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A new Nanotechnology for Translational Medicine


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A whole new world in Biology is exposed. Cells can now be "dissected" into nanometre thin "slices" while at the same time the composition and 3-D ultrastructure of each "slice", using Nano Scanning …

A whole new world in Biology is exposed. Cells can now be "dissected" into nanometre thin "slices" while at the same time the composition and 3-D ultrastructure of each "slice", using Nano Scanning Auger Microscopy, are determined
Prof. J.L.F. Kock (Ph.D.)
Department of Microbial, Biochemical and Food Biotechnology
University of the Free State
P. O. Box 339
Bloemfontein 9300
South Africa

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  • 1. Audio Text A new Nanotechnology for Translational Medicine Invited video lecture for Translational Biomedicine Prof. Lodewyk Kock and Dr. Chantel Swart Department of Microbial, Biochemical and Food Biotechnology
  • 2. Talk 1 Dear Earthling, Stop. You might think that you have stumbled on this postcard by chance. This is not so. You have been chosen... Unlike many messages you have received until today, this is one message that cannot be ignored. You see, this postcard from the future has a secret code which, once you understand it, it will ensure that you will never view life in one dimension again... The hieroglyphic code at the back of this postcard is an invitation to enter the fascinating futuristic world of a special type of nanotechnology. A world only limited by the boundaries of your imagination. This is a world where cells are “dissected” into nanometre thin “slices.” We can then determine the composition and 3-D ultrastructure of each slice, using Nano Scanning Auger Microscopy. What a way to create an explosion in 3-D cell information. Now, let’s go for the first change in lenses. Please put on your 3-D glasses and join us for a journey into the future. Make sure you buckle up – there is an exciting adventure ahead. Enjoy the ride. Best wishes. The Nanotechnology Team
  • 3. Talk 2 The Nanotechnology Team consists of the following members: Front: Prof. Lodewyk Kock (Dept. Microbial, Biochemical and Food Biotechnology). Back (from left to right): Prof. Hendrik Swart (Dept. of Physics), Prof. Pieter van Wyk (Centre for Microscopy), Dr. Carlien Pohl (Dept. Microbial, Biochemical and Food Biotechnology), Dr. Chantel Swart (Dept. Microbial, Biochemical and Food Biotechnology) and Dr. Lisa Coetsee (Dept. of Physics).
  • 4. Talk 3 Welcome, Ladies and Gentlemen. My name is Lodewyk Kock and I am excited to be one of your guides on this journey. As we depart, we have the benefit of viewing the first application of the nanotechnology known as Nano Scanning Auger Microscopy to Biology, which, in future, may also impact on Translational Medicine. Accompanying me on our journey as main host, is Dr. Chantel Swart who is at present a Post Doctoral Fellow in my research group.
  • 5. Talk 4 Hallo Fellow Travellers. I am Chantel Swart and it will be my privilege to introduce you to this technology. What you will experience represents part of research that was performed during my Ph.D. under the main supervision of Professor Lodewyk Kock. So without any further ado, let’s start translating the hieroglyphs.
  • 6. Hieroglyph 1 Firstly, let us have a look at the meaning of Nano Scanning Auger Microscopy. In this lecture I will focus on the integral parts of this nanotechnology, which are Scanning Electron Microscopy (SEM), Auger Electron Spectroscopy (AES) and Scanning Auger Microscopy (SAM). Of course these are combined with an etching device using Argon. Let’s decipher these elements.
  • 7. Hieroglyph 2 The first part of this nanotechnology involves SEM. The first SEM image was obtained by Max Knoll in 1935. Since then, many breakthroughs were achieved on this front. The SEM works on the following principle: (i) an electron gun bombards the sample in vacuum with electrons, known as an electron beam, (ii) the electrons collide with the sample that is covered with gold to make it more electron conductive and (iii) electrons are then scattered from the sample and detected by a Secondary Electron Detector (SED) that converts the signal into an image that we observe on a computer screen.
