Soft X-Ray Resistance of D. radiodurans
H. Fujisaki1, S. Takahashi1, H. Kondo1, Y. Kobayashi2, H. Watanabe2
1
Nikon Corporation, Tsukuba Research Laboratory,
5-9-1 Tokodai, Tsukuba, Ibaraki 300-26, Japan
E-mail: fujisaki@tsukubagw.nikon.co.jp
2
Japan Atomic Energy Research Institute,
Department of Radiation Research for Environment and Resources,
1233 Watanukicho, Takasaki, Gunma 370-12, Japan
Abstract.Biological samples are thought to be damaged by exposure to soft-Xrays during X-ray microscope observation. All available reports, although few,
concerning experimental examination of biological samples in water have
concluded that it is difficult to continuously observe such samples while
preserving their functions [1-3].
The soft-X-ray resistance of Deinococcus radiodurans, a type of hard-radiationresistant bacteria, was investigated in the "water window" wavelength region
usually used in X-ray microscopy. The sample was exposed to soft-X-rays using
an apparatus which included a laser-plasma X-ray source with an yttrium target
and a titanium membrane filter, a Wolter-type X-ray focusing mirror, and a
vacuum-tight sample cell with a silicon-nitride membrane as an X-ray
transmitting window. D. radiodurans cells kept their dye exclusion ability even
when exposed to soft-X-rays up to 4 ∴108 photons/µm2, whereas our previouslyreported myofibril and yeast cell samples, were damaged when exposed to soft-Xrays higher than ~106 photons/µm2 [3].
According to our previous calculations [3], a soft-X-ray flux of 6 ∴107
photons/µm2 is required in order to obtain a resolution of 50 nm when imaging a
biological sample in water. Therefore, based on the results of our present
experiment, when D. radiodurans is used as a sample for soft-x-ray biological
study, seven such high resolution X-ray images of a single sample may be
obtained without damaging biological function.
1 Methods
The same apparatus as employed in Ref. 3 was used to expose D. radiodurans cells to
soft-X-rays. Nd:YAG laser light, with output kept at 1 J throughout the experiment
(although it could be increased up to 2 J), was focused on the target plate to generate a
plasma. In this experiment, an yttrium plate was used as a target instead of the
previously used tungsten plate to achieve a stronger soft-X-ray source according to
Kondo and Tomie [4]. Though this was an expensive replacement, the present
apparatus was able to expose the sample to soft X-rays of a total flux of 2 ∞ 107
photons /µm2 per each 8 ns exposure at up to 10 Hz, greatly improved from 3 ∞ 105
photons /µm2, when the filter consisted of two pieces of 0.5 µm-thick titanium
membrane. The X-ray spectrum was similar to the previous one. X-rays emitted by
the plasma were focused on the sample in the sample cell by a Wolter mirror
condenser (∞ 1/4). A Wolter mirror objective (∞ 32) and a CCD image detector were
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used with a He-Ne laser light aligned coaxially with the Nd:YAG laser light to
determine the position of the focused area of the X-ray on the sample prior to
exposure. Inside of the vacuum-tight sample cell, the sample solution was held
between a 0.5 mm-thick glass plate and a 0.4 mm-thick silicon plate, each 10 mm
square. The silicon plate had a 0.2 mm square X-ray-transparent window made of 0.1
µm-thick silicon nitride. 10 µm-thick polymethylmethacrylate spacers on the silicon
plate determined the thickness of the sample solution.
After exposure to soft-X-rays, the D. radiodurans cells were observed under a
Nikon fluorescence microscope. Since only the dead cells stained with propidium
iodine were visible under the fluorescence microscope, a transmitting light was
introduced so that living, unstained cells could also be made visible. A video strainer
(Hamamatsu Photonics) was used to increase contrast and improve image quality.
Usually, 60 frames of CCD (Hitachi) output were processed by the video strainer in
order to generate a background-subtracted, averaged image, which was input into a
personal computer.
2 Results and Discussion
D. radiodurans cells were exposed to soft-X-rays in 3 µM propidium iodide and 10
mM phosphate buffer at pH 6.8, and observed under a fluorescence microscope with
transmitting light in addition to illumination for fluorescence. An example of this is
shown in Fig. 1. Images (a) and (b) were obtained, respectively, before and 2 hrs after
a total X-ray exposure of 5.8 ∞ 108 photons/µm2 at the center of the silicon nitride
window, i.e., the center of each image. Image (c) was acquired with the same
microscope without illumination for fluorescence, to confirm that the sample was not
contaminated by materials visible by fluorescence but not visible by transmitted light.
The soft-X-ray exposure was completed with 29 shots of 1-J-Nd:YAG laser light at 1
Hz, using a filter consisting of two pieces of 0.5 µm-thick titanium membrane and an
yttrium target. The 2 hrs taken for the complete staining of the D. radiodurans cells
after their exposure to X-rays is thought not to be the time the cells survived, but
rather the time required for the absorption and accumulation of the dye molecules
from the surrounding solution into the cells (refer to the discussion in Ref. 3 for
staining yeast cells with trypan blue).
Figure 2 shows the survival curve of D. radiodurans cells exposed to various
photon flux of X-rays. Our previously reported results for yeast cells [3] are also
shown for comparison. In our experiments exposing myofibrils to various photon flux
of X-rays [3], a survival curve similar to that of yeast cells was obtained, showing a
shoulder at the exposure of 1 ∞ 105 photons/µm2. This was consistent with results
reported in Refs. 1 and 2. The survival curve of D. radiodurans cells shows a shoulder
at the exposure of 4 ∞ 108 photons/µm2, which is 4000-fold higher than in the case of
yeast cells and myofibrils and seven-fold higher than the exposure required for
imaging biological samples in water at a resolution of 0.05 µm [3]. This implies that
we can obtain seven high resolution images of D. radiodurans cells while maintaining
their living state and, consequently, their biological functions.
Soft X-Ray Resistance of D. radiodurans
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Although the reason why D. radiodurans is so highly resistant to soft-X-ray
irradiation remains to be further investigated, this may be good news for X-ray
microscopists.
Fig. 1. Images of D. radiodurans cells obtained with both fluorescence and transmitted light (a)
before and (b) 2 hrs after exposure at the center of the silicon nitride window to soft X-rays of
5.8 ∞ 108 photons/µm2 in 3 µM propidium iodide. (c) The same sample observed without
illumination for fluorescence. Each image shows the entire silicon nitride window of 0.2 mm
square.
Fig. 2. Survival curves of yeast cells (solid circles) and D. radiodurans cells (open circles)
exposed to various photon flux of X-rays.
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