Study On The Applications Of Hard X-ray Microscopy And Nano-CT In Cellular Imaging

X-ray microscopy and nano-CT, which can examine internal structures of whole cells in three-dimensional (3D), have many advantages compared with conventional cellular imaging techniques. Because of its larger depth of focus and higher penetration power, hard X-ray microscopy can overcome the limitation of soft x-ray microscopy which has difficulty in imaging thick cells. Thus, this technique has the potential to become a useful tool for cellular imaging. The major difficulty of hard X-ray microscopy in cellular imaging is the low imaging contrast. We developed a novel sample preparation method for imaging cells under hard x-ray energy range. Through using this sample preparation method and the Zernike phase contrast technique, we successfully imaged the fission yeast, Schizosaccharomyces pombe in 2D and 3D at 8keV. The images taken at this energy displayed sufficient contrast for observing cellular ultra-structures. Plenty of useful information in biology can be obtained from the imaging results. This study demonstrated the feasibility of cellular imaging using hard X-ray microscopy.The main works in the thesis are described as the following:1. Summarized cellular imaging and X-ray microscopy. The new techniques in X-ray microscopy such as Zernike phase contrast and nano-CT were introduced. The advantages, current situation and potential of X-ray microscopy in cellular imaging were analyzed. In particular, the unique advantages of hard X-ray microscopy were analyzed, demonstrating the significance of developing ability and methodology of cellular imaging using hard X-ray microscopy and nano-CT.2. Study of methodology of cellular imaging using hard X-ray microscopy. A series of sample preparation processes including fixation, staining, dehydration and drying were developed. Taken at 8 keV phase contrast mode, the fission yeast cells prepared by these procedures displayed sufficient contrast for examining celluar ultra-structures. By using nano-CT technique, subcellular structural organizations of whole cells were visualized in 3D at the resolution better than 100 nm. Comparison between phase contrast and absorption contrast was made, indicating the obvious advantage of phase contrast for cellular imaging at hard X-ray energy range. In addition, various factors affecting the imaging results were also analyzed and discussed.3. Study of the biology of cellular imaging using hard X-ray microscopy. The imaging results of fission yeast cells were analyzed and discussed with respect to biology. The layered structures of cell walls were analyzed, demonstrating the origin and composition of the three layers. Intracellular organelles were segmented in 3D and they were further assigned. Two kinds of cells, which have obvious different structural organizations, were analyzed, demonstrating that they were in different cellular phases, that is, the logarithmic growth phase and the stationary phase. And the differences in their subcellular structures were quantitatively measured.