Atsushi Ito’s research while affiliated with Tokai University and other places

Background: The soft X-ray projection microscope has been developed for high resolution imaging of hydrated bio-specimens. Image blurring due to X-ray diffraction can be corrected by an iteration procedure. The correction is not efficient enough for all images, especially for low contrast chromosome images. Objective: The purpose of this study is to improve X-ray imaging techniques using a finer pinhole and reducing capture time, as well as to improve image correction methods. A method of specimen staining prior to the imaging was tested in order to capture images with high contrasts. The efficiency of the iteration procedure and its combined version with an image enhancement method was also assessed. Methods: In image correction, we used the iteration procedure and its combined version with an image enhancement technique. To capture higher contrast images, we stained chromosome specimens with the Platinum blue (Pt-blue) prior to the imaging. Results: The iteration procedure combined with image enhancement corrected the chromosome images with 329 or lower magnification effectively. Using the Pt-blue staining for the chromosome, images with high contrast have been captured and successfully corrected. Conclusions: The image enhancement technique combining contrast enhancement and noise removal together was effective to obtain higher contrast images. As a result, the chromosome images with 329 or lower times magnification were corrected effectively. With Pt-blue staining, chromosome images with contrasts of 2.5 times higher than unstained case could be captured and corrected by the iteration procedure.

We examined lethal damages of X rays induced by direct and indirect actions, in terms of double-strand break (DSB) repair susceptibility using two kinds of repair-deficient Chinese hamster ovary (CHO) cell lines. These CHO mutants (51D1 and xrs6) are genetically deficient in one of the two important DNA repair pathways after genotoxic injury [homologous recombination (HR) and non-homologous end binding (NHEJ) pathways, respectively]. The contribution of indirect action on cell killing can be estimated by applying the maximum level of dimethylsulfoxide (DMSO) to get rid of OH radicals. To control the proportion of direct and indirect actions in lethal damage, we irradiated CHO mutant cells under aerobic and anoxic conditions. The contributions of indirect action on HR-defective 51D1 cells were 76% and 57% under aerobic and anoxic conditions, respectively. Interestingly, these percentages were similar to those of the wild-type cells even if the radiosensitivity was different. However, the contributions of indirect action to cell killing on NHEJ-defective xrs6 cells were 52% and 33% under aerobic and anoxic conditions, respectively. Cell killing by indirect action was significantly affected by the oxygen concentration and the DSB repair pathways but was not correlated with radiosensitivity. These results suggest that the lethal damage induced by direct action is mostly repaired by NHEJ repair pathway since killing of NHEJ-defective cells has significantly higher contribution by the direct action. In other words, the HR repair pathway may not effectively repair the DSB by direct action in place of the NHEJ repair pathway. We conclude that the type of DSB produced by direct action is different from that of DSB induced by indirect action.

High LET radiation is highly expected to control hypoxic fraction in tumor tissue that shows radiation resistance to low LET radiation, because oxygen effect decreases with increasing LET. Due to the availability of various ions and LETs, HIMAC has produced many outcomes from basic to clinical studies. In the basic studies, the mechanism of decreasing oxygen effect with increasing LET was experimentally examined based on the previously proposed “oxygen in the track” model. In addition, in vitro cellular and in vivo transplanted tumor studies demonstrated the LET dependent feature of DNA damage produced under hypoxic condition. For the effort to connect basic studies to clinical trials, measurement of LET dependence of OER (Oxygen Enhancement Ratio) was extended to various oxygen concentrations to mimic oxygen environment in tumors. Finally clinical research using carbon ions for cervical cancer patients demonstrated the effective control of hypoxic fraction.

It is well known that neutrons have more damaging effects, with high relative biological effectiveness or a radiation weighting factor depending on neutron energy, compared to low-LET ionizing radiations. In the current work, we evaluated the contribution of the indirect effect induced by radicals for the purpose of studying the mechanisms of fast neutron effects or mechanisms of boron neutron capture therapy (BNCT) using relatively lower energy neutrons. Plasmid pBR322 DNA with a supercoiled structure was irradiated with fast neutrons (1-10 MeV) in the reactor core of the YAYOI research reactor at the University of Tokyo, and with thermalized neutrons passing through a phantom made of acrylic resin to simulate a human body. The single- and double-strand breaks (SSBs and DSBs) of the plasmid were evaluated from the numbers of its open circular and linear forms, respectively, detected using agarose gel electrophoresis. The number of supercoiled forms decreased exponentially with the absorbed dose of fast neutrons. The decrease was inhibited by dimethyl sulfoxide (DMSO) in a concentration-dependent manner. The contributions of the indirect effect to DNA strand breaks by fast neutrons and thermalized neutrons were calculated to be 50-65% which, while lower than the value for X rays, still accounts for the majority of the DNA strand breaks. In the case of thermalized neutrons, SSB and DSB yields were increased by the addition of boric acid. Moreover, an increased 10B isotope concentration was found to increase the DSB/SSB ratio.

