Study On Reconstruction Method Of Soft X-ray Imaging And Organelles Identification For Cells

Soft X-ray Nano-CT plays an important role in biology,medicine,material science,etc.Soft X-ray imaging in water window can probe cellular structures without dehydration,heavy metal staining,tissue sectioning or plastic embedment etc.By rapid cryogenic freezing,specimen cell in near native state can be preserved and observed non-intrusively.So soft X-ray Nano-CT is very suitable for cell imaging.Processing steps are required to obtain valid information from the cryogenic specimen cell in Soft X-ray Nano-CT,including pre-processing,3-D reconstruction,image processing,calculation and analysis.In the pre-processing step of projection data,problems such as noise,bad pixels,uneven brightness and deviation of rotation axis can be corrected.Generally,it’s difficult to identify subcellular structures in the 3-D reconstruction of specimen cell.If using the parallel auxiliary imaging modalities,for example fluorescence microcopy,this will affect the near native state of the specimen cell.Therefore,a method of identifying subcellular structure based on the linear absorption coefficient(LAC)of Soft X-ray was proposed,and it establishes the foundation for the post-processing and analysis of reconstruction.Moreover,spatial limitation of the specimen carrier will cause limited-angle range of projection,which will affect the 3-D reconstruction data.In this dissertation,a discrete tomography algorithm for multi-grey level material was proposed,which doesn’t need the prior knowledge of sample’s grey level and manual segmentation,and explored the possibility of high-quality reconstruction.The main researches of this dissertation are as follows:1.We summarized the characteristic of the interaction between X-ray and substance,and introduced the Soft X-ray Nano-CT imaging.A few representative examples of researches were excerpted to illustrate the application scope and achievement of Soft X-ray Nano-CT.2.According to the features of data collection in the Soft X-ray Nano-CT device,a set of data pre-processing procedures,including line integral conversion of projection,bad pixels and noise removal,high-precision alignment and equalization of brightness for the cryogenic sample were proposed.To align the rotation axis precisely,a method of high-precision alignment based on multiple fiducial markers was proposed.Based on the characteristic of the background ice layer,an equalization method of projection brightness for the cryogenic sample was proposed,which makes the brightness of the cell projection more equalized.After the pre-processing procedures on the projection data,3-D reconstruction can be obtained by the modified total variation based iterative reconstruction algorithm.3.According to the interaction between soft X-ray and substance structure,a method of identifying the organelle for Soft X-ray Nano-CT was proposed,which is based on the linear absorption coefficient(LAC)of the subcellular structures.Two cells of Candida utilis were experimented by Soft X-ray Nano-CT imaging,which verified the proposed method.Analysis on the saptial structure of the identified organelles was also carried out.4.In order to overcome the limitation of the discrete algebraic reconstruction technique for multi-grey sample,a modified discrete tomography reconstruction algorithm for restoring the ’missing wedge’ artifact was proposed,which doesn’t need the prior knowledge of the grey level of sample and the appropriate manual segmentation.By coupling with the over-segmentation,boundary extraction and mathematical morphology operation,’missing wedge’ artifact areas can be located and extracted,and high-quality reconstruction can be obtained.A series of simulation experiments were carried out,which showed better performance by comparison with other algorithms.Soft X-ray projection data were used to testify the proposed algorithm,and the result still shows the higher quality of the reconstruction.