Observation of the Inner Structures of Sea-Urchin Eggs by Projection X-Ray Microscopy

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1 Observation of the Inner Structures of Sea-Urchin Eggs by Projection X-Ray Microscopy K. Yada 1, K. Shinohara 2, Y. Hamaguchi 3 1 Aomori Public College, Aomori , Japan 2 Faculty of Medicine, The University of Tokyo, Tokyo 113, Japan 3 Faculty of Science, Tokyo Institute of Technology, Tokyo 152, Japan Abstract. The biological application of projection X-ray microscopy has been continued using the imaging X-rays at the wavelength range of 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 LaB 6 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 MgCl 2, 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 P- 40. 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

II - 256 K. Yada et al. microscope observation after drying without further treatment. Two kinds of drying methods, the natural drying and the critical point drying methods, were tried.

1, where (a) is taken by light microscopy and (b) by projection X-ray microscopy with Au target at 10 kv.

2 II K. Yada et al. 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 X- ray 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.

Observation of the Inner Structures of Sea-Urchin

3 Observation of the Inner Structures of Sea-Urchin Eggs II 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.

II - 258 K. Yada et al. 3.2 Stereo Observation Fig. 5. Stereo pair of a sea-urchin egg which is covered with a fertilization envelope.

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

$So, it is desirable to restore the defocused image by image processing to the infocused one restoring the effect of Fresnel diffraction.$ We already reported two kinds of image processing, the deconvolution treatment using Wiener filter [2] and the weak amplitude approximation [1].

We already reported two kinds of image processing, the deconvolution treatment using Wiener filter [2] and the weak amplitude approximation [1].

4 II K. Yada et al. 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.

5 4 Conclusion Observation of the Inner Structures of Sea-Urchin Eggs II 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).

S200 Course LECTURE 1 TEM

S200 Course LECTURE 1 TEM Development of Electron Microscopy 1897 Discovery of the electron (J.J. Thompson) 1924 Particle and wave theory (L. de Broglie) 1926 Electromagnetic Lens (H. Busch) 1932 Construction