Synergy of SEM and Ultramicrotomy

SBEM Spreading from Life Sciences to Materials Science

  • Fig.1: 3D-reconstruction of neurons in the grasshopper’s visual system. The different colors correlate with different single neurons. At the bottom a typical block-face with several cells can be seen. The white rectangle indicates the region of the detail shown in the upper left corner (with part of the block-face at a higher z-value compared to the overview image). The location of an input synapse onto the yellow and orange cell is depicted in light green (white arrow). It is identified by the presynaptic density which can be clearly recognized on SBEM micrographs. The presynaptic cell (white asterisk) is not reconstructed. The rear edge of the micrograph has a total length of 25 µm.
  • Fig.2: 3D-reconstruction of the main crack (blue) and the crack distribution (light blue) after a tensile test and rubber particles (yellow); (a) original SEM images (inverted), (b) segmented slices, (c) the corresponding 3D-reconstruction and (d) the distance map of the filler particles, where the grey levels correlate to the spatial distances between the objects; reconstructed volume: 20.5 x 20.5 x 10.0 µm3.
  • Fig.3: (a) The block-face of an aluminum specimen. (b) A segmentation technique can be used for the 3D-reconstruction (unit: microns) delivering e.g. the distribution of fine (blue) and coarse (grey) precipitates. (c) Setup of the SBEM system: an ultramicrotome is installed in the specimen chamber of the microscope.

Serial block-face scanning electron microscopy (SBFSEM, SBEM) is a well established method in life sciences. The combination of ultramicrotomy and scanning electron microscopy enables imaging with high resolution and the reconstruction of big volumes compared to other methods.  Recent publications, where computer simulations were performed additionally to volumetric information about different materials, show that this method will contribute to material sciences, technology and industry services.

Serial Block Face Scanning Electron Microscopy, SBEM

In 2004 a groundbreaking paper was published by Denk and Horstmann [1] introducing a new technique called serial block-face scanning electron microscopy, abbreviated SBFSEM or SBEM. Here an ultramicrotome is located in the specimen chamber of a scanning electron microscope that enables the investigation of electrically non-conductive samples, either a variable-pressure scanning electron microscope (VPSEM) or an environmental scanning electron microscope (ESEM). Automated slicing and imaging of the block-face of a specimen is performed using a diamond knife for cutting. This serial imaging leads to a stack of micrographs which can be used for the 3D-reconstruction of specimens. The advantage of the use of a diamond knife in comparison to e.g. an ion beam which would be available in a focused ion beam microscope (FIB) is the fact that no implantation of ions into the material takes place and additionally a comparatively bigger sample volume can be investigated in an adequate time [2].

A Great Impact on Life Sciences

The first decade of SBEM was primarily characterized by publications in the field of life sciences. Especially in neuroscience two main advantages of the method can be combined: imaging with SEM resolution and investigating volumes of interest with macroscopic dimensions. This is possible since cuboids of about 0.6 x 0.6 x 0.6 mm3 can be cut by the microtome. As an example a 3D-reconstruction of a bundle of neurons of the visual system of a grasshopper is shown in figure 1. Here different colors indicate different single neurons.

Even synaptic connections can be identified which is vital for the understanding of the neurons’ functionality. In neuroscience, not only the authentic 3D-reconstruction of several neurons is relevant but also their precise connectivity. For this purpose special computer tools were developed in the community in order to shorten the time of investigation [3]. Below the bundle of neurons a typical block-face of the specimen can be seen, where the different biological structures, the cell membranes and the cell organelles were made visible by using heavy metal staining. This pretreatment increases not only the compositional contrast delivered by backscattered electrons (BSE), but hardens also the material in order to make it sliceable at room temperature. Figure 1 correlates to a paper where the connectivity within the grasshopper’s visual system was described [4]. There is high demand for this method, not only in neuroscience but generally in life sciences. Concerning the preparation techniques and the number of publications SBEM can be regarded as well established in life sciences.

SBEM of Soft Matter Materials

In 2009 first results in material sciences were presented in [5], describing applications on different soft matter materials. Again compositional contrast was used either due to different phases in the material or due to specimen preparation.

As an example figure 2 shows several steps in the 3D-analysis of a polymer blend (isotactic polypropylene/ethylene propylene rubber particles) after a tensile test. Here a staining protocol similar to life sciences had to be applied using ruthenium tetroxide, which enables the differentiation between the polypropylene matrix and the rubber particles. Figure 2a shows the slices of the block faces with particles and cracks, figure 2b the binarized images after applying a segmentation technique. The resulting 3D-reconstruction in figure 2c shows the cracks developing in the polymer blend as well as the crack surfaces and the position of the cracks related to the distribution of the particles. Additionally, further analyses like the distance map of the particles can easily be calculated (fig. 2d).

In [2] and [5] investigations on other types of soft matter materials are listed such as embedded paper, embedded membranes and materials including particles like talcum filled polypropylene (compositional contrast due to different phases) or foam-like structures like polymer monoliths (heavy metal staining).

SBEM of Metals and Alloys

In a recent paper [6] it was outlined that George E. Thompson from the University of Manchester and co-workers “have been at the vanguard of developing workflows to investigate the corrosion behavior of aluminum and magnesium, the influence of organic coatings on corrosion pathways, the distribution of pigments in the paint, and the distribution of intermetallics during casting or forming” (literally from [6]). In this paper several examples are described and special aspects of cutting metals with a diamond knife are mentioned. Although limits of the method are also discussed in [6] it is concluded that SBEM is capable of generating 3D-images of different engineering materials at nanoscale resolution over large volumes.

