Development of a Stereo Imaging System
for Soft X-Ray Contact Microscopy
of Living Biological Material
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A.D. Stead , R. Bagby , D. Neely , A.M. Page , S. Rondot , E. Wolfrum , T.W. Ford
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Biological Sciences, Royal Holloway (Univ. of London), Egham, UK
2
Dept. of Zoology, Univ. Tennessee, Knoxville, TN, USA
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Rutherford Appleton Lab. Chilton, Didcot, UK
Abstract. Single-shot, short-pulse, soft X-ray contact microscopy (SXCM)
has been used to image living cells with spatial resolutions between those of
light and electron microscopy with exposures requiring only a few
nanoseconds. However, there is loss of spatial information in the Z-direction.
By using two simultaneous soft X-ray emissions, twin images of biological
specimens were obtained in which the separation of the images was
proportional to the height above the photoresist surface. In order to get good
separation the specimen must be placed several microns above the photoresist
surface and resolution is therefore sacrificed. In order to overcome this
problem grooved perspex was prepared with uniform, equally-spaced, rightangled V-grooves providing surfaces for two separate contact images. In this
case the plasma sources were normal to the respective faces of the V-grooves.
Moist, living mushroom spores or diatoms were mounted behind a silicon
nitride window to ensure that they remained moist and at atmospheric pressure
at the time of imaging. Overlapping double images resulted from specimens on
flat areas and discrete stereo images from specimens in the grooves.
1 Introduction
The attraction of imaging biological specimens with soft X-rays in the "water
window" (2.3–4.4 nm) is that it is possible to discriminate between structural features
of the cell which have a high carbon content and the cytoplasm which is relatively
carbon-dilute [1]. The technique needs a high-brightness X-ray source and suitable
detector and these requirements were not available when the prospects of X-ray
microscopy were first discussed in the 1960's [2]. Recently laser plasma and
synchrotron sources have been coupled with sensitive detectors such as polymethylmethacrylate (PMMA) and back-thinned cooled CCD cameras to make the technique
more viable.
The major goal of soft X-ray microscopy for the biologist is the ability to image
living material without the need for either chemical fixation or tissue freezing, both of
which can introduce artifacts [3-5]. However, unless the images are made with a either
a very low dose of X-rays, or with exposure to the X-rays being very short, then
radiation damage or thermal expansion will occur and alter the specimen before its
image is recorded [6-8]. Synchrotron sources have the ability to alter wavelength, but
the soft X-ray flux is low and exposure times are long. To date the only sources that
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can produce sufficient soft X-ray fluence for the single-shot, short-duration exposures
required to image living specimens are the pulse plasma sources (Z-pinch and laserproduced). Such images have been produced using soft X-ray contact microscopy
(SXCM) in which the specimen is placed in contact with a photosensitive resist usually PMMA. Exposure to X-rays results in bond breakage in the PMMA molecule
[9] and, after chemical development, the image in the resist can be viewed by
scanning electron microscopy or atomic force microscopy (AFM). The topography of
the image corresponds to the X-ray absorption by the specimen which reflects the
original specimen carbon density.
Although SXCM is able to give images of living biological specimens with
resolution superior to that obtained with light microscopy, the resolution of structures
deep within the specimen is compromised by the recorded image being an absorption
map of everything in line with the X-ray path. Superimposition of separate, spherical
structures in different XY planes can lead to the impression of a larger, multi-lobed
structure as imaged at the plane of the photoresist. The thicker the specimen, the more
superimposition becomes a problem. In light microscopy the problem can be
overcome by assigning Z-coordinates to separate XY planes, as is now done routinely
with confocal light microscopes. However, with X-rays this cannot yet be done with
sufficient resolution as the numerical aperture of the focussing optics (usually zone
plates) is too low. An alternative approach has been to record several different views
with the specimen rotated between each image. Such tomographic studies can produce
high resolution images of test objects [10] but as yet have not encompassed living
biological specimens. Furthermore, the need to take several images increases the
radiation received by the specimen to well beyond that which might be tolerated by
even the most radiation-insensitive biological specimens. Similarly X-ray holography,
which has been used to image biological specimens [11] precludes the possibility of
imaging living material without radiation-induced damage since the imaging times,
and radiation doses, are excessive. One technique that has not been explored is stereo
imaging, in which a pair of images are made from different perspectives. The two
images are then used to determine the relative positions of two structures in threedimensional space, usually with sufficient Z-axis resolution to determine whether two
structures are actually joined or merely appear to be joined in single images because
of superimposition of structures. Both light and electron microscopists [12] have
endeavoured to avoid the loss of 3D information by the use of stereo pairs. Stereo
pairs have also enhanced the information available for X-ray projection microscopy
[13] and such stereo images, taken by X-ray projection microscopy show detail not
seen by light microscopy [14]. Since SXCM has been able to provide better resolution
than X-ray projection microscopy, single-shot, laser-pulsed, stereo SXCM should give
the best 3D resolution of all the currently available techniques. Moreover it has the
advantage over other stereo X-ray methods published so far that it may be used on
living cells to capture details before the energy of the X-rays distorts the structure.
