Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

11A,12A/2003G326

Improvement of soft X-ray projection CT microscope
Tatsuo SHIINA1*, Tsuyoshi Suzuki1, Yosuke TSUGE1, Toshio HONDA1,Hideyuki YOSHIMURA2, Yasuhito KINJO3, and Atsushi ITO4
1
Faculty of Engineering, Chiba Univ., Chiba-shi, Chiba, 263-8522, Japan
2
School of science and Technology, Meiji Univ., Kawasaki-shi, Kanagawa, 214-8571, Japan
3
Tokyo Metropolitan Industrial Technol. Res. Inst., Setagaya-ku, Tokyo 158-0081, Japan
4
School of Engineering, Tokai Univ., Hiratsuka-shi, Kanagawa, 259-1292, Japan
Introduction
Since soft X-ray is in the spectral region of“Water-window”, it is dominant in the observation of aliving cell. In other word, soft X-ray gives a good contrast
for wet specimens. In a projection microscopy, thespecimen should be brought close to the light source that islocated at the position of a pinhole with a diameter of
1-5µmφ when the microscope raises its magnification.
Therefore, the authors have exercised the ingenuity to holdand rotate a specimen close to the pinhole.
In this study, the magnification and specimen holder
were improved on the precedent soft X-ray projection
microscope[1]. The lateral magnification of the microscope
was fixed at x107 or x134. As an X-ray CCD camera was a
back-illuminated type of 512x512 pixels (24.8µm/pixel),
its field of view became 118µm or 95µm square at theabove magnifications. The experimental setup is shown in
Fig. 1. The experiment was conducted at the beam line 11A
and 12A. Monochromatic soft X-ray was used at thewavelengths of 15 - 25 angstrom (0.83 - 0.50 keV inenergy). The specimen, the rotation stage, and the imaging
area of the CCD camera were in vacuum. The specimen
was bonded on the tip of the rotating shaft and observed
under the low vacuum of 10-4 Torr. An X-Y stage can becontrolled from the outside of the vacuum.
Reconstructed CT images
Each projection image was integrated in its intensity for
2 – 5 minutes to improve the signal-to-noise ratio. It took3-4 hours to obtain a set of projection images. The
projection image blurs because of fringes caused by
Fresnel’s diffraction. This study eliminates such fringes on
the projection image with the iteration process, which
repeats the calculation between Fresnel and inverse Fresnel
transformation. Figure2 shows the CT image of a glass
capillary reconstructed by the filtered back projection fromthe projection images at an interval of 3 degrees all roundthe specimen. The outer diameter of the capillary wasestimated as about 6 µm, while inner diameter was about
4.2 µm. We confirmed that the pipe structure was clearlyreconstructed with enough contrast by using the iterationprocess. CT reconstructed image of HeLa cells were also
shown in Fig. 3. The HeLa cells were cultivated on a mylar
film on a single-hole mesh for 12 hours. Cells were fixedwith glutaraldehyde and then dried; (a) Air-drying and (b)Critical point drying. Because the single-hole mesh wasslightly listed, the specimen was away from the rotation
axis. As the mesh’s edge shadowed the X-ray light, theprojection images could not be obtained in the angularrange of about 100 degrees in total. The lacked images
were interpolated with the dummy data at the cell’s
position in their angles. In the CT reconstructed image, theinternal structure could not be clearly visualized because of
the Fresnel’s diffraction, while the nucleus and otherintracellular structures were recognized.

Fig. 1 Soft X-ray projection CT microscope

Fig. 2 CT image ofglass capillary with
iteration process.

Fig. 3 CT image of HeLa cells without iteration process.
(a) Air-drying (b) Critical point drying.
References[1]T. Shiina et al., PF Activity Rep. #22 Part B, 231 (2004)*shiina@faculty.chiba-u.jp

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