X-Ray Microscopy in Aarhus
Joanna Abraham1, Robin Medenwaldt1, Erik Uggerhøj1, P. Guttmann2, T. Hjort3,
J. Jensenius3, T. Vorup-Jensen3, F.Vollrath 4, E. Søgaard5, J. Tyge Møller 6
1 ISA, Institute for Storage Ring Facilities, University of Aarhus, DK-8000, Denmark
E-mail: jabraham@dfi.aau.dk or: robin@dfi.aau.dk (R. Medenwaldt)
2 Forschungseinrichtung Röntgenphysik, Georg-August-Universität Göttingen and Berliner
Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH (BESSY), Germany
3 Department of Medical Microbiology and Immunology, University of Aarhus,
DK-8000, Denmark
4 Department of Zoology, University of Aarhus, DK-8000, Denmark
5 Department of Chemistry, University of Aalborg, Esbjerg, Denmark
6 Department of Geomorphology, University of Aarhus, DK-8000, Denmark
Abstract. We have seen an ever increasing number of collaborative projects
since the start of the Aarhus XM in 1992, with such diverse materials as human
spermatozoa, freshwater micro-organisms, metal-induced cysts, spider orb silk,
iron-precipitating bacteria, and sludge collected from water purification filters.
1 The Microscope
The Aarhus X-ray microscope [1] has been in operation since 1992 and has evolved to
be part of a user facility at ISA. It operates biannually for ten weeks, which is
sufficient (though not optimum) for extensive studies of objects in selected scientific
projects, some of which are described below. The majority of those projects involve
investigations of wet samples from fields in biology, medicine, and soil sciences.
Objects are illuminated by synchrotron radiation focused by a condenser zone
plate (the Göttingen KZP 7 type) through a monochromizing pinhole. Although the
usual wavelength is 2.4 nm, the configuration allows a continuous wavelength change
throughout the whole water window. A second zone plate images the object on a CCD
camera (Photometrics). The CCD chip (Tectronix) is peltier cooled, thinned and back
illuminated with 1024 by 1024 pixels of 24 µm size. In combination with a micro zone
plate of 30 nm outermost zone width, the achievable resolution is 30 nm at an X-ray
magnification of 1600. Some micro zone plates for the Aarhus XM have been
fabricated in Göttingen [2]. Since 1996, however, micro zone plates made by Steven
Spector from Stony Brook have been in use. All zone plates are germanium structures
on silicon backings [3].
Objects are located under atmospheric pressure surrounded by helium gas in
order to minimise X-ray absorption. For dry samples, almost any kind of holder can be
mounted in the microscope. Wet samples are placed between two silicon foils of
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150 nm thickness in a special chamber that is sealed with o-rings. With flexible tubes
and syringes, liquids can be pumped in and out, thereby adjusting the layer thickness
of the medium and/or exchanging the medium. With typical liquid layer thicknesses of
5-15 µm, samples can be kept in the chamber for many hours without drying out.
For object finding and prefocusing, there are several options. A light
microscope with an x-y stage calibrated with an x-y stage in the XM is used to get an
overview of the sample, the image of which can be shown on a video screen and can
be stored on video or in a computer. After placement of the object in the XM, the
holder can be rotated for prefocusing in a video microscope. The prefocused position
matches the necessary final position in the X-ray beam within a micron. The X-ray
micrograph can be recorded on the CCD or through a two stage micro channel plate in
combination with a phosphor screen and video camera. The latter option is used for
adjustment and alignment and when dynamic, real-time effects are made visible with
X rays. Although the resolution in this case is only 150 nm and the noise is high, this
option is often used to survey larger areas around objects or when looking at living,
moving samples such as sperms. These have to be immobilised before imaging, which
can be done by irradiating them for a second.
Imaging times of wet samples are typically 10-20 s. On this timescale, the
mechanical stability of the XM is high. The set-up, where the frame of the microscope
is rigidly bolted to a vibration damping table resting on pneumatic vibration isolators,
has shown that vibrations are below the detectable limit and thus have negligible
influence on the imaging properties of the XM.
2 The Projects
2.1 Spider Silk
Spider silk is extremely strong, weight for weight it is stronger than nylon or steel. It is
extremely elastic more so than any commercially made rubber. It can be extended
many times its own length but contracts easily and immediately to its former length.
However, it has been little studied unlike the cocoon silk of moths such as Bombyx
mori, which has a long history (at least 5000 years). The cocoon silk of Bombyx mori
has played an important role in industry and consequently has been extensively
studied. The wealth of information available on cocoon silk made it an obvious
starting point in the study of spider silk. As genetical techniques advanced, it became
clear that spider silk evolved independently and that there was no likely ancestor with
the cocoon silk. Now it has been shown that dragline spider silk has a very different
structure from that of the silkworm cocoon silk. This is not surprising when one
considers that spider silk is mechanically far superior.