  • 8. Hieroglyph 3 Another integral part of this nanotechnology is AES. To understand this better, I will first discuss the Auger effect by means of a schematic representation. Here, we have the various orbitals in a specific metal atom, ranging from the inner shell or 1s orbital to the outer shell or 2p orbital. Ef represents the fermi level, below this level is the atom and above this level is the environment. E represents the energy released by an Auger electron as it is ejected from the outer shell. Auger electrons are electrons that are released due to the Auger effect. The Auger effect involves the following: An incident beam causes an electron in the inner shell to become excited. This electron is then ejected from the inner shell leaving an empty space. The resultant vacancy is soon filled by an electron from one of the outer shells. This electron releases energy in the process of relaxation. The energy is transferred to an electron in the outer shell and this electron is then ejected from the atom. We call these Auger electrons. Each element has a specific Auger profile and this is then used to identify the elements based on these energy profiles.
  • 9. Hieroglyph 4 A schematic representation of an AES working chamber is shown. At the top we can observe the Auger optics where the electron gun is situated. We can also see the sample in the working chamber through a viewport. The machine can be equipped with a sputter gun as well as various leak valves for the inlet and outlet of gases. A mass spectrometer can be used to determine which gases are present.
  • 10. Hieroglyph 5 The last integral part of this nanotechnology is SAM. This works on the same principle as AES, yet instead of determining the elements in one small target area, the electron beam or nanoprobe scans across the whole sample surface. The element composition is determined in that area while scanning. Different colours can then be assigned to different elements to give a selectively coloured element map as illustrated. Here copper was labeled in red, iron in green and sulphur in blue.
  • 11. Hieroglyph 6 This nanotechnology is therefore a combination of SEM, AES, SAM as well as an Argon etching gun. This will from now on be called Nanoprobe analysis. This allows targeted etching of samples, along with simultaneous element analysis and SEM imaging. The viewport of the apparatus (PHI 700 Nanoprobe) is shown, where samples can be viewed in the working chamber. This is similar to the AES. Next in line is the introductory chamber, where the samples are placed before entering the working chamber. We also observe the ion gun that uses Argon to etch the samples. Shown at the top of the instrument is the electron gun as well as the different detectors similar to that of the SEM.
  • 12. Hieroglyph 7 This nanotechnology therefore has three different functions. Let us start with the SEM mode. An electron beam of 12nm in diameter scans across the sample. Secondary electrons will be emitted and detected by an SED. This signal is converted to an image to yield a picture as shown. Here we can see two asci of the yeast Nadsonia fulvescens attached to two mother cells respectively. The wrinkled ascus is due to the shrinking of the ascus wall to tightly fit around the spiny protuberances of the ascospore.
  • 13. Hieroglyph 8 The second function is the etching of the sample using an Argon gun. During this process, the sample is bombarded with Argon. The Argon may etch the sample at a rate of 27nm per minute. Therefore, after an etching cycle of one minute, on a specific area a surface layer with a thickness of 27nm will be removed. Thus, after every etching cycle, a specific area of the sample will only be a few nanometres smaller. After etching, we again use the SEM function to obtain an image. Now we can clearly see how the asci were etched to reveal a solid ascospore structure inside.
  • 14. Hieroglyph 9 The third function is element analysis. Here the electron beam or nanoprobe of 12nm in diameter will focus directly on a specific area or target of interest. One can choose these areas and more than one area can be analysed. Auger electrons will be ejected from the bombarded spot and will be detected by a detector. In this target area the various elements in the sample will be analysed by measuring the number of Auger electrons at different kinetic energies. An Auger profile will be obtained. The various elements will be determined depending on their specific Auger profile and the data will be created in the form of a graph. The APPH will be determined and a depth profile will be constructed. On this graph we can see a typical depth profile in which the various lines represent various elements after a series of etchings have occurred.
