In life science research fields, it is important to obtain the information on the structure of biological cells and protein distribution in cells for elucidation of cell function and pathogenic mechanism and for evaluation of drug in the drug discovery. In material science research fields, light element materials such as carbon composites and functional polymer materials attract attention from both academia and industry as next generation materials. Their structural analysis is necessary to understand the structure-function relationship.
Soft x-rays with energies below several keV are useful for observing biological specimens and light element materials since they are well absorbed by materials composed of light elements with low atomic number. A soft x-ray 3D microscope is an apparatus to visualize three-dimensional structure of samples.
We have been developing a compact soft x-ray microscope which enables high-resolution observation of 3D fine structures in a regular laboratory room combining an electron-impact x-ray source and grazing incidence mirror optics. The x-ray mirrors which we fabricate have high utilization efficiency of x-rays in comparison with other x-ray optics and can provide imaging in different x-ray energies.
X-ray photons emitted from the x-ray source are collected and focused on the sample by the condenser mirror, and the transmitted x-ray image is magnified by the objective mirror. The magnified image is detected by a CCD camera with sensitivity to x-rays. The sample is rotated with rotating stage and the 3D image of the sample is obtained by tomographic reconstruction.
Fig.1 Schematic drawing of 3D x-ray microscope
Fig.2 X-ray mirrors Fig.3 Photograph of 3D x-ray microscope
■Observation of biological specimen in near native state and light element materials is possible
We constructed two x-ray microscopes operating at different x-ray energy regions. One is the microscope using so-called “water-window” energy range from 284 eV to 543 ev. In this range, the different absorption coefficient between water and protein gives good contrast in the image. The water window energy range is suitable for imaging of biological specimen in near-native state. The other is the microscope using 1.8 keV x-rays which can provide large absorption contrasts for light material elements.
■High resolution at the cellular level (several hundred nanometers or less) is achieved.
Our x-ray optics is wolter type I mirror composed of hyperboloidal and ellipsoidal inner surfaces. The image qualities of the microscope rely on accuracy of fabricating the mirror. Currently, the surface roughness and figure error are around 2 nm and less than 0.1 μm, respectively. In evaluation of the resolution limit using an x-ray resolution chart, we confirmed the 200 nm-sacle structures are resolved as shown in Fig.5.
■Multi-energy observation is possible
Since the x-ray mirrors has no chromatic aberration, they can provide imaging in the same way with x-rays of different x-ray energies. We can obtain different contrast images for same amples with various x-ray energies.
■Small enough to fit in a laboratory
Most of x-ray microscopes has developed at synchrotron-based facilities. The performance of the x-ray microscope is excellent, but they are large (about 100 m × 100 m) and their accessibility is limited. In our x-ray microscope, the distance from the x-ray source to the CCD is 3.4 m, so that our x-ray microscope can be placed in a regular laboratory room.
Fig.4 X-ray absorption spectra of water, protein and polymer materials
Fig.5 Evaluation of resolution limit
1. 3D inner structure ・・・dehydrated mouse kidney slice (provided by the Research Center for Advanced Science and Technology of the University of Tokyo)
We observed glomerulus in a 5 μm thickness dehydrated mouse kidney slice. The reconstructed slice image with 1-μm spacing are shown in Fig.7. The different structure between the reconstructed slice images can be seen.
Fig.6 Optical microscope image（left）Transmission x-ray image（right)
Fig.7 Images of dehydrated mouse kidney slice
2. Observation of biological samples in a frozen hydrated state・・・yeast cells
We tried imaging of biological samples embedded in ice. A water droplet containing yeast cells is confined between two silicon substrates with silicon nitride windows (Fig.8) and cooled on a cryogenic stage. Fig.9 shows an image of yeast cells in ice with thickness of several tens of micrometer. High contrast image of samples is obtained by using water-window x-ray. This result demonstrates applicability to imaging of frozen hydrated sample.
Fig.8 Sample holder
Fig.9 X-ray transmission image of yeast cells in ice
Polystyrene beads were observed with oxygen Kα (525 eV) x-rays and with carbon Kα (277 eV) x-rays. We can obtain different contrast images because the x-ray absorption of carbon at oxygen Kα is higher than that at carbon Kα.
Fig.10 With oxygen Kα (525 eV) x-rays (left),
With carbon Kα (277 eV) x-rays (right)
We observed a morning glory pollen, whose contents were removed by pretreatment process. The hollow structure inside the pollen and the shape of the inner wall can be seen.
Fig.11 X-ray transmission image
Fig.12 Reconstructed slice image
In order to demonstrate the capability of our x-ray microscope as useful tool in the life science fields and materials science & engineering fields, we will improve the performance of our 3D x-ray microscope and observe various samples in the fields.
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