Light microscopy and scanning electron microscopy studies suggest a skincore microstructure, but others disagree. Some hypothesise that one strand consists of
a fibril structure, others say not. These and many other contradictory findings have
created a gap in the information that is now beginning to be addressed by taking
advantage of new techniques.
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Fritz Vollrath from the Department of Zoology hypothesises that the thread
of the Nephila spider silk has a previously unsuspected structural organisation
consisting of a structured fibril wall surrounding a fibrilless core that can explain its
extraordinary tensile strength [4]. We have started a project in this direction and thus
have some preliminary results. Fig.1 shows threads of Nephila spider silk, where some
of the fibrils are visible at the ends of a broken thread. The protein polymers are
densely packed and absorbency is high, which demonstrates the problems involved in
its study. The question about a possible empty core inside this particular silk could be
answered in an easy way with X-ray microscopy, simply by comparing the X-ray
transmission of the silk with theoretical values. Thereby we showed that the thread
was not hollow. Future investigations are planned and will involve different
preparation techniques and manipulation of the silk. For example, by treating the silk
with urea solutions, thus causing the silk to swell.
A different kind of silk, the hackled spider silk (Fig. 2), is much thinner than
Nephila silk and consists of a network.
Fig. 1. X-ray micrograph of Nephila spider silk. λ=2.4 nm, t=10s
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Fig. 2. X-ray micrograph of hackled spider silk. λ=2.4 nm, t=2s
2.2 Colloidal Chemistry
The water treatment works in and around Esbjerg, Denmark, receive high levels of
iron that has made its way into the water system. The conventional method of
removing this iron from the water has been chemical, but now it is becoming clear that
a biological approach is far more efficient. In Esbjerg, they have four water
purification plants, three of which use the chemical method and one uses ironprecipitating bacteria. We are examining samples of sludge from these plants with the
aim of gaining a better understanding of how the sludge is structured.
Despite their known efficiency in iron removal, the microbes responsible
have been little studied. At Astrup water purification works, Esbjerg, the organism
responsible is Leptothrix, a bacteria known for its iron precipitation abilities.
However, the biology of such organisms is less well understood. Fig. 3a shows an XM
image of these bacteria taken from the water treatment plant, Esbjerg.
Also, at the Department of Geomorphology, Aarhus University, the process
of iron precipitation in Danish wetlands (probably connected with the occurrence of
bacteria) is being studied. Natural spring water with a high content of iron has been
imaged with the Aarhus XM (Fig. 3b). These samples show bacteria and the shells in
which they inhabit.
X-Ray Microscopy in Aarhus
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Fig. 3. X-ray micrographs of the sheaths of iron-precipitating bacteria Leptothrix.
Left (a): The microbe responsible for iron removal at Esbjerg water purification works.
Right (b): Bacteria seen in a sample taken from Danish wetlands. λ=2.4 nm, t=20s and t=60s
2.3 Spermatozoa
Often in the past it was the woman who was persecuted if a couple remained childless,
and even today there is a stigma attached to the situation, be it voluntary or not.
However, in recent years, it is male infertility that has been in the spotlight with
controversial claims that sperm counts have declined in recent years and that male
infertility has increased. It is not surprising then, that there is much work to elucidate
the mechanisms and processes involved in the developmental stages of the
spermatozoon.
The sperm is a highly specialised cell. The head is packed with genetic
information and an acrosomal vesicle containing hydrolytic enzymes that will help the
sperm penetrate the egg’s outer coat and so fertilise it. Mitochondria are strategically
placed at the base of the tail where they can effectively power the flagellum (Fig. 4a).
Sperm maturation is associated with a series of changes in the membranes
surrounding the head region. When the sperm are deposited into the vagina they do not
have the ability to fertilise an egg. However, by the time they reach the egg in the
oviduct they will have acquired the capacity to fertilise. Little is known about the
mechanism of capacitation and so far no morphological changes have been observed
during this process. It has been reported that the tail motion changes after capacitation
which lead us, together with the Department of Medical Microbiology and
Immunology at the University of Aarhus, to study mitochondrion morphology through
the developmental process.
Sperm are fragile, they have a single plasma membrane which is easily
damaged by conventional electron microscopy preparation techniques. With the XM
we could look at fully-hydrated sperm that had intact membrane structures.
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Fig. 4. X-ray micrographs of spermatozoa taken with the Göttingen XM at BESSY.
Top (a): Fresh ejaculated sperm. Note the densely packed mitochondria.