  • 15. Talk 5 In the past this nanotechnology was specifically used for semi- conductors and some other materials, excluding biological material (Hochella et al., 1986). This is due to the fact that the preparation technique for biological samples was not yet developed.
  • 16. Hieroglyph 10 In the study this nanotechnology was applied for the first time to biological material.
  • 17. Hieroglyph 11 I would now like to introduce you to the yeast Nadsonia fulvescens that was analysed by this nanotechnology. The yeast has a unique life cycle yielding an ascus or birth sac on one side of the mother cell. Each ascus contains a single offspring or ascospore. Upon maturity these ascospores are surrounded by spiny protuberances and contain melanin that colours it brown when cultured on Petri dishes (Swart et al., 2010a).
  • 18. Talk 6 During the formation of matured asci, the mother cell releases all of her contents as is demonstrated by the moving “clay model” animation using Confocal Laser Scanning Microscopy. Scattered fluorescing compounds in red and green represents the released cytoplasm while the attached ascus can be seen as a red fluorescing uneven-shaped cell (Swart et al., 2010a).
  • 19. Hieroglyph 12 This yeast was used in the following experiments (Swart et al., 2010b):
  • 20. Hieroglyph 13 Cells of the yeast were treated with the antifungal fluconazole, causing malformation of the ascospore. This will serve as our model for applying this nanotechnology. These cells were subjected to light microscopy first. Next the cells were prepared for SEM. The process was quite challenging since the samples that we prepared for the SEM also had to be compatible with this nanotechnology without clogging the apparatus with moisture. Therefore the samples had to be completely dehydrated to achieve high vacuum. Furthermore, the cells had to handle a 20kV electron beam, where normally we use a 5kV beam for normal SEM. We also had to evaluate the artefacts caused by SEM preparation such as dehydration of the cells. The samples were then viewed with an SEM (20kV beam). Lastly, the cells prepared for SEM were subjected to this nanotechnology (SEM, AES, SAM and etching) to determine the 3-D architecture and element composition.
  • 21. Hieroglyph 14 Cells were spread over an YM agar plate to form a homogenous lawn. A test strip containing a concentration gradient of fluconazole was then overlayed on the plate and incubated at 25 ºC until a white zone with no mature asci, and a brown zone with mature asci, could be observed.
  • 22. Hieroglyph 15 Cells from different zones were then viewed with a light microscope to determine the morphology and effect of fluconazole without any sample preparation steps, such as dehydration that could lead to artefacts.
  • 23. Hieroglyph 16 Again cells from the two different zones were scraped off and subjected to SEM sample preparation. This includes fixation, followed by critical point drying, mounting on stubs, sputter coating with gold and then viewing of the samples with normal SEM (Van Wyk and Wingfield, 1991). Here the challenge was to completely dehydrate the samples to safely use in this nanotechnology with minimum artefact formation.
  • 24. Hieroglyph 17 Next the samples prepared for SEM were subjected to nanoprobe analysis (SEM, AES, SAM and etching). Consequently, the cells were imaged, etched and element analysis was performed.
  • 25. Hieroglyph 18 Let us now look at the results obtained.
  • 26. Hieroglyph 19 A characteristic of this yeast is that the sexual stage or ascospores produce a brown colour on the plate due to melanin production (Kurtzman and Fell, 1998). If no mature ascospores are formed, the growth will remain white. Therefore, after incubation three zones can be observed on the plate. A transparent zone (here indicated in blue) where no growth could be observed, a white zone where only asexual growth occurred and a brown zone where asexual and sexual growth occurred. Light microscopy indicated the effect of fluconazole on the ascospore development of this yeast. In the brown zone we can clearly observe a large, mature ascospore with spiny protuberances in the ascus. In the white zone however, a smaller, smooth immature ascospore that seems to be a hollow ring-like structure, can be observed in the ascus. These samples were further evaluated with normal SEM and this nanotechnology.
