Performance of a Laboratory X-Ray Microscope,
Using Z-Pinch-Generated Plasmas,
for Soft X-Ray Contact Microscopy
of Living Biological Specimens
T. W. Ford1, A. M. Page1, S. Rondot1, R. Lebert2, K. Bergmann2, W. Neff3,
C. Gavrilescu4, A. D. Stead1
1
Division of Biology, School of Biological Sciences, Royal Holloway,
University of London, Egham Hill, Egham, Surrey TW20 0EX, UK
2
Lehrstuhl für Lasertechnik, Steinbachstrasse 15, D-52074 Aachen, Germany
3
Fraunhofer Institüt für Lasertechnik,
Steinbachstrasse 15, D-52074 Aachen, Germany
4
AL. I. Cuza University of Iasi, Copou 11, 6600-Iasi, Romania
Abstract. The use of high energy lasers for generating plasmas for soft X-ray
contact microscopy (SXCM) of living biological specimens is now well
established. However, such systems are only available at national laboratories
where SXCM is carried out in competition with other users which can create
problems when using living specimens. An ideal system would be contained
within a normal laboratory. Using the pinch-plasma X-ray source developed at
the Institut für Lasertechnik in Aachen, a laboratory-scale soft X-ray contact
microscope has been assembled. A selection of images of living specimens are
presented here to illustrate the performance of such a microscope.
1 Introduction
Imaging of living cells has only been possible using light microscopy which, despite
recent improvements to the basic system (confocal scanning microscopy; image
enhancement), has a resolution which is essentially limited by the source of illumination.
The maximum resolution possible with visible light systems is around 250nm. Electron
microscopy significantly improves the resolution to 2nm for biological material.
However, to achieve this, ultrathin sections are required which necessitates fixing,
dehydrating and embedding the material before sectioning. These processes can
introduce ultrastructural artefacts into the image [1],[2]. By using X-rays, whole living
cells can be examined at a resolution superior to that of light microscopy and by
irradiating the cells with soft X-rays in the so-called ’water-window’ (2.3-4.4nm) a degree
of contrast is introduced due to differential absorption of this radiation by carbon and
oxygen.
Soft X-ray contact microscopy (SXCM) uses the impact of a high energy laser onto a
suitable metal target to generate a plasma rich in soft X-rays. We have been using the
Nd-glass laser (VULCAN) housed at the Rutherford Appleton Laboratory (RAL) in the
U.K. This produces in the region of 8-12J laser energy (1053nm) onto target resulting in
measured doses of soft X-rays in the water window of around 50-100mJ.cm-2. The living
specimen is mounted in an environmental sample holder which maintains atmospheric
pressure and normal humid, aerobic conditions until the laser shot is fired. This system
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has produced good images of a range of biological specimens including details of cell
ultrastructure [3–4]. However, VULCAN, due to its size, is a multi-user facility with two
or three target areas operating in competition. Whilst this usually poses no serious
difficulty for physics experiments, imaging of living material can be problematical. Even
though the sample is in an environmental holder it will not remain in a healthy condition
indefinitely. Loss of water and a continuously reducing oxygen concentration can cause
ultrastructural changes. Ideally specimens should be imaged as soon as they have been
installed in the target chamber. This cannot be guaranteed with a multi-user facility such
as VULCAN. In addition, the possibility of examining ultrastructural changes at
frequent, regular time periods following experimental manipulation of the sample is
virtually impossible.
For useful biological X-ray microscopy a dedicated instrument within the laboratory
is required, as with all other forms of microscopy. This requires a reduction in scale of
the equipment, in particular of the system for generating soft X-rays. One solution is a
laboratory-scale X-ray microscope such as that constructed and housed at the Fraunhofer
Institut Lehrstuhl für Lasertechnik in Aachen.
2 Imaging System at Aachen
This microscope uses a Z-pinch plasma focus device to generate a plasma, the X-ray
emission of which is then focused by two condenser mirrors onto the specimen [5]. By
using 150Pa argon in the discharge chamber (1.8kJ bank energy) and 120Pa oxygen as
the absorber gas in the beamline, the emission spectrum of argon is predominately in the
water window i.e.2-4nm. Other gases can be used in the discharge chamber e.g. nitrogen
or acetylene to produce different emission spectra.
Due its relatively small size this imaging system can be housed in a normal laboratory
providing the operators with easy, regular and continual access. The sample holder used
for the work at RAL could be accommodated within the beamline of this microscope so
ensuring that the living cells are held under normal environmental conditions up to the
time of the laser shot.
Following exposure to water window soft X-rays, the photoresist was removed and
developed in a mixture of methyl isobutyl ketone (MIBK) in isopropyl alcohol (IPA).
This was usually a 50:50 mixture. Development of the image was monitored using
interference light microscopy and development continued until clear images of the cells
could be seen. The resist was then scanned using a Burleigh ARIS 3300 atomic force
microscope to produce a high resolution readout of the images. If necessary, the resist
could be returned to the MIBK:IPA for further development.
3 Results
One of the main claims of SXCM is that it can produce high resolution images of living
cells. This has been possible with a large national facility such as that at RAL but
whether such images could be obtained with a smaller laboratory-scale microscope was
uncertain. We have imaged a number of unicellular organisms at RAL and a selection of
these have also been imaged at Aachen. Some of these images are presented here.
