Internal Structures of Small Animals and Tissues
Studied by Projection X-Ray Microscopy
S. Kumagai, T. Horikoshi, H. Chiba, K. Takahashi,
D. Shoutsu, H. Yoshimura, T. Mitsui
Department of Physics, School of Science and Technology, Meiji University,
Higashimita, Tama-ku, Kawasaki 214-71, Japan
E-mail: hyoshi@isc.meiji.ac.jp
Abstract. The internal structure of optically opaque biological samples were
studied with a projection X-ray microscope. Critical-point drying and Golgistaining were applied to enhance contrast. The morphology of insect muscle
fibers and stellate cells in the liver tissue were clearly observed. We also observed the metamorphosis of Drosophila melanogaster, from pupa to adult, in
the living state.
1 Introduction
Our projection X-ray micro-scope utilizes a small X-ray light source which is produced by a focused electron beam at a thin metal target. This microscope features
very simple operation [1, 2, 3]. Since there are no optical elements (e.g. zone plate)
focusing depth of this micro-scope is theoretically infinite. The sample room of the
microscope is separated by a metal target film against a vacuum area of electron
optics (Fig. 1), and therefore samples can be studied in a air atmosphere. Considering
these advantages, we con-structed a projection X-ray microscope to prove its
application for studying entomology and histology.
Fig. 1. Schematic of projection X-ray microscope.
The electron beam was focused by optics of a SEM on the metal target. The distance ratio between target-object (D0) and target-film (D1) determines the magnification (D1/D0). If D0=100 µm and D1=30 mm, the magnification is 300.
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2 Materials and Methods
2.1 X-Ray Microscope
The projection X-ray microscope was constructed by modifying a scanning electron
microscope (SEM), HITACHI S-2500CX, and equipping it with an LaB6 cathode for
a more intense and fine electron beam than that with a conventional tungsten filament.
The X-ray wavelength can be selected by choosing the appropriate acceleration
voltage and metal target material [3]. We generally used a gold (Au) target with a
thickness between 0.2 µm and 1 µm at an acceleration voltage of 7 kV to 20 kV.
Under these conditions, the X-rays components were the characteristic X-rays of gold,
La (0.13 nm, 9.1 keV) and Ma (0.58 nm, 2.1 keV), with continuous X-ray. The
configuration around the sample room of this microscope is shown in Figure 1. The
magnified projection images were recorded on electron microscope negative film
(Kodak 4489). The resolution of this microscope is limited by the size of the electron
beam, which diffuses into a metal target, and the Fresnel diffraction. For our
experiments, the resolution limit was close to 0.1 µm, examined by the structure of a
diatom.
Fig. 2. X-ray micrograph of an ant treated with critical-point drying.
The focused electron beam with an acceleration voltage of 10 kV was irradiated
on a 0.2 µm Au target. The sample was put in a helium gas atmosphere and exposed
to X-ray for 10 minutes. Inset is the magnified image of the leg.
2.2 Sample Preparation
Critical-point drying. This method is widely used in scanning electron microscopy.
To remove water in the organ, water was substituted at first by an organic solvent
(e.g. ethanol) and then the solvent was substituted by liquid carbon dioxide. Carbon
dioxide was removed without crossing phases between liquid and gas, and thus
avoiding the effects of surface tension. In this way, the structure of the sample was
preserved better than with natural drying.
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Golgi-staining. This staining method was developed for transmission electron
microscopy and optical microscopy [4]. The tissue blocks were fixed in 4%
paraformaldehyde and were immersed in a mixture of 2% osminium tetroxide and
2.5% potassium bichromate for 2 or 3 days at room temperature. After a rinse in
0.75% silver nitrate solution, the tissues blocks were incubated in the same solution
for 2 or 3 days. The blocks were rapidly dehydrated, embedded in paraffin, and cut
into 50 µm sections. The some special cells in the tissue were selectively stained with
silver.
3 Results
3.1 X-Ray Micrographs of Small Animals Treated with Critical-Point Drying
An X-ray micrograph of an ant treated with the critical-point drying method is
shown in Fig. 2. The muscle fiber construction in legs were clearly observed. Figure
3 is an X-ray micrograph of an Echiniscus japonicus (water bear) which lives in moss
and moves very slowly. After contraction, its primitive muscle system extends the
muscle fiber with body liquid internal pressure without antagonist. The arrow in
Fig. 3 points to a muscle fiber.
The sample was treated with critical-point drying. The arrow shows the single
muscle bundle (0.3 µm Au film target, 10 kV acceleration voltage, 7 min exposure
time in a helium gas).
Fig. 3. X-ray micrograph of Echiniscus japonicus (water bear).
3.2 Stellate Cells of Porcine Liver by Golgi-Staining
Using the projection microscope, it is easy to take stereo-pair micrographs up to 100
µm thick sections. The stereo-pair of a stained stellate cell is shown in Figure 4. The
three dimensional network of bile canaliculi and a stellate cell surrounding the blood
vessel (it was not stained) were clearly observed.
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Fig. 4. Stereo-pair of Golgi-stained liver tissue section.
The upper networks are bile canaliculi. The stellate cell which coil around a capillary
vessel (not stained) can be seen in the lower part (0.2 µm Au film target, 15 kV
acceleration voltage, 20 min exposure time in an air atmosphere). The growth of the
pupa using the same living sample after (A) 2 hours and (B) 39 hours of pupa
formation. The adult Drosophila (C) treated with critical-point drying (1 µm Au film
target, 10 kV acceleration voltage, 10 min exposure time in an air atmosphere).
Fig. 5. Metamorphosis of Drosophila melanogaster.
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3.3 Metamorphosis of Drosophila melanogaster
The differentiation process from pupa to the adult state was inspected using an X-ray
microscope on the same living sample. The organs in the pupa started to melt and
formed a bubble in the body 2 hours after formation of the pupa (Fig. 5A). The
differentiation completed after about 37 hours (Fig. 5B). We examined 15 pupas and
successfully observed the growth to adulthood for 3 of them after several ten- minute
X-ray exposures. Figure 5C is an adult Drosophila treated by critical-point drying.
Thick flight muscle fibers and thin muscle fibers in the legs can be clearly seen.
4 Summary
A projection microscope is a useful tool to investigate the inner structure of small
animals or tissue sections. Although the resolution of the projection microscope is
slightly lower than a microscope that uses zone plate optics, our microscope has
several advantages such as a large focusing depth, wide viewing angle, and simple
operation. To further the advantages of projection microscope, we are planning to
image three dimensional internal structure of thick samples by using a CCD camera.
Low contrast is a common problem for biological samples, but can be improved with
Golgi-staining and critical-point drying. We also intend to examine milder contrast
enhancement techniques, such as iodine or gold decoration, for studying living
biological samples.
Acknowledgements
The authors thank Prof. K. Yada, Aomori Public College, for advise during construction of our X-ray microscope. Echiniscus japonicus were supplied by Prof. K. Utsugi, Tokyo Woman’s Medical College, porcine liver tissue sections by Prof. K. Wake,
Tokyo Medical and Dental University, and Drosophila melanogaster by Prof. Y. Hotta,
University of Tokyo. The authors appreciate both the supplied samples and helpful
discussions.
References
1
W. C. Nixon, Proc. Roy. Soc. A232, 475 (1955).
2
S. P. Newberry and S. E. Summer, Proc. Int. Conf. Electron Microscopy, London
305 (Royal microscopical Soc. 1954).
3
K. Yada and S. Takahashi, J. Electron Microscopy 38, 321 (1989).
4
K. Wake and T. Sato, Cell Tissue Res. 273, 227 (1993).