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 by 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 This presentation has been delivered to "Online Medical Conference" at http://conferences.medicalia.org

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