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

Audio Text

Dear Earthling,Stop.You might think that you have stumbled on this postcard by chance. This is notso.You have been chosen...Unlike many messages you have received until today, this is one message thatcannot 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 onedimension again... The hieroglyphic code at the back of this postcard is aninvitation to enter the fascinating futuristic world of a special type ofnanotechnology. A world only limited by the boundaries of your imagination.This is a world where cells are “dissected” into nanometre thin “slices.” We canthen determine the composition and 3-D ultrastructure of each slice, using NanoScanning Auger Microscopy. What a way to create an explosion in 3-D cellinformation.Now, let’s go for the first change in lenses. Please put on your 3-D glasses andjoin us for a journey into the future. Make sure you buckle up – there is anexciting adventure ahead.Enjoy the ride.Best wishes.The Nanotechnology Team

Talk 1

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).

Talk 2

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Hieroglyph 10

In the study this nanotechnology was applied for the first

time to biological material.

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).

Hieroglyph 11

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).

Talk 6

Hieroglyph 12

This yeast was used in the following experiments (Swart et

al., 2010b):

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.

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.

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.

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.

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.

Hieroglyph 18

Let us now look at the results obtained.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Hieroglyph 28

The question now arises: will it be possible to apply

SAM to yeasts in order to observe cell inclusions in

different colours?

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.

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!

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).

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.

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!

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.

Talk 8

Dear Fellow Futurist,

As scientists it has been our quest to bring you a slice of the hithertounseen. Did you notice that the postcard in your hand has turned into amap? From seemingly indecipherable hieroglyphs into the unravellingof this nanotechnology? We hope that you share in the excitement wefeel. We will keep on pressing forward and we would like for you toremain with us on this journey...

Please feel free to contact us at the e-mail address indicated elsewhere. Inthe meantime we will be busy chasing the gigantic Blue Morphobutterfly in the idyllic surroundings of Amazonia in our quest to findunique fungi for eventual Nano Scanning Auger Microscopyapplications.

Hope to hear from you soon.Until we meet again.The Nanotechnology Team