Soft X-ray microscopy was applied to study the quantitative distribution of DNA, RNA, histone, and proteins other than histone (represented by BSA) in mammalian cells, apoptotic nuclei, and a chromosome at spatial resolutions of 100 to 400 nm. The relative distribution of closely related molecules, such as DNA and RNA, was discriminated by the singular value decomposition (SVD) method using aXis2000 software. Quantities of nucleic acids and proteins were evaluated using characteristic absorption properties due to the 1s–π * transition of N=C in nucleic acids and amide in proteins, respectively, in the absorption spectra at the nitrogen K absorption edge. The results showed that DNA and histone were located in the nucleus. By contrast, RNA was clearly discriminated and found mainly in the cytoplasm. Interestingly, in a chromosome image, DNA and histone were found in the center, surrounded by RNA and proteins other than histone. The amount of DNA in the chromosome was estimated to be 0.73 pg, and the content of RNA, histone, and proteins other than histone, relative to DNA, was 0.48, 0.28, and 4.04, respectively. The method we present in this study could be a powerful approach for the quantitative molecular mapping of biological samples at high resolution.

In this study, soft X-ray spectromicroscopy was applied to investigate and analyze the distribution of DNA and RNA in a mammalian cell at the spatial resolution of 400 nm. The relative distribution of DNA and RNA was examined by the SVD (singular value decomposition) method in aXis2000 program using combined full spectra of DNA and RNA at the absorption edge regions of carbon, nitrogen and oxygen. The absorption of nucleic acid was evaluated using 1s-π* transitions in the NEXAFS spectra at the nitrogen K absorption edge and distributed to DNA and RNA according to the relative level obtained above. The study results revealed the usefulness of the SVD method to discriminate closely related molecules such as DNA and RNA.

3-Dimensional Chemical Structures of an Isolated Cell Nucleus by a Scanning Transmission X-ray Microscope - Volume 24 Supplement - Takuji Ohigashi, Atsushi Ito, Kunio Shinohara, Shigenobu Tone, Yuichi Inagaki, Hayato Yuzawa, Nobuhiro Kosugi

Soft X-ray microscopy has been developed for high resolution imaging of hydrated biological specimens due to the availability of water window region. In particular, a projection type microscope has advantages in wide viewing area, easy zooming function and easy extensibility to CT. The blur of projection image due to the diffraction of X-rays, which eventually reduces spatial resolution, could be corrected by an iteration procedure, i.e., repetition of Fresnel and inverse Fresnel transformations. However, it was found that the correction is not sufficiently effective for all images, especially for images with low contrast. In this study, first, grayscale distribution of the projection images was analyzed to reveal the reason for the insufficient correction. The result showed that the insufficient correction is due to high background noises. Therefore, a noise removal method prior to the iteration procedure was applied and found to be effective. Thus, some chromosome images with magnification of 329 times or lower which had not been correctable solely by the iteration procedure became successfully correctable after noise removal in combination with our contrast enhancement method whose effect was evaluated as effective in the previous study.

Soft X-ray microscopy has been developed for high resolution imaging of hydrated biological specimens due to the availability of water window region. In particular, a projection type microscopy has advantages in wide viewing area, easy zooming function and easy extensibility to computed tomography (CT). The blur of projection image due to the Fresnel diffraction of X-rays, which eventually reduces spatial resolution, could be corrected by an iteration procedure, i.e., repetition of Fresnel and inverse Fresnel transformations. However, it was found that the correction is not enough to be effective for all images, especially for images with low contrast. In order to improve the effectiveness of image correction by computer processing, we in this study evaluated the influence of background noise in the iteration procedure through a simulation study. In the study, images of model specimen with known morphology were used as a substitute for the chromosome images, one of the targets of our microscope. Under the condition that artificial noise was distributed on the images randomly, we introduced two different parameters to evaluate noise effects according to each situation where the iteration procedure was not successful, and proposed an upper limit of the noise within which the effective iteration procedure for the chromosome images was possible. The study indicated that applying the new simulation and noise evaluation method was useful for image processing where background noises cannot be ignored compared with specimen images.