Imaging Soft X-Ray Microscopy with Zone Plates
in Parallel Use of Optical Microscope
for Wet Bio-Specimens in Air at UVSOR
Norio Watanabe1, Atsuhiko Hirai2, Kuniko Takemoto3, Yoshio Shimanuki4, Mieko
Taniguchi5, Eric Anderson6, David Attwood6, D. Kern7, Sumito Shimizu8, Hiroshi
Nagata8, Kenzo Kawasaki4, Sadao Aoki1, Yasuyuki Nakayama2, Hiroshi Kihara3
1
4
6
Institute of Applied Physics, University of Tsukuba, Tennoudai 1-1-1, Tsukuba,
Ibaraki, 305, Japan, E-mail: watanabe@kirz.bk.tsukuba.ac.jp
2
Department of Physics, Ritsumeikan University, Kusatsu, Shiga 525-77, Japan
3
Physics Laboratory, Kansai Medical University, Hirakata, Osaka 573, Japan
Department of Oral Anatomy, Tsurumi University, Tsurumi, Yokohama 230, Japan
5
Department of Physics, Nagoya University, Chikusa-ku, Nagoya 464, Japan
Center of X-ray Optics, Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA
7
IBM T. J. Watson Research Center, Yorktown Heights, NY 10598, USA
8
Nikon Corp. Nishi-ooi, Shinagawa-ku, Tokyo 140, Japan
Abstract. Soft X-ray microscope, a specimen holder of which was placed in
air, was constructed at UVSOR, synchrotron radiation facility at Institute for
Molecular Science, Japan. This made possible to investigate a specimen
without impairing the vacuum of the microscope and to prefocus a specimen
with an optical microscope incorporated in the microscope. Dry and wet
specimens could be observed at a wavelength of 0.94 nm.
1 Introduction
We have been developing a soft X-ray microscope with zone plates to observe
hydrated biological specimens with higher resolution than that of an optical
microscope. Our previous report showed that a 63 nm line and space pattern could be
resolved at a wavelength of 3.2 nm, and dry and wet biological specimen could be
observed[1].
In 1995, the soft X-ray microscope was improved in the following points. (a) A
specimens stage was put in air gap, which was separated from condenser and
objective vacuum chambers by SiN windows. This made possible to investigate a
specimens without breaking the vacuum of the beam line. (b) An optical microscope
was set to adjust and prefocus a specimens. (c) A cooled CCD camera system was
used as a detector. This type of a soft X-ray microscope was first developed by
Göttingen X-ray microscope groups [2]. This report describes our new microscope
chamber and the results of imaging experiments.
2 Optical System
The optical system was the same type as the Göttingen X-ray microscope [2].
Figure 1 shows the optical system of the microscope. Synchrotron radiation from the
bending magnet source BL8A at UVSOR (750 MeV, 200 mA, Institute for Molecular
Science, Okazaki, Japan) was used. Soft X-rays from the source were monochroma-
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N. Watanabe et al.
tized by a condenser zone plate (CZP) and incident on a specimen. The image of a
specimen was enlarged by an objective zone plate (OZP), and focused on a cooled
backside illuminated CCD camera (Astromed Corp. CCD: SITe Corp. SI502A). To
prevent zero-th order radiation of the OZP from reaching the imaging area on the
detector, the image of a specimen was focused on the off-axis area outside of the
zero-th order radiation.
Focusing tests were performed at wavelengths of 1.3 nm, 2.4 nm, and 3.2 nm.
1
Images were observed at only 1.3 nm in wavelength . Then, images were observed at
a wavelength of about 1 nm. The synchrotron source has the electron beam size of
σx=0.39 mm (horizontal) and σy=0.26 mm (vertical) [3]. Assuming the diameter of
the source to be 0.80 mm, the source image size at a pinhole plane was calculated to
be 120 µm (first order) or to be 32 µm (third order) at 1.3 nm in wavelength. The
third order radiation of the CZP was mainly used to reduce the source image size at
the pinhole plane, and to improve the monochromaticity. The monochromaticity λ/∆λ
of the linear monochromator was calculated to be 67 from the relationship λ/∆λ
=D/2d, where D is a diameter of a CZP and d is the diameter of the source image at
the pinhole plane because the diameter of the source image size was larger than that
of the pinhole[4]. The fourth order radiation of 0.94 nm in wavelength was strongly
contaminated into the third order radiation. It could not be removed, and soft X-rays
of both wavelengths were used for imaging.
Fig. 1. Optical system of soft X-ray microscope at UVSOR
1
After the experiments, the quantum efficiency of the CCD was measured. The efficiency was
several percent at a wavelength longer than 2 nm. It is too low for a backside illuminated CCD
with no coat. This seems to be main reason that images could not be observed at a wavelength
longer than 2 nm.
