Abstract
Three-dimensional information is much easier to understand than a set of two-dimensional images. Therefore a layman is thrilled by the pseudo-3D image taken in a scanning electron microscope (SEM) while, when seeing a transmission electron micrograph, his imagination is challenged. First approaches to gain insight in the third dimension were to make serial microtome sections of a region of interest (ROI) and then building a model of the object. Serial microtome sectioning is a tedious and skill-demanding work and therefore seldom done. In the last two decades with the increase of computer power, sophisticated display options, and the development of new instruments, an SEM with a built-in microtome as well as a focused ion beam scanning electron microscope (FIB-SEM), serial sectioning, and 3D analysis has become far easier and faster.
Due to the relief like topology of the microtome trimmed block face of resin-embedded tissue, the ROI can be searched in the secondary electron mode, and at the selected spot, the ROI is prepared with the ion beam for 3D analysis. For FIB-SEM tomography, a thin slice is removed with the ion beam and the newly exposed face is imaged with the electron beam, usually by recording the backscattered electrons. The process, also called “slice and view,” is repeated until the desired volume is imaged.
As FIB-SEM allows 3D imaging of biological fine structure at high resolution of only small volumes, it is crucial to perform slice and view at carefully selected spots. Finding the region of interest is therefore a prerequisite for meaningful imaging. Thin layer plastification of biofilms offers direct access to the original sample surface and allows the selection of an ROI for site-specific FIB-SEM tomography just by its pronounced topographic features.
Key words
- Focused ion beam scanning electron microscope (FIB-SEM)
- Tomography
- Resin embedding
- Targeted preparation
- Biofilm
- Cell-substrate interface
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Acknowledgments
We would like to thank Dr. Roger Wepf, EMEZ, ETH Zürich, for his general support. We cherish the cooperation with PD Dr. Heike Hall, d-MATL, ETH Zürich, who passed away last year. We thank Dr. Thomas Hefti, Thommen Medical AG, Waldenburg, Switzerland, for the osteoclast-on-bone cultures and Dr. Tessa Lühmann, Pharmazie, Universität Würzburg, for her work on the FIB-SEM. The authors also acknowledge the financial support by the Faculty of Biology and Medicine of the University of Lausanne and Prof. E. Welker, DNF, UNIL, for submitting the R'Equip grant 316030_128692 to the Swiss National Science Foundation and for blowing new life into EMF. We also would like to thank Prof. D. Mastronarde, University of Colorado, Boulder, for the IMOD list and the uncountable hints and help for the IMOD software; Dr. Chengge Jiao, FEI Company, Eindhoven, the Netherlands, for the FIB instructions and the suggestion of the optimized sample geometry; and last but not least Dr. Céline Loussert, EMF, UNIL, for samples, valuable discussions, and her encouragement.
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Kizilyaprak, C., Bittermann, A.G., Daraspe, J., Humbel, B.M. (2014). FIB-SEM Tomography in Biology. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_24
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DOI: https://doi.org/10.1007/978-1-62703-776-1_24
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