Abstract
Serial block face SEM (SBFSEM) relies on particular fixation and embedding protocols introducing high amounts of different heavy metals into the sample to generate conductivity and sufficient contrast for imaging with back-scattered electrons. We describe two different preparation protocols to increase either overall metal content or to specifically enhance contrast of endomembrane systems. For samples containing large voids or lumina, such as kidney glomeruli or plant vacuoles this is not sufficient. They are charging, nevertheless, due to the absence in these lumina of molecules where heavy metals would bind in cytoplasm. We demonstrate how this structure-dependent charging can be alleviated by gas injection into the SEM chamber—highly localized to the vicinity of the sample surface. This is called focal charge compensation or focal CC. In addition, we explain in detail how to get resin blocks ready for imaging in an SEM using the GATAN 3view system.
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Abbreviations
- CC:
-
Charge compensation
- CNTs:
-
Carbon nanotubes
- SEM:
-
scanning electron microscope
- EHT:
-
Electrical high tension
- FIB:
-
Focused ion beam
- TEM:
-
Transmission electron microscopy
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Unger, AK., Neujahr, R., Hawes, C., Hummel, E. (2020). Improving Serial Block Face SEM by Focal Charge Compensation. In: Wacker, I., Hummel, E., Burgold, S., Schröder, R. (eds) Volume Microscopy . Neuromethods, vol 155. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0691-9_9
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DOI: https://doi.org/10.1007/978-1-0716-0691-9_9
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-0691-9
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