  • 27. Hieroglyph 20 SEM on the cells from the brown zone indicated an ascus attached to the mother cell. Here, the ascus wall shrunk around the spiny protuberances hence the wrinkled appearance of the ascus when viewed with SEM. This could not be observed in the light micrograph as well as Transmission Electron Microscopy (TEM) micrograph. The dehydration of the cells during SEM preparation caused an artefact that can be seen as the shrinking of the ascus wall around the spiny protuberances.
  • 28. Hieroglyph 21 Cells obtained from the white zone are quite different in appearance from those obtained from the brown zone. We already observed a smooth walled immature ascus with light microscopy. This structure was confirmed by SEM. This indicates that there were no spiny protuberances surrounding the immature ascospore.
  • 29. Hieroglyph 22 The cells were further subjected to this nanotechnology to determine the 3-D architecture of the cells obtained from the white and brown zones respectively. In this instance we demonstrate different animations of what we expect to see as etching proceeds into the asci. Firstly in the brown zone, we expect to observe a mature ascospore with crunched spiny protuberances and a solid structure inside an ascus. As soon as etching starts, we expect to see wrinkled spiny protuberances surrounding the ascospore. As etching proceeds into the ascus, we expect to observe a solid ascospore structure with surrounding wrinkled protuberances. For the white zone, we expect to see a smooth walled ascus. As etching starts we should observe a sphere without any protuberances that disintegrates with further etching to disclose a hollow structure.
  • 30. Hieroglyph 23 Figure (a) indicates asci obtained from the brown zone, the wrinkled appearance again due to the shrinking of the ascus wall around the spiny ascospore protuberances. As etching proceeds, we observe the wrinkled protuberances in figure (b). Even further etching, to about 1030nm into the ascus, discloses a solid ascospore structure (figure c) with surrounding protuberances, as expected. For the white zone, a smooth walled ascus is observed in figure (d). As expected, etching exposed a sphere. Figure (f) shows the disintegration of this spherical structure to disclose, as expected a hollow structure. This again indicates the effect that fluconazole has on spore development in this yeast.
  • 31. Hieroglyph 24 The video demonstrates how etching proceeds through the ascus obtained from the brown zone. Here the ascus wall is etched off to show crunched spiny protuberances. Even further etching reveals a solid ascospore structure surrounded by these protuberances.
  • 32. Hieroglyph 25 The video clip shows how etching proceeds through the ascus obtained from the white zone. As etching starts, the ascus wall is etched away to reveal a sphere-like structure. Further etching reveals that this structure is in fact a hollow sphere, again indicating the effect of fluconazole on ascus formation in this yeast.
  • 33. Hieroglyph 26 Cells from the white and brown zones were also subjected to element analysis. Various targets were chosen as indicated by 1 to 4 in figure (a) and 1 and 2 in figure (c). Figure (b) depicts the element analysis for target 3 in figure (a). Here we observe various elements including carbon, oxygen, gold and osmium. The graph indicates a high C/O ratio, this could be due to melanin deposits that give the mature ascospores its brown colour. Melanin has a high C/O ratio. Figure (d) indicates the element analysis of target 2 in figure (c). Here, once again, we see the various elements. F (fluorine) is indicative of fluconazole used in the treatment of the cells. It was possible to follow the dispersal of this element throughout the cell with this nanotechnology. Notice that the C/O ratio is lower, probably due to the absence of melanin and also the presence of low C/O intensity ratio compounds such as chitosan. Further element analysis should be performed to determine the exact composition and reasons for the variation in element ratios. The presence of gold (Au) and osmium (Os) can be ascribed to the sample preparation techniques used.
  • 34. Hieroglyph 27 After every etching an element analysis was performed showing that the intensities of the various elements vary as etching continues. This pulsing effect can be ascribed to etching through the different organelles and other inclusions in different areas of the cell as illustrated in the drawing.
  • 35. Hieroglyph 28 The question now arises: will it be possible to apply SAM to yeasts in order to observe cell inclusions in different colours?
