Serial Block-Face Scanning Electron Microscopy (SBFSEM) is one the technique which provides true insight into the composition of the volume of different materials. The volume imaging method helps to understand not only the structure but the function as well. SBFSEM is based on the combination of an in situ ultramicrotomy and electron microscopy which can be turned into the powerful tool for high resolution imaging of large volumes. Tissue and cell biology represents the traditional domain of the method but its application within materials sciences is becoming more apparent. It has been successfully employed in the observation of polymers, composite materials, membranes, metals etc. [1].

Integration and automation of the complete process of the data acquisition; and subsequent data processing represent the challenging task. The method is destructive in its very nature and there are potentially many factors which can influence the cutting and imaging properties. Teneo VS^TM is based on refined SBFSEM and designed for fully automatic data acquisition on stained resin embedded biological samples [2]. It combines hardware and software components into one integrated system. The in situ ultramicrotome, placed on the SEM stage, cuts the specimens into thin slices. The exposed block-face is scanned with the electron beam and the backscatter signal collected. Alternate slicing and imaging end up with a series of two dimensional images which come from different depth of the sample. Generally the depth resolution is limited by the thinnest slice thickness which can be cut by a diamond knife. To overcome this limitation virtual slicing was introduced [3]. A series of images at different accelerating voltages is acquired and processed. By their proper selection different depth emission profiles are created. Combination of both approaches allows shifting of the in-depth resolution towards nanometer level and to achieve isotropic voxel size. By using these methods in combination with thicker physical slices means more reliable section thickness and artefact control. 

Imaging of the stained, resin embedded samples is challenging. Both cutting properties and electrical conductivity have to be considered. To neutralize the charge built up on the sample surface low vacuum option is available including the dedicated backscatter detector. Nevertheless clever management of the signal can extend the applicability of the high vacuum mode to a broader range of samples. For Teneo VS such a unique dedicated extension is available to suppress the noise of a charging sample in image formation.

Fig. 1 shows the application of Teneo VS system to volume reconstruction of a polymer blend (isotactic polypropylene/ethylene propylene rubber particles) after a tensile test. Part of the fracture zone was visualized to trace the propagation of cracks through the bulk. Three dimensional field of micro-cracks and a distribution, structure of the filler particles can be studied at high resolution. The crack has been enhanced by RuO₄ vapor staining.

As it can be seen here the advancements despite having been engineered for life sciences can be applied directly to other materials. It is hoped that the advances and enhancements which are enjoyed by life sciences with this technique can be fully realized by other sectors interested in volume microscopy.

Acknowledgement

We would like to thank to Dr. Armin Zankel (FELMI-ZFE Graz, Austria) who kindly provided us with samples; Technology Agency of the Czech Republic, project TE01020118 for funding.

References

[1] Zankel A. et al., Journal of Microscopy, vol. 233(1), pp. 140-148 (2009).

[2] Hovorka M. et al., MC 2015 – Microscopy Conference 2015, Göttingen, Germany.

[3] F. Boughorbel et al., SEM Imaging Method, Patent US 8,232,523 B2, 31st July 2012.

Figures:

Fig. 1. Volume reconstruction of a part of the fracture zone in a polymer blend after tensile test. Data acquired by physical slicing at low vacuum mode (50 Pa) to suppress charging. The block-face was imaged in BSE mode at 3.5 kV accelerating voltage, 0.1 nA beam current, 3 μs dwell time, pixel size of 15 nm x 15 nm, nominal thickness of a physical cut was 50 nm. The visualization shows a part (100 μm x 52 μm x 10 μm) of the acquired volume (122 μm x 122 μm x 10 μm); see the inset with an indicated selection from the complete dataset. Data were processed and visualized in Amira software. Specimen courtesy of FELMI-ZFE Graz.

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