Bottom (b): Capacitated sperm. Note the less dense mitochondria. λ=2.4 nm, t=2s
X-Ray Microscopy in Aarhus
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Figure 4a shows sperm from fresh ejaculate. Important to note are the mitochondria, densely packed around the base of the flagellum. In Fig. 4b of a capacitated
sperm, note that the mitochondria are now not as densely packed and are vacuolated
with an increased volume. This observation has not been reported previously but could
be linked to the increased tail movements seen in the capacitated sperm.
2.4 Filamentous Blue-Green Algae
Filamentous blue-green algae are primary producers, using sunlight for photosynthesis. They have an important role to play in the lake ecosystem. However, some
species can become a problem if nutrient levels rise above a critical level. When they
grow in very large numbers, toxic blooms can be formed, choking the lake and
ultimately killing the other organisms present. Fig. 5 shows a blue-green algae seen in
a sample of lake water collected from Aarhus University lake. Note that sensitive
structures such as the mucilaginous sheath are fully hydrated and intact. Fig. 6 shows a
pennate diatom.
Fig. 5. X-ray micrograph of blue-green algae, note the fully-hydrated mucilaginous sheath.
λ=2.4 nm, t=8s
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Fig. 6. X-ray micrograph of Pennate diatom. λ=2.4 nm, t=12s
2.5 Protozoa and Encystment
Protozoa are single-celled or colonial, eukaryotic organisms. The cells vary
considerably in size but are usually 1-250 µm in diameter. They are a diverse group,
both morphologically and in their environmental adaptations, so have occupied a wide
range of ecological niches. In recent years, research using protozoa has flourished not
only to forward knowledge of the protozoa themselves, but because biologists have
recognised that these organisms provide excellent subjects for studying biological
phenomena at the cellular level. This increased research activity has primarily been
aimed at elucidating the structure and understanding the functioning of protozoa as
cells. There is a great deal of interest in planktonic protozoa and their functional role
in both marine and freshwater environments. World-wide, protozoa form a significant
part of planktonic biomass and, more important, have a major role in the flow of
energy and recycling of nutrients. So far, ultrastructural studies of micro-organisms
have been limited to conventional electron microscopy, which despite its high
resolution capabilities, also produces many artefacts during sample preparation.
Encystment (cyst formation) is a stage in the life cycle of many invertebrates
used to avoid adverse conditions. Generally, encystment is induced by starvation,
depletion of oxygen, increased salinity or dehydration. The cyst may survive in the
dormant state for many years. Excystment takes place once conditions become
favourable again. Figure 7 shows a protozoan cyst taken from a mixed culture of
protozoa.
X-Ray Microscopy in Aarhus
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Fig. 7. X-ray micrograph of a protozoan cyst which has an undulating outer membrane.
λ=2.4 nm, t=16s
Metals such as copper can be toxic to protozoa even at low concentrations [5].
Once the metal enters the cell, it can be accumulated and disrupt the elemental
distributions within the cell [6]. Therefore, some species exploit the avoidance
mechanism of encystment in order to survive during elevated external levels of
copper. Chilomonas paramecium starts to form a cyst within minutes of exposure to
elevated external levels of copper. Metal-induced encystment (cryptobiosis) of
flagellated protozoa has been studied here at ISA by LM and XM. Chilomonas
paramecium is approximately 20 µm long and 8 µm wide in favourable conditions.
However, the cell becomes more spherical within 10 minutes of copper exposure.
When the cell is centrifuged and resuspended in nutrient poor medium the cell also
forms a cyst. The rate of encystment can be followed by measuring the cell
dimensions. When exposed to copper the process is much faster. Fig. 8a shows an XM
image of an untreated cell. The cell appears very dense with many organelles. After
only 10 minutes in a solution with 10 ppm copper, the cells lose their cellular integrity
and round-up (Fig. 8b). This project aims to study structural changes during the
process of encystment in real time, to quantify the rate of metal-induced cyst wall
formation and examine the structural organisation within the cell during this process.
Further studies will determine if the process is metal dependent.
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Fig. 8. X-ray micrographs of Chilomonas paramecium. Top (a): untreated cell,
bottom (b): cell after 35 min. exposure to copper. λ=2.4 nm, t=12s
X-Ray Microscopy in Aarhus
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3 Conclusions
As a user facility, the Aarhus XM continues to be developed and improved, whilst the
number of collaborative research projects steadily increases each year. In the
forthcoming synchrotron radiation period, it is planned to incorporate phase contrast
and stereo X-ray microscopy.
Acknowledgements
We thank the Göttingen XM group led by Prof. G. Schmahl and the St. Brook group
by Prof. J. Kirz for their advisary and practical help.
References
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6
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