Synchrotron Radiation-based Biological Response Studies Of Single Cell Using FTIR Microspectroscopy

This dissertation includes two parts:(1) synchrotron radiation-based Fourier transform infrared (FTIR) microspectroscopy studyding the radiation responses of single cells;(2) preliminary investigation of single celled chlamydomonas using soft x-ray transmission imaging technique.Part1:Synchrotron radiation-based FTIR microspectroscopy studying the radiation responses of single cells.This part mainly included improving synchrotron radiation soft X-rays microprobe apparatus (radiation platform) to make sure it’s suitable for the investigations of radiation responses of live animal cells. The combination of comet assay and synchrotron FTIR microspectroscopy (SFTIR MSP) technique was used to detect the radiobiological responses and to testify the feasibility of the radiation platform. Meanwhile, the SFTIR MSP was demonstrated to be advanced and favorable in detecting radiation responses. The SFTIR MSP was used to systematic study the radiation responses of colonic cancer qualitivally and quantitavely. Furthermore, the SFTIR MSP was utilized to investigate the effect of single-gene (p53) difference on the cell metabolism at single cell level.The1st chapter introduced the brief history and progress of radiation biology, the interaction of X-rays with matters and the mechanism of radiation responses. Additionally, the advantages of synchrotron radiation soft X-rays were presented, especially the’water window’wavelength of synchrotron soft X-rays was really unique and helpful for the investigations of radiation biology. Besides, the mechanism of infrared spectroscopy and the applications in bio-medical research area of FTIR spectroscopy were introduced. Also the feasibility of synchrotron-based Fourier transform IR microspectroscopy in the radiation biological researches was discussed.The2nd chapter introduced the synchrotron-based soft X-rays radiation platform for the radiation studies of animal cells, including synchrotron soft X-rays microprobe and life-support system for animal cells.In the3rd chapter, the soft X-rays radiation platform mentioned above was applied to study the radiation response of classic cancer cell line—HeLa cell. The soft X-rays with defined energy of oxygen element K-shell (Ok) absorption edge in cells were used to irradiate the target cells. The conventional single-cell gel electrophoresis was utilized to detect the radiation response, demonstrating the radiation platform that we established was suitable for radiation studies of animal cells. And the single-cell SFTIR MSP technique was tentatively used to characterize the radiation effects. The results showed this technique can perform the radiation studies of single cells by monitoring the changes of cellular multi-components simultaneously at single molecular level.In the4th chapter, the soft X-rays with defined energy at Ok absorption edge was selected to carry out the radiation study of colonic cancer HCT116cells without p53gene under different radiation doses. The SFTIR MSP was used to qualitatively and semiquantatively analyze the radiation responses. The results showed the low-dosed soft X-rays at Ok edge damage the HCT116cells by changing the contents of DNA, protein and lipids, and their structures and conformations after radiation, which were reflected by the intensity increments of typical absorption peaks, and the shifts of corresponding absorption bands. And the absorption of amine group contributed more to the multi-band of amine and hydroxyl groups after radiation. In amide I band, the content of a helix and the β turns decreased significantly, the contents of P sheet and random coil structures increased by about1.6%and3%, respectively. All of these above demonstrated the radiation inducing the disorder increase of proteins. The results of lipid absorption indicated soft X-rays inducing the change of chain length of lipids, and the content increase of cellular CH3group. In studying the dose effect, the absorption area ratio of amide Ⅰ and amide Ⅱ bands, and the relative content of DNA to protein decreased with radiation dose increasing.In the5th chapter, the SFTIR MSP was used to acquire the absorption spectra of single cells with and without p53gene. The analytical results showed the relative content of DNA to proteins and the content of secondary structures of protein change in the spectrum of p53gene-absence cell, compared to the spectrum of cell with p53gene. Also the structure and cotent of lipids changed, indicating the p53gene-absence evidently changed the metabolism of cells. Our research work demonstrated the SFTIR MSP is potential in single-gene diagnose, providing a supplementary method for medical diagnosing and single-gene detection.Part II:the preliminary investigation of chlamydomonas reinhardtii using soft X-rays transmission imaging technique.The full-field transmission X-ray microscopy at Soft X-rays microscopic endstation in National Synchrotron Radiation Laboratory was used to perform the preliminary imaging study of chlamydomonas reinhardtii cells to explore the experimental methods. The transmission imaging of entire chlamydomonas reinhardtii cell was carried out with soft X-rays with energy near K-shell absorption edge of oxygen element. Also the different methods to fix cells and to dehydrate were employed to optimize the microscopic imaging method, by comparing the imaging results.