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3.1 Chlamydomonas
This is a unicellular green alga which can swim by using two equal length flagella
inserted, and anchored by flagellar roots, at the anterior end of the cell. Cells are
typically 5–10 µm in diameter and most of the cell volume is occupied by a large, cupshaped chloroplast. Within the basal end of the chloroplast is a dense, spherical
aggregation of protein called the pyrenoid. The cell nucleus is contained within the "cup"
of the chloroplast. The whole cell is covered by a cell wall which, in the case of
Chlamydomonas, is not cellulose but a glycoprotein mixture.
Figures 1 & 2. AFM readout of SXCM images of living cells of Chlamydomonas produced
using either the microscope at Aachen (1) or the imaging system at the Rutherford Appleton
Laboratory (2). CW-Cell Wall; F-Flagellum; S-Spherical Inclusion.
AFM readout of SXCM images of living Chlamydomonas reveals an ovoid cell of
around 8µm in diameter (Fig. 1). Two prominent, equal flagella are seen to issue from
the anterior end of the cell through a relatively carbon-dilute area of the cell (in
comparison with the main cell body). No internal detail of the main part of the cell can
be distinguished. Images of this alga obtained using the system at RAL routinely show
spherical inclusions in the anterior end of the cell cytoplasm in addition to a much clearer
image of the flagella and cell covering (Fig. 2). This is probably due to the higher soft Xray fluence obtainable at RAL.
3.2 Tetraselmis
This is also a unicellular green alga but, unlike Chlamydomonas, has four equal size
flagella inserted at the anterior end of the cell. Once again there is a large cup-shaped
chloroplast occupying most the cell volume and containing a basal pyrenoid. The nucleus
is centrally located. The Tetraselmis cell is enclosed by a polysaccharide theca consisting
of galactose and uronic acid units.
AFM readout of SXCM images of living Tetraselmis reveals an elongated cell
approximately 12µm long by 5µm wide (Fig. 3). The flagellar position at the anterior
end of the cell is just visible but no detail can be observed. Likewise there is no structural
detail visible in the main body of the cell. This resist has been developed in 50% MIBK
in IPA for 6 minutes. By continuing development for another 10 minutes the location of
the four flagella can be seen (Fig. 4). However, this rather excessive period of
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development causes a significant increase in the surface roughness of the resist so
obscuring any improved detail. Once again, the low fluence of soft X-rays results in an
image of poor quality with no observable detail within the cell body.
Figures 3 & 4. AFM readout of SXCM images of living cells of Tetraselmis following development of the photoresist for either 6 minutes (3) or 16 minutes (4). Increased surfaceroughness
and noise is obvious after prolonged periods of development. F-Flagellum.
3.3 Phytomonas
This single-celled protozoan is a parasite of plants. The spindle-shaped cell has a corset
of microtubules under the plasma membrane which define and maintain the shape of the
cell. The organism swims by means of a single, long flagellum which arises from a
flagellar pocket at the anterior end of the cell.
Figures 5 & 6. AFM readout of SXCM ages of living cells of Phytomonas. The patterning visible
at higher magnification is probably AFM scan noise (6). F-Flagellum.
SXCM images of living Phytomonas cells revealed by AFM show long cigar-shaped
bodies with a single flagellum issuing from the anterior end of the cell (Fig. 5). Cell
dimensions are approximately 12µm long by 2µm wide. A higher magnification scan
shows the position and dimensions of the flagellum rather clearer, though little detail of
the cell ultrastructure can be seen (Fig. 6). Previous imaging of this flagellate at RAL
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showed the cross-hatching of the cell surface due to overlap of the helical arrangement of
cytoskeletal microtubules. Whilst the image reproduced here provides hints of similar
structures, it is possible that the pattern observed results from the AFM scan rather than
components of the cell.
4 Conclusions
The laboratory X-ray microscope at Aachen is a reliable and accessible system for
imaging living cells. Images of three living, unicellular, motile organisms of up to 12µm
in size have been produced which show some detail of the morphology of the cell.
However, details of the internal structure of the cells could not be seen in these examples
presumably due to insufficient soft X-ray fluence resulting from non-optimal operating
conditions of the instrument for SXCM e.g. condenser optics generated point-like
(20µm) illumination resulting in too high a fluence at the centre of the sample and too
low at the periphery. In addition, alignment was not always accurately adjusted and the
distance between source and sample was much greater than at RAL. Correction of these
will produce superior imaging conditions in the future.
This microscope provides an ideal system for the biologist since it can be housed and
operated within a conventional laboratory setting with all necessary ancillary equipment
to hand. As a dedicated instrument it allows immediate and continual access so allowing
experimentation to take place with sequential, timed imaging.
Acknowledgements
Provision of funding for this collaborative work through an EU Network is gratefully
acknowledged as is funding from the EPSRC (UK) for purchase of the atomic force
microscope (Grant GR/K23522). We are also grateful to Stephen Janes for assistance
with producing the figures for this paper.
References
1
B. Mersey and M.E. McCully, J. Microscop. 114, 49 (1978).
2
S.G.W. Kaminskyj, S.L. Jackson, and I.B. Heath, J. Microscop. 167, 153 (1992).
3
T.W. Ford, R.A. Cotton, A.M. Page, and A.D. Stead, in X-Ray Microscopy IV (Institute of
Microelectronics Technology, Chernogolovka, Russia 1994).
4
A.D. Stead, R.A. Cotton, A.M. Page, J.A. Goode, J.G. Duckett, and T.W. Ford, in X-Ray
Microscopy IV (Institute of Microelectronics Technology, Chernogolovka, Russia (1994).
5
K. Bergmann, R. Lebert, and W. Neff, this volume.