Imaging Soft X-Ray Microscopy with Zone Plates
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3 Microscope Chamber
Figure 2 shows a schematic diagram of the microscope chamber. The microscope was
separated to two vacuum parts, a CZP chamber and an OZP chamber. A specimen
stage was placed in air. The CZP chamber could be moved back and forward along
the optical axis with a pneumatic cylinder, the air gap between the two chambers
could be changed from several hundred microns to several millimeters and a
specimen could be easily changed. These stage was placed on a rotating stage and the
soft X-ray microscope and an optical microscope could be switched by rotating the
stage with a pneumatic cylinder. The optical microscope could be used for searching
and prefocusing a specimen.
Figure 3 shows a schematic of the specimen holder. SiN windows of 0.1 µm
thickness and 200 by 200 µm area were used for vacuum seals. These windows were
stuck on frames by adhesive (Torr Seal, Varian Corp.). The pinhole was also stuck on
the frame of the SiN window outside of the vacuum chamber. The pinhole was
changed to new one with a period of about one month because it was closed with
contamination.
Fig. 2. Top view of the microscope chamber
Fig. 3. Schematic of the specimen stage in air
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N. Watanabe et al.
4 Results and Discussion
Figs. 5 and 6 show images of Cu #2000 mesh at wavelengths of 1.3 nm (the third
order radiation of the CZP) and 0.94 nm (the fourth order). These images were taken
under the same CZP position and the OZP position was only changed along the
optical axis to focus soft X-ray images of 1.3 nm and 0.94 nm in wavelength,
respectively. The resolutions were estimated from the edge profiles of these images.
The width of the intensity rise from 10 to 90 % was 0.26 µm at a wavelength of 1.3
nm, and 0.50 µm at 0.94 nm. Fig. 6 shows an image of a Ni zone plate as a specimen
at a wavelength of 0.94 nm. The specimen had the same specification as the OZP. The
diameter of the first zone was 3 µm. The finest resolvable zone width was 0.14 µm.
These resolutions were worse compared with a theoretical one of the OZP (1.22 × the
outermost zone width = 55 nm). It was considered to be due to the low
monochromaticity and the higher order radiation of the CZP. To obtain images
without chromatic aberration, it is necessary to use quasimonochromatic soft X-rays
of monochromaticity λ/∆λ =277, which is the zone number of the OZP. However, the
theoretical monochromaticity was 67 in this experiment. Superposition of several
order radiation of the CZP caused degradation of the image quality. The
superimposed mesh image (central bright rectangle area) in Fig. 6 is an image focused
with the fourth order radiation of 0.94 nm in wavelength.
Fig. 4. (a) Cu #2000 mesh image at 1.3 nm
in wavelength. (b) The intensity profile of
the image at the white bar.
Fig. 5. (a) Cu #2000 mesh image at 0.94
nm in wavelength. (b) The intensity
Profile of the image at the white bar.
Imaging Soft X-Ray Microscopy with Zone Plates
Fig. 6. Image of a zone plate as a
Specimen. Wavelength: 0.94 nm.
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Fig. 7. Image of diatoms at 0.94 nm.
Using a specimen holder with polyimide foils, several wet biological specimens
could be observed at 0.94 nm in wavelength[5]. Fig. 7 shows an image of a diatom,
which was collected from the sea, and killed by sulfuric acid, and observed.
Acknowledgments
The authors are grateful to the help and encouragement by Dr. T. Kinoshita,
E. Nakamura and other staffs of the Institute for Molecular Science.
References
N. Watanabe, S. Aoki, Y. Shimanuki, K. Kawasaki, M. Taniguchi, E. Anderson,
D. Attwood, D. Kern, S. Shimizu, H. Nagata, and H. Kihara, in X-ray
Microscopy IV, eds. V. V. Aristov, and A. I. Eriko, (Bogorodskii Pechatnik
Publishing Company, Chernogolovka, Moscow region, Russia 1994), p.333.
B. Niemann, G. Schneider, P. Guttmann, D. Rudolph, and G. Schmahl, in X-ray
Microscopy IV, eds. V. V. Aristov, and A. I. Eriko, (Bogorodskii Pechatnik
Publishing Company, Chernogolovka, Moscow region, Russia 1994), p.66.
UVSOR Activity Report 1995 (1996)
B. Niemann, D. Rudolph, and G. Schmahl, Opt. Commun. 12, 160 (1994).
K. Takemoto, N. Watanabe, A. Hirai, Y. Nakayama, and H. Kihara, this
conference.