Recently stereo X-ray images using X-ray transmission microscopy have been
produced [15] but this required two sequential views taken after the specimen had
been tilted.
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In order to achieve stereo SXCM it is necessary to obtain two views from different
perspectives simultaneously so as to avoid natural or radiation-induced movement; the
rotation of the specimen, as occurs during tomographic studies, is not a viable
alternative. Therefore, this paper reports the manufacture of contoured photoresists
with V-shaped grooves in which the biological specimen can be placed so that the
specimen can be irradiated simultaneously from two separate soft X-ray sources each
o
at 90 to one of the surfaces of the groove.
2 Material and Methods
2.1 Soft X-Ray Source
Soft X-rays were produced from laser plasma sources by the interaction of the laser
(Nd:glass at Rutherford Appleton Lab.) with an yttrium target. The experimental
layout is shown in Fig. 1. The laser ( =1.053 m) energy varied from 10–20J per beam.
The optimum specimen to target distance was c.13mm and the soft X-ray fluence was
-2
estimated at between 50 and 150 mJ.cm at the resist surface.
Fig. 1. The experimental set up for simultanously imaging with two beams.
2.2 V-Grooved Photoresist
Commercial-grade perspex sheeting 1mm thick (Goodfellows, UK), which has the
same chemical structure as the PMMA photoresists usually used for SXCM, was used
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to produce the V-grooves. The 90 edge of a piece of cut plate glass was used to
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emboss a series of parallel 90 V-grooves, this produced grooves from 5-7 m wide at
the top and 3-5 m deep. The desired groove depth was obtained by varying both the
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embossing time and the ambient temperature. The approximately 90 profile of the
finished grooves was verified by light microscopy, and the roughness of the finished
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grooves was found to be about the same, or better, than the original flat surface of the
perspex by AFM (atomic force microscopy).
2.3 Spun PMMA Photoresist
It was proposed to use 1 m thick PMMA on silicon wafers to calibrate exposures with
the biological specimens prior to starting the more difficult stereo shots with the Vgrooved resists. However, it was found that their sensitivity was about an order of
magnitude less than that of the perspex sheeting. It was also found that at the angle
o
used for stereo shots (45 to a flat surface), the increased path length of the X-rays
through the water-immersed specimen made it very difficult to get sufficient exposure
through the specimen to the photoresist, especially when the laser beam energy to an
X-ray target was halved with the stereo setup.
2.4 Biological Specimens
Previous SXCM images of living cells on PMMA resists suggest several specimens
whose structure is sufficiently complex that stereo images would be useful. However,
when fully immersed many specimens ended up on the flat surfaces between the Vgrooves. The Si3N4 windows (FaSTec, UK) could not be placed too near the surface of
the V-grooved resist lest the specimens break the window and the extra depth of water
required to prevent breaking the window increased the X-ray path length so that
exposure of the resist at the bottom of the V-grooves was impossible. Mushroom
(Agaricus campestris) spores and diatoms (Navicula sp.) were chosen as mushroom
spores are able to withstand desiccation over long periods as part of the life cycle of a
mushroom, and freshwater diatoms have a very ordered structure in their silica
skeletons. The exposed photosensitive resists were chemically developed with 50%
methylisobutyl ketone:iso propyl alcohol and examined initially by DIC light
microscopy (Leica) and finally, when the development depth was sufficient, by AFM
(Park Scientific or Burleigh).
3 Results and Discussion
3.1 Comparison of Perspex and Spun PMMA as SXCM Resists
Initial images suggest that "perspex" is an extremely sensitive photoresist material but
it has surface scratches and roughness which make it unacceptable for SXCM.
Measurements of the surface area roughness indicate that figures of 100nm are typical
for the exposed surface of commercially available perspex, by comparison spun
PMMA has a surface roughness of 10nm. Despite this it was possible to produce cell
images as long as the development time for the resist was minimal. The embossing of
perspex therefore offers one way to create the necessary grooved surface for stereo
imaging. The use of glass knives with differing angles facilitates the production of
grooves with differing angles, whilst the duration of embossing and the temperature of
the photoresist, affect the depth of the grooves. The surface of the grooves produced in
this way appears to be a considerable improvement over the unembossed surface (Fig.
2). However, during development of the exposed groove surface, surface cracks
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appear more often than in the spun PMMA, indicating a difference in the "grain-size"
of the two materials which gives the desirable property of increased sensitivity but,
undesirably, lower resolution. It may, however, be possible to get higher resolution Vgroove surfaces by embossing thick-spun PMMA layers or by increasing temperatures
during the embossing of perspex.