  • 36. Hieroglyph 29 So far, colour SAM maps have been constructed of the surface of an ascospore and also after a single etching procedure. Here we can see gold (Au) in green, before etching starts. As the gold is etched away the carbon (C) can be seen in blue as well as some oxygen (O) in red. Further studies should be conducted to obtain a SAM colour map after etching has proceeded into the ascospore. When this is done, one would probably be able to observe the different elements of the spiny protuberances as well as the cell inclusions in the ascospore.
  • 37. Hieroglyph 30 To conclude, this nanotechnology was found to be applicable as a research tool to biological material, yet it is still in its infancy and its full potential should now be evaluated. Furthermore, the possibility of visualizing the 3-D structure of cell inclusions as well as cell metabolism should be assessed using SEM and Argon etching as well as SEM, Argon etching and SAM in combination with the use of element ratio comparisons and tagged probes that target cell inclusions or enzymes. A drawback to this technique is that there are only a few modern such apparatus available worldwide and it is expensive!
  • 38. Hieroglyph 31 Some examples of engineering performed by means of this nanotechnology so far. An ascus tip of the yeast Dipodascopsis uninucleata is shown. The element composition of a single microfibrillar fibre in this structure could be determined. Here, the ascus was etched until it became “transparent” and the spores inside the ascus became visible. After release, the spores were again observed with nano-scale ridges on their surfaces (Olivier et al., 2011). These ridges are said to aid in effective liberation of spores from a narrow, bottle-neck ascus tip (Kock et al., 1999, 2007). An ascus after liberation of ascospores is shown. Another micrograph shows the ascospores of a specific Lipomyces strain obtained from the Amazon. In this case the ascus wall is etched away, revealing the ascospores (Maartens et al., 2011). Another yeast was grown on deteriorated toxic oils causing warty protuberances to be produced. Using this nanotechnology we could prove that these warts were part of the cell wall (Leeuw et al., 2010).
  • 39. Hieroglyph 32 A movie is shown that simulates movement and release of a sickle-shaped fungus spore from an elongated birth sac (ascus) (Kock et al., 2004). Such spore release is a mode of fungal dispersal and infection. It would be interesting to assess the influence of antifungals on such types of spore dispersal by using this nanotechnology. In addition, the metabolic fate of the antifungal may be followed throughout the fungus life cycle.
  • 40. Talk 7 It is clear that this nanotechnology opens up a whole new world of 3-D ultrastructure combined with element research of biological materials. Fungal dispersal mechanisms which are important in fungal infections can now be visualised and studied in detail while the effects of different antifungal agents on fungal dispersal are exposed. Even the metabolic fate of these drugs can be monitored via element analysis throughout the cell. Even more exciting is the fact that this technology may in a similar way visualise human cells in 3-D ultrastructural mode while determining the element composition of the whole cell. Just think what application this may have in early cancer cell detection and other metabolic cell changes, to just touch on the tip of the iceberg! Even the quality of drug composition and metabolic fate may be screened by using this nanotechnology!
  • 41. Hieroglyph 33 We have now come to the end of our journey. Thank you for your participation and attendance. If you are interested, the highlights can be found in summarised format from the poster on display. This can also be obtained from the authors in PDF format, free of charge.
  • 42. Talk 8 Dear Fellow Futurist, As scientists it has been our quest to bring you a slice of the hitherto unseen. Did you notice that the postcard in your hand has turned into a map? From seemingly indecipherable hieroglyphs into the unravelling of this nanotechnology? We hope that you share in the excitement we feel. We will keep on pressing forward and we would like for you to remain with us on this journey... Please feel free to contact us at the e-mail address indicated elsewhere. In the meantime we will be busy chasing the gigantic Blue Morpho butterfly in the idyllic surroundings of Amazonia in our quest to find unique fungi for eventual Nano Scanning Auger Microscopy applications. Hope to hear from you soon. Until we meet again. The Nanotechnology Team