As an example, figure 3a shows the block face of an aluminum specimen. The compositional contrast due to regions of different chemistry enables a 3D-reconstruction (fig. 3b) of a sample volume showing the distribution of fine and coarse precipitates. In figure 3c the setup of the SBEM system is shown. While imaging is typically done with the BSE detector, the combination of SBEM and energy dispersive x-ray spectroscopy (EDS) was first published as 3D-elemental mapping in [2] and [7] (see also the online film with the title “3D elemental mapping in the ESEM”). Here an aluminum-copper alloy specimen of the type EN AW 2024 T351 was investigated and different precipitates (e.g. Mg2Si, Al2CuMg) were reconstructed from 3D-elemental maps of the elements Al, Cu, Mg, Mn, and Si.

An Established Method with a Promising Future

More than a decade after the publication of [1] SBEM can be regarded as well established in life sciences. However, recent publications show that this method is even promising in the field of materials science. It is not only useful to get 3D-reconstructions of volumes of interest but also to apply mathematical models [8] and furthermore computer simulations [9, 10]. Additionally the combination with other methods like EDS or Electron backscatter diffraction (EBSD) will contribute to science, technology and industry services.

Acknowledgements
Figure 1 was made within the framework of a project funded by the Styrian government (HTI:SmApp 2012-2016). Thanks to Selina Haingartner, Manuel Paller, Sanja Šimić, and Ilse Letofsky-Papst for graphical support and Peter Poelt and Gerd Leitinger for fruitful suggestions.
References

References

[1] Winfried Denk and Heinz Horstmann: Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure, PLoS Biol 2(11): e329 (2004) DOI 10.1371/journal.pbio.0020329.

[2] Armin Zankel, Julian Wagner and Peter Poelt: Serial sectioning methods for 3D investigations in materials science, Micron 62, 66–78 (2014) DOI 10.1016/j.micron.2014.03.002.

[3] Stefan Wernitznig, Mariella Sele, Martin Urschler, Armin Zankel, Peter Poelt, Claire Rind and Gerd Leitinger: Optimizing the 3D-reconstruction technique for serial block-face scanning electron microscopy, J. Neurosci. Methods 264, 16–24 (2016) DOI 10.1016/j.jneumeth.2016.02.019.

[4] Stefan Wernitznig, Claire Rind, Peter Poelt, Armin Zankel, Elisabeth Pritz, Dagmar Kolb, Elisabeth Bock and Gerd Leitinger: Synaptic Connections of First-Stage Visual Neurons in the Locust Schistocerca gregaria Extend Evolution of Tetrad Synapses Back 200 Million Years, Journal of Comparative Neurology 523, 298–312 (2015) DOI 10.1002/cne.23682.

[5] Armin Zankel, Bernd Kraus, Peter Poelt, Miroslava Schaffer and Elisabeth Ingolic: Ultramicrotomy in the ESEM, a versatile method for materials and life sciences, J. Microsc. 233, 140–148 (2009) DOI 10.1111/j.1365-2818.2008.03104.x.

[6] Teruo Hashimoto, George E. Thompson, Xiaorong Zhou and Philip J. Withers: 3D imaging by serial block face scanning electron microscopy for materials science using ultramicrotomy, Ultramicroscopy 163, 6–18 (2016) DOI 10.1016/j.ultramic.2016.01.005.

[7] Armin Zankel, Herbert Reingruber and Hartmuth Schröttner: 3D Elemental Mapping in the ESEM, Imaging & Microscopy 2, 35–37 (2011)
http://www.imaging-git.com/science/electron-and-ion-microscopy/3d-elemental-mapping-esem-combination-serial-block-face-sem-and-

[8] Tibor Muellner, Armin Zankel, Yongqin Lv, Frantisek Svec, Alexandra Höltzel and Ulrich Tallarek: Assessing Structural Correlations and Heterogeneity Length Scales in Functional Porous Polymers from Physical Reconstructions, Advanced Materials 27, 6009–6013 (2015) DOI 10.1002/adma.201502332

[9] Harun Koku, Robert S. Maier, Kirk J. Czymmek, Mark R. Schure and Abraham M. Lenhoff: Modeling of flow in a polymeric chromatographic monolith, J. Chromatogr. A 1218, 3466–3475 (2011)                                         DOI 10.1016/j.chroma.2011.03.064

[10] Christian Jungreuthmayer, Petra Steppert, Gerhard Sekot, Armin Zankel,Herbert Reingruber, Jürgen Zanghellini, Alois Jungbauer: The 3D pore structure and fluid dynamics simulation of macroporousmonoliths: High permeability due to alternating channel width, J. Chromatogr. A 1425, 141–149 (2015) DOI 10.1016/j.chroma.2015.11.026

Authors
Dr. Armin Zankel
MSc Manfred Nachtnebel

Graz University of Technology
NAWI Graz
Institute for Electron Microscopy & Nanoanalysis
and Center for Electron Microscopy
Graz, Austria
www.felmi-zfe.at

Dr. Stefan Wernitznig
Institute of Cell Biology, Histology and Embryology
Research Unit Electron Microscopic Techniques
Graz, Austria
and Institute of Neuroscience
Centre for Behaviour and Evolution
Newcastle University
Newcastle upon Tyne, United Kingdom

Register now!

The latest information directly via newsletter.

To prevent automated spam submissions leave this field empty.