Observation of the Inner Structures of Sea-Urchin Eggs
by Projection X-Ray Microscopy
K. Yada1, K. Shinohara2, Y. Hamaguchi3
1
Aomori Public College, Aomori 030-01, Japan
Faculty of Medicine, The University of Tokyo, Tokyo 113, Japan
3
Faculty of Science, Tokyo Institute of Technology, Tokyo 152, Japan
2
Abstract. The biological application of projection X-ray microscopy has been
continued using the imaging X-rays at the wavelength range of 0.5 - 1 nm in
order to ascertain whether the inner structures of the mitotic apparatus in seaurchin eggs are detectable or not. As a result, it is found that spindle fibers,
astral rays, chromosomes, and centrosomes of the mitotic apparatus are well
recognized in the sample prepared by critical point drying using Au targets,
while no structures are recognized in the case of the natural drying. These
structures are clearly seen with 3-D stereo pair. Image processing is
successfully tried to improve the image resolution and contrast.
1 Introduction
It was reported that projection X-ray microscopy is useful to reveal the inner
structures with structural variety in HeLa cells (about 10 µm in diameter) at the
mitotic phase. An image processing technique [1] which was effective to improve
resolution and contrast of projection microscope image was applied to the case of the
sea-urchin eggs.
2 Method and Materials
A projection X-ray microscope whose resolution reaches to a level better than 0.1 µm,
converted from a commercial scanning electron microscope, Akashi DS-130, working
with LaB6 cathode was used throughout the experiment. Ge and Au were selected as
the target materials which yield imaging X-rays of 1.05 nm and 0.58 nm in wavelength
respectively when 10 kV accelerating voltage is used.
The samples were prepared as follows: the fertilized eggs of the sea-urchin,
Hemicentrocus pulcherrimus, developed to metaphase or anaphase were lysed and
extracted for 1 hour in Mes-glycerol (MEG) buffer {0.55 mM MgCl2, 10 mM
glycoletherdiamine tetraacetic acid (EGTA), 25 % glycerol, 2.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM Mes-KOH, pH 6.8} containing 1 % Nonidet P40. Microtubules in the eggs lysed in MEG buffer were stable for many hours, and
thereby we called the microtubules in the lysed eggs a stabilized microtubules in order
to distinguish those microtubules from native microtubules in living cells and fixed
ones in the lysed and fixed eggs. The lysed eggs were attached to 0.1 % polylysinecoated SiN thin membrane. These eggs attached to the SiN membrane were fixed in
methanol at –20°C by replacing the MEG buffer with methanol and used for X-ray
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microscope observation after drying without further treatment. Two kinds of drying
methods, the natural drying and the critical point drying methods, were tried.
3 Results and Discussion
3.1 Differences Due to Drying Methods
Images of sea-urchin eggs by natural drying are shown in Fig. 1, where (a) is taken by
light microscopy and (b) by projection X-ray microscopy with Au target at 10 kV. It is
seen that no inner structure is recognized in the X-ray image for the case of the natural
drying, though faint structures are seen in the light microscope image.
Fig. 1. Sea-urchin eggs by natural drying taken with light (a) and X-ray (b).
Figure 2 shows images of a sample prepared by critical point drying, where (a) is Xray image taken with Au target at 10 kV and (b) scanning electron image at 3 kV
without conducting coating. Inner structures in relation to the mitotic apparatus are
now recognized in (a) as marked with arrows, where MP represents mitotic pole, Ch
chromosome. It is seen that sea-urchin eggs have rather rough surface structures,
which will be superposed to the inner structures in the case of X-ray image and might
disturb the recognition capability of the inner structures. It was also found that Au
target is more suitable than Ge target for sea-urchin egg because transparency of the
imaging X-ray from Ge target is insufficient to observe the details of inner structures
in such a big sea-urchin egg. Figure 3 shows an enlarged X-ray image of the same seaurchin egg as above to see the mitotic apparatus more easily. Figure 4 shows another
example of X-ray image of a critical point dried sea-urchin egg partially covered by
so-called fertilization envelop, where the mitotic apparatus such as astral rays, spindle
fibers, chromosomes are more clearly seen perhaps because of smoother surface
structures of the egg. It is seen that individual chromosomes are just going to be
identifiable. On the other hand, individual microtubules are so thin (20–30 nm in
diameter) that it is far below the resolution of the present X-ray microscope but it
seems that a kind of bundle effect makes it visible.
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Fig. 2. X-ray image (a) and SEM image (b) of a critical point dryed sea-urchin egg.
Fig. 3. Enlarged image showing the mitotic apparatus taken with Au target at 10 kV.
P: mitotic pole, Ch: chromosome.
Fig. 4. Enlarged image showing the mitotic apparatus, where inner structures are seen
more clearly because of the smoother surface structures.
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3.2 Stereo Observation
Fig. 5. Stereo pair of a sea-urchin egg which is covered with a fertilization envelope.
A stereo-pair of an egg is shown in Fig. 5, where the egg is covered by the fertilization
envelope or the hyaline layer. The envelope is expanding to a fairly wide area but
structureless as compared with the surface structure of the egg and the surface
structure itself seems to be fairly smooth when covered with the fertilization envelope.
3.3 Image Processing
A point projection image is essentially defocused one. So, it is desirable to restore the
defocused image by image processing to the infocused one restoring the effect of
Fresnel diffraction. Consequently, resolution and contrast will be improved. We
already reported two kinds of image processing, the deconvolution treatment using
Wiener filter [2] and the weak amplitude approximation [1]. The latter was here tried
for the case of sea-urchin egg using the same algorithm [1]. In Fig. 6 (a) is the original
but digitized image and (b) the processed one by assuming a suitable defocus amount.
Considerable improvement in resolution and contrast is seen in (b).
Fig. 6. An example of image processing. (a): original, (b): processed image,
where resolution and contrast are considerably improved.
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4 Conclusion
1. The structures in the mitotic apparatus such as mitotic poles, astral rays, spindle
fibers, and chromosomes were recognized in the critical point dried sea-urchin
eggs by projection X-ray microscopy.
2. The mitotic apparatus can be stereoscopically observed by stereo pair.
3. Image processing was successfully applied to improve the image resolution and
contrast.
Acknowledgements
The present authors want to express sincere thanks to Dr. Qingxin Ru, Basic Structure
Group, ERATO, Res. Devel. Corp. Japan, for his valuable suggestions and technical
assistance to the image processing.
References
1
K. Yada and K. Shinohara, X-ray Microscopy IV, Proc. XRM ’93
Chernogolovka, Russia 171 (1993).
2
K. Yada, S. Takahashi and T. Tanji, Proc. 3rd Beijin Conf. and Exhib. Instrum.
Analysis A-7 (1989).