(Pt 24):5748-57. doi: 10.1242/jcs.139139.
Roque H, Saurya S, Pratt MB, Johnson E, Raff JW. (2018) Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation. PLoS Genet. 14(2):e1007198. doi: 10.1371/journal.pgen.1007198.
Peterzan MA, Clarke WT, Lygate CA, Lake HA, Lau JYC, Miller JJ, Johnson E, Rayner JJ, Hundertmark MJ, Sayeed R, Petrou M, Krasopoulos G, Srivastava V, Neubauer S, Rodgers CT, Rider OJ. (2020). Cardiac Energetics in Patients With Aortic Stenosis and Preserved Versus Reduced Ejection Fraction. Circulation. 141(24):1971-1985. doi: 10.1161/CIRCULATIONAHA.119.043450.
Kleinnijenhuis, M., Johnson, E., Mollink, J., Jbabdi, S. and Miller, K.L., (2020). A semi-automated approach to dense segmentation of 3D white matter electron microscopy.

Serial block-face scanning electron microscopy

What is SBF-SEM?

How does SBF-SEM work?

What tissues or cells can be imaged by SBFSEM?

Publications