3.2 Basis of Stereo X-Ray Imaging
By considering two X-ray images it is possible to demonstrate both how contact
microscopy could lead to inaccuracies and how stereo imaging would overcome this.
Consider the image on the left hand groove face (Fig. 3) and it is quite likely that the
two small dense structures would appear as one larger structure. However when
imaged with a source at right angles to this the image obtained corresponds to that on
the right hand groove face. It is now clear that the two small dense structures are
indeed separate and a reconstruction from these two images clearly shows the position
that these would occupy within a sphere of less absorbing material.
Fig. 2. AFM of groove used for stereo imaging in
unexposed PMMA.
Fig. 3. Theoretical basis of stereo
imaging; the two hypothetical images
on each side of the groove can be used
to reconstruct the original.
3.3 Stereo SXCM of Specimens on Horizontal Surfaces
An important part of the experiment was to demonstrate that one could obtain two
equal, orthogonal soft X-ray beams which were at right angles to the V-grooves.
Examination of specimen images on the top surfaces (between the V-grooves) parallel
to the plane of the Si3N4 window showed this had been accomplished. Since the top of
the grooved resist was some distance away from the window, typically 20-25 m, only
the middle portion of the resist received X-rays from both targets, whilst the end
regions were shaded by their respective soft X-ray-opaque window edges. Thus, at one
end of the resist, one ought to see shadows of the specimen extending away from one
X-ray source, and at the opposite end the shadows on the resist should extend in the
opposite direction. In the middle portion, where X-rays from both sources struck the
same specimen, there ought to be two shadows for each specimen, each of which
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points away from its X-ray source. Such images were obtained, and whilst those of
mushroom spores (Fig. 4) showed no internal detail, those of diatoms show the
substructure of the frustule (Fig. 5). Therefore all of the anticipated conditions were
met.
3.4 Stereo SXCM of Specimens in the V-Grooves
Mounting specimens in the grooves is difficult, the fresh basidiospores are sticky,
making it impossible to dust them onto the resists. Suspending them in water allowed
a better dispersion, and, being heavier than water, they fell into the grooves and onto
the horizontal surfaces. However, as the suspension dried the surface tension of the
water pulled the spores from the grooves onto the horizontal surfaces. When enough
water had evaporated for the exposure to soft X-rays, few, if any, of the spores were in
the grooves.
Fig. 4. Paired images of a mushroom spore
on the flat surface of perspex.
Fig. 5. Paired images of diatoms. In this case
the specimen was several m from the resist
surface and the two images are well separated.
By withdrawing water from the ends of the grooves with tissue it was possible to
ensure that some spores remained in the grooves. It was easier to ensure that the
diatoms remained in the grooves since these organisms secrete a sticky mucilage-like
material which caused the cells to adhere quite strongly to whatever surface they were
in contact with. Therefore, once positioned in the grooves, the cells mostly remained
there.
By exposing the grooved photoresists to two soft X-ray sources, each orthogonal to
the sides of the groove, it was possible to produce two images of the same specimen
with a single 2nsec laser shot (Fig. 6). Ideally these images should be studied using
AFM to produce two high resolution images which can then form the basis of the
production of the stereo pair. For most AFM’s, however, the range of movement in the
Z-direction is approximately 1/10 of the XY scan size, thus obtaining images with a
small scan head is not possible but this difficulty should be surmountable. At the
present time, therefore, we can demonstrate, by LM, that it is possible to make stereo
pairs of living biological specimens with SXCM.
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Fig. 6. Paired images in groove.
By intoducing a timing slide into one of the laser beam paths it is possible to delay
one of the X-ray sources, in doing time resolved studies on the effects of X-ray
radiation are possible and are reported in another paper in this volume [16].
There are several aspects of this work which require further attention before the
technique could be considered routine, firstly a more suitable material for making
high-resolution V-grooved resists should be found, the use of thick, spun-coatings
would seem the most appropriate. Secondly the presence of biological material on the
upper, horizontal resist surface must be eliminated so that the distance between the
Si3N4 window and the bottom of the grooves can be minimised. Lastly a method for
the examination of the images on the sides of the grooves needs to be perfected.
We hope that our demonstration of stereo SXCM with living biological specimens
will attract other biologists and hasten the technological developments necessary to
make this technique more available and easier to perform.
Acknowledgments
The authors would like to thank the University of Tennessee and AIST, Japan for
provision of funds for travel and accommodation; and Unilever plc for continued
financial support. We are very grateful for the assistance provided by the staff at RAL
in particular Richard Durrant and Oliver Thomas and the staff of target preparation,
laser operations and engineering.
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