The application of the transmission electron microscope (TEM) in essence may be regarded as one of the first instruments used to visualize molecular structures at nano-analysis level inside biological cells, although not always recognized as such. These biological structures ranged over the decades since 1939 from membranes and many other inclusions specific of cell morphology to virus structures. Different preparation techniques on cells led to different observations and conclusions. This can vary between negative staining, e.g. extracted DNA, potassium permanganate (oxidative) or a double fixation technique (reductive and oxidative), dehydration, embedding, sectioning and a variety of heavy metal staining techniques. The identification and interpretation of cellular components thus solely depend on such specific techniques. Any technique of the aforementioned is known and daily applied in electron microscopy laboratories, therefore no specific references are presented.

            Many published TEM images pertaining to specifically yeast (Ascomycetes) research, show structures in cells that were not described in either results or discussions [1]. The latest research on fermentative yeast cells highlighted the occurrence of gas in the cell cytoplasm appearing as bubbles (Fig. 1 and Fig. 2). Using TEM only, similar structures were observed in the past in yeast cells [2] but never defined, nonetheless as gas bubbles. In almost all cases the absence of membranes around such structures led many researchers either to ignore it, probably because studies covered other cell phenomena, or confusingly and incorrectly defined as lipids. The formation of gas (carbon dioxide) in yeast cells is important in the brewing and bakers industry, e.g. for the perfect leavening of bread. Therefore the importance of studies to understand gas formation, retention and release by yeast cells may help in developing improved yeast strains.

            Scanning electron microscope (SEM) techniques, however, cannot reveal internal structures of cells sufficiently. Nano scanning Auger microscopy (NanoSAM), a nano-analysis technique not typically developed for biological material, was applied in an attempt to clarify this phenomena of prominent membrane-less structures in fermenting yeast cells [3]. The NanoSAM has the additional advantage over SEM to blast the cells with an argon-gun. This process allows controlled erosion of cells and can reveal internal components such as gas bubbles. Elemental analysis can also be done at nano-levels.

            The exact same set of samples can afterwards be transferred to a high resolution field emission gun SEM to examine cell structure of bubbles in detail (Fig. 3). NanoSAM analysis of nano-areas in the exposed areas of the cells was performed to reveal chemical composition to determine the presence of zinc (Fig. 4). Zinc was added to the growth medium, which reacts to carbonic acid in cells related to fermentation gas release. Compared to TEM examination of these bubble-like structures the distribution, size and appearance correspond well to observations with this novel application of the NanoSAM in biology. Comparatively, lipids are not expected to appear as vacant structures as gas bubbles observed after argon gun erosion in the NanoSAM.

            Conclusively, this review emphasizes the importance to search and find applications such as NanoSAM with typical applications in physics research, which can then be used to reveal missing links with current and past research in other fields such as biology. Furthermore, it can also be emphasized that many areas now uncovered, can lead to other hidden phenomena in biological cells above and beyond the yeast group. Other techniques such as time-of-flight secondary ion mass spectrometry (TOF-SIMS), is also able to provide elemental, chemical state, and molecular information not only from surfaces of solid materials but from biological organisms as well. Current research in this field has already been started by this research group.

References

[1] Wilson, C.L. et al (1970). Phytopathology 60, 216-227.

[2] Van der Walt, J.P. and von Arx (1985). South African Journal of Science 81, 156-159.

[3] Swart, C.W. et al (2012). FEMS Yeast Research 12, 867-869.

Acknowledgement

The Authors wish to thank you University of the Free State (UFS) and the National Research Foundation (NRF) of South Africa for funding the project. Ms. H. Grobler, Laboratory Manager of the Laboratory for Microscopy for SEM and Dr. E. Coetzee-Hugo, Department of Physics, UFS, for NanoSAM analysis.

Figures:

Fig. 1 TEM of yeast cell showing electron lucent structures without membranes defined as gas bubbles. (Scale bar = 500nm).

Fig. 2 TEM enlargement of gas bubbles in yeast cell. Smaller gas bubble nuclei observed expanding from and linked to larger gas nucleus (right area above the scale bar). (Scale bar = 200nm).

Fig. 3 SEM of yeast cells after NanoSAM argon blasting to expose inner cell area. Similar oriented structures detected (as observed by TEM), which are void of any substance and appear "see-through", confirmed as gas bubbles. (Scale bar = 1 micron).

Fig. 4 NanoSAM colour map of yeast (zinc=red, gold=green and carbon=blue). The zinc "galvanized" areas represent the associated carbon dioxide of fermentation in the vicinity of the bubbles. (Scale bar = 1 micron).

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