X-Ray Microscopy System
with an Electronic Zooming Tube
K. Shinohara1, 2, A. Ito3, H. Nakano2, T. Honda4, K. Yada5
1
Radiation Research Institute, Faculty of Medicine, The University of Tokyo,
Tokyo 113, Japan, E-mail: kshino@m.u-tokyo.ac.jp
2
Department of Radiation Research,
Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan
3
Department of Nuclear Engineering, School of Engineering,
Tokai University, Kanagawa 259-12, Japan
4
Department of Image Science, Faculty of Engineering,
Chiba University, Chiba 263, Japan
5
Aomori Public College, Aomori 030, Japan
Abstract. An X-ray microscopy system is presented using an electronic
zooming tube as a two dimensional X-ray detector based on previous works
on the X-ray contact microscopy of dried human cells in a wide wavelength
range of X-rays and on the X-ray holographic microscopy of dried human
cells. The system is designed to work for a contact microscopy and for a
holographic microscopy, and is arranged as a user-friendly system. The
system was tested for a resolution, X-ray attenuation curve by aluminum, and
efficiency of the detection of photons at the energy of 2472.7 eV. The results
showed that the resolution was about 1 µm, that photon counting method was
suitable for a wide dynamic range, and that the efficiency of the system was
2.6x10-3. Imaging phosphorus in a cell was successful by subtraction of
images at the energy on (2152.8 eV) the peak in XANES profile of DNA and
below (2145.3 eV) K-absorption edge of phosphorus. The present results
suggest that the system is successful but remains to be improved.
1 Introduction
Electronic zooming tube is a two dimensional detector of soft X-rays with a resolution
higher than 0.5 µm [1]. The detector may be directly applicable to an X-ray
microscopy system as an X-ray contact microscopy or an X-ray holography with the
following features: (1) No optical elements are required for imaging specimens at the
resolution of the detector; (2) Images obtained with different wavelengths can be
directly compared; and (3) the detector is applicable to a wide wavelength range of
X-rays even shorter than 1 nm down to 0.1 nm [1].
With an electronic zooming tube, we have succeeded in X-ray contact microscopy
of dried human cells with various wavelengths of X-rays from 1.5 - 10 nm which
include K-absorption edges of carbon, nitrogen and oxygen, and L-absorption edges
of iron, calcium and sulfur [2]. The data were analyzed for the relative fraction of
elements in local areas of cells. The results suggest that the system may be applicable
to imagines of chemical natures in biomolecules using a specific absorption peak in
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K. Shinohara et al.
XANES profiles. It has been demonstrated that the detector is also applicable to an Xray holographic microscopy in combination with coherent X-rays [3].
In the present communication, a new type of X-ray microscopy system with an
electronic zooming tube is presented based on the experimental results mentioned
above. The system is designed to work for an X-ray contact microscopy and for an Xray holographic microscopy, and is arranged as a user-friendly system.
2 X-Ray Microscopy System
The major modifications had been performed at the photocathode part of an electronic
zooming tube. Figure 1 illustrates a structure of a specimen holder and holder heads
(one for contact microscopy and the other for holographic microscopy). The
photocathode was arranged to be easy mounting system as a part of the specimen
holder.
Fig. 1. Specimen holder (a), and holder heads for contact microscopy (b) and holography (c).
X-Ray Microscopy System with an Electronic Zooming Tube
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3 Test of the System
3.1 Resolution
Resolution of the present system was studied at the photon energy of 2472.7 eV with
the edge pattern of EM-grid made of copper (Fig. 2). The resolution defined as the
distance to show 10 - 90 % in the intensity profile of edge pattern was 0.98 µm when
100 nm thick gold film was used as photocathode and the magnification of the system
was 210 corresponding to 0.246 µm/pixel.
Fig. 2. Resolution of the present system. Profile of relative photon intensity along the gray line
in the inserted picture of EM-grid was presented. The distance between two vertical white lines
indicates the resolution.
3.2 Efficiency of the Detection of Photons
Beam size was restricted by a pinhole of 0.1 mm in diameter. Then the total photons
were detected by a silicon photodiode (AXUV-100, International Radiation
Detectors). In this condition, total beam area was covered by the photocathode in the
electronic zooming tube. Therefore, the total photons obtained by the electronic
zooming tube was estimated by summing up the photons detected in each pixel. The
measurement was performed at the photon energy of 2472.7 eV.
For example, total photons detected by the zooming tube operated at photon
counting mode was 2.09 photons/mA ring current/sec under the condition that incident
X-rays were attenuated by aluminum of 14 µm thickness. At the same time, total
current in photodiode was 8.81x10-5 nA/mA ring current corresponding to 807.4
photons/mA ring current/sec because the conversion factor of photodiode was
1 electron/3.63 eV from IRD (International Radiation Detector) data sheet. Therefore,
the efficiency was 2.6x10-3. Similar efficiency was obtained when different photon
fluxes were used by inserting aluminum of various thickness.
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K. Shinohara et al.
3.3 X-Ray Attenuation by Aluminum
Figure 3 shows the relative intensity of transmitted X-rays at the energy of 2472.7 eV
with respect to the thickness of aluminum. The results by photodiode were well
coincided with those by the electronic zooming tube when the data were obtained by
the photon counting method. On the other hand, the data was removed from the
expected line of attenuation curve within 2 orders of magnitude when the integration
mode was used. The results strongly recommended the use of photon counting method
for the analysis of data with a wide dynamic range.
Fig. 3. Attenuation curve of X-rays by aluminum sheet. Transmitted X-rays through 1 mmφ or
0.1 mmφ pinhole were measured . Intensity was normalized at that of aluminum.
4 Imaging Phosphorus in a Cell
Phosphorus distribution in a cell was obtained by dividing a cellular image on the
absorption peak in XANES of DNA at the phosphorus K-absorption edge by that
below the absorption peak. XANES profile of DNA is shown in another paper by us in
this volume. HeLa cells were cultured on the reverse side of SiN thin film and dried.
On the front side of SiN Au was then coated with the thickness of 100 nm. Photon
energies were adopted 2152.8 eV for the energy on the absorption peak and 2145.3
eV for that below the absorption peak. Panel (a) and (b) in Fig. 4 show these images
of cells. Phosphorus distribution was obtained as a ratio image shown in panel (c).
This picture clearly indicated the preferential distribution in nuclear areas in cells.
5 Conclusions
1) Operation of the system, and handling of specimens and photocathodes were
greatly improved.
2) Phosphorus mapping of cells shows the preferential location of phosphorus in a
nucleus.
3) The system needs further improvement of dynamic range, resolution and
efficiency to study detailed difference in the local distribution of elements in a
cell.
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Fig. 4. Images of HeLa cells. (a) On the absorption peak (2152.8 eV),
(b) below the peak (2152.3 eV), (c) Ratio image.
Acknowledgments
This work was performed under the approval of the Photon Factory Advisory
Committee (Proposal nos. 93G319, 95G283 and 95G284), and partly supported by a
Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science,
Sports and Culture.
References
1 K. Kinoshita, T. Matsumura, Y. Inagaki, N. Hirai, M. Sugiyama, H. Kihara,
N. Watanabe, and Y. Shimanuki, Proc. SPIE 1741, 287 (1992).
2 A. Ito, K. Shinohara, H. Nakano, T. Matsumura, and K. Kinoshita,
J. Microsc. 181, 54 (1996).
3 K. Shinohara, A. Ito, H. Nakano, I. Kodama, T. Honda, T. Matsumura,
and K. Kinoshita, J. Synchrotron Rad. 3, 35 (1996).