| Three-dimensional(3D)imaging gives an insight to understand the relationship between the structure and the function of biological cells and materials.An achieving dynamical 3D imaging deepens our understanding of the cell function and process.The current 3D imaging method involves different process suitable for various applications.The details are presented as follows:1.Multiple equal angle projections of the sample are measured by rotating the sample followed by application of interpolation.Or multiple equal slope projections of the sample are measured by rotating the sample followed by application of real space and Fourier space iterative algorithm.The above method has a wide range of applications in electron microscopy imaging and x-ray tomography.2.Measuring the sample at different depths by focusing the laser beam can produce 3D imaging and it is widely used in confocal microscopy.3.Recently,multiple scattering methods have been developed in scanning coherent diffractive imaging.4.A series of projections obtained by using a large amount of the same samples at different angle are combined into a 3D image.This method is successfully applied to obtain the 3D imaging using free electron laser and cryo-elecron microscopy.These different 3D imaging methods have its limitations and advantages.For example,3D imaging using equal angle projections relies on rotating samples.It usually needs dozens or hundreds of projections.On the other hand,this method can acquire a complete structure of the sample.In 3D imaging with equal slope,the angle of projection is reduced largely,which helps to reduce the radiation damage due to x-ray or electron beam.In confocal 3D imaging the laser beam can be focused at different depths of the sample by adjusting the optical path,forming a series of images along the optical path.Simply,3D image can be obtained without any changes in the position of the sample i.e.,true nature of the sample.In the process of scanning coherent diffraction imaging,a large amount of redundant data is obtained.Under the condition of multiple scattering,the 3D images can be acquired by iterative algorithm.The limitation of these approaches is it consumes a lot of time to collect the large amounts of data because of multiple angles and,multiple depths,or multiple overlapping positions,which gradually tails off suitability for dynamic 3D imaging.Recently,single orientation 3D coherent diffraction imaging has been developed to overcome the limitation in sample damging and biological cell imaging.It provides new possibilities to achieve dynamic 3D imaging of cell and other materials.This is a 3D imaging process based on CDI.The coherent x-ray diffraction imaging is a lensless imaging technique that uses a phase recovery algorithm to obtain the image of sample structure by measuring the coherent diffraction signal.Since the consistant growth and the development in the field of x-ray crystallography,the application of CDI is progressively increased and can be applied to acquire high-resolution imaging of not only crystalline materials but also non-crystalline materials which is difficult to grow as crystals.In recent years,two-dimensional or three-dimensional high resolutions,high contrast imaging of biological samples such as viruses,bacteria,cells and bone tissue has been obtained by CDI.Further,since the CDI can obtain the density information,a quantitative analysis of the sample structure can be realized.Moreover,in coherent diffraction imaging,when the distance between the sample and the CCD detector is very close,the diffraction pattern can be projected onto the Ewald sphere.If the diffraction information is enough,it is possible to obtain the 3D image of the sample by iterative algorithm in the 3D real space and the 3D Fourier space.Single orientation 3D coherent diffraction imaging is first demonstrated using a soft x-ray source,and then demonstrated using a visible laser source in acquiring the image of phase objects.In both experiments,voids were mistakenly considered as samples,later,a thin and weak-phase object were used.In addition,because of reduced data volume compared with of other 3D imaging methods,it is difficult to reconstruct the 3D-coherent diffraction images in one direction.The single orientation 3D-coherent diffraction imaging is still in its infant stages and debatable.The use of this method to achieve the 3D imaging of thick samples has not yet been verified.This thesis focuses the problems of the single orientation 3D-coherent diffraction imaging.In order to verify the method of single orientation 3D-coherent diffraction imaging,we set up a variety of laser coherent diffraction imaging systems in the laboratory.Using this imaging system,we propose and implement a single orientation 3D coherent diffraction imaging of phase objects and thick samples,as well as a single orientation 3D imaging of polychromatic light.The experimental and simulation details and the results are briefed as follows:Experimental Details:1.The optical path of the sample was optimized based on the reference sample.The ratio of the focal length of the lens was adjusted to capture the micron sized sample with a size of 100 microns.The focal length covers most of the mammalian cells.2.The light path was detected and optimized with small crystal particles.3.The microscopic imaging of the coherent diffraction was performed by placing silica spheres on one side of the slide.The single direction 3D coherent diffraction imaging with a wavelength of 543 nm was performed.The sample was prepared by placing 20?m silica spheres on both sides of a 100 nm silicon nitride film.4.The single direction 3D coherent diffraction imaging with two wavelengths of 543 nm and 432nm was investigated.For this measurement,the sample was prepared by placing 10?m silica spheres on both sides of a 30 nm silicon nitride film.Simulation Details:1.The difference map program and simulation for planar reconstruction of phase object was performed.The plane image was projected on Ewald sphere.2.The simulation program of multi-wavelength single orientation 3D imaging of cells was completed,including for different diffraction angles.The data loss of the center spot of the diffraction image reinforces the noises.The reconstruction work is:1.The reliability of single orientation 3D imaging reconstruction:Since the silicon spheres used in the experiment were thicker than the laser wavelength and not the weak phase object,the diffraction image was assymmetric.The reconstruction work was more difficult with limited data acquird from large amount of the samples.We added the curvature corrected high numerical aperture diffraction image in the Fourier space to improve the convergence speed and reliability of the reconstruction.2.Multi wavelength single orientation 3D-coherent diffraction imaging:To achieve a dual wavelength reconstruction of the single orientation 3D-coherent diffraction image,than the one wavelength single orientation coherent diffraction three dimensional imaging can get more longitudinal three dimensional information,The Ewald sphere was used in half of the Fourier space during all the 3D reconstruction..The innovation of this paper is as follows:1.The single orientation 3D coherent diffraction imaging of thick samples was successfully verified for the first time.The sample consists of several silica spheres placed on both sides of the silicon nitride membrane.2.The Fourier projection of beam direction was used to enhance the speed and stability of image convergence process.The high numerical aperture diffraction pattern was corrected as the middle layer of Fourier space,which provides lateral constraints,in order to improve the quality of 3D image reconstructions.3.The single orientation 3D coherent diffraction imaging for two wavelengths,such as 543nm,green light and 432nm,blue light was realized first.The single orientation 3D coherent diffraction imaging for red,green and blue wavelengths of cells was designed and simulated,which provides a reference for 3D dynamic imaging of biological cells and materials.In conclusion,the aim of this thesis is to explore the experimental conditions of single orientation 3D coherent diffraction imaging method and stable reconstruction algorithms for the investigation of biological samples.The 3D imaging of thick samples at the single orientation was realized by this method.This 3D imaging method can have more possibility to further apply for dynamic imaging of biological cells and materials.However,due to the limitation of current experimental conditions such as long measurement time and low resolution achieved by CCD,multiple wavelength dynamic 3D imaging can not be achieved.Simulation results of single orientation 3D imaging of biological cells by red,green and blue lasers provide a reference for the scanning of Fourier space.The suitability of this method with x-ray and free electron laser will be investigated in future.The main imaging indicators are to improve the spatial resolution and temporal resolution. |
PDF Full Text Request