Research On Digital Holographic Microtomography For Tiny Organisms

In the field of biomedical research, the imaging and analysis measurement equipment is an important tool for observing morphology and life status of cells and biological tissues. The microscopy apparatus is the most familiar imaging instrument in the life science research.However, the conventional optical microscope can not meet the development trend of the scientific research, because it can only get a 2-D image of the object wave intensity distribution.Optical microscopy tomography is a kind of technique to achieve 3-D imaging of the internal structure of the sample, but typical technology such as confocal laser scanning microscopy and optical coherence tomography is based on measuring the intensity of light, so the imaging contrast is low for phase type biological samples and low scattering samples, and they also have the disadvantage of slow speed in imaging due to the point by point scanning confocal technology. Therefore, for the urgent demand of biomedical research, it is necessary to grope for a novel optical microscopic imaging technique that can achieve non-interference, high resolution and quantitative analysis.Digital holographic microtomography is a new type of 3-D refractive index imaging technology which is put forward and developed in recent years. It is the outcome of the combination of digital holographic microscopy and CT technology. Based on the projection reconstruction principle of CT technology, the 3-D structure of the phase-type object can be reconstructed by using digital holographic microscopy complex amplitude data under various observation angles. Comparing with other tomography techniques, digital holographic microtomography has the advantages of non-destructive, non-contact, high-resolution,high-contrast, full-field imaging and auto-focus. The successful development of this technique will provide an advanced and nondestructive detection and research tool for life science, clinical medicine, biotechnology, new materials and other high-tech fields. So the study of digital holographic microtomography is of important scientific significance and application value.First, the dissertation carried out the researches on the optimization of reconstructed image quality based on the principle of digital holographic microscopy, which is a crucial problem in determining the quality of tomographic image. The main contents include the following aspects:In order to be able to directly recording high quality digital hologram, the optimization ofstructural parameters of the recording configuration was studied, the imaging resolution of three kinds of typical recording system was theoretically analyzed and compared, and through the experimental results proved that the digital image-plane holographic microscopy is an optimization imaging system; The main distortion of numerical representation of wavefront in digital holographic microscopy was analyzed, and the aberration correction methods that satisfy the application requirement of digital holographic microtomography were put forward; The auto-focusing methods in the reconstruction of the digital hologram were studied, and an criterion function based on accumulated edge detection was selected. The feasibility and reliability of the above methods were verified by experiment.A digital holographic microtomography system was established to realize 3-D RI distribution reconstruction. On the basis of experimental apparatus of off-axis digital holographic microscopy, the light path environment of the specimen was designed and configurated to meet the tomographic reconstruction theory. The location mark of the specimen was designed and the corresponding methods to correct the displacement errors of sample were proposed. An automatic angle rotation device developed on the basis of the SCM was added to the experimental apparatus of off-axis digital holographic microscopy, and the related interactive control procedures were developed by Matlab to realize the joint control of the specimen rotation and the digital hologram acquisition.Based on the Fourier projection slice theorem, the theoretical model of digital holographic microscopy tomography was thoroughly studied. The basic principle and computer implementation steps of the tomographic reconstruction of 3-D RI distribution by using digital holographic microscopy projection data were described in detail. The reconstruction accuracy,calculation efficiency and anti-noise robustness of the algorithm were analyzed and optimize the algorithm. The numerical simulation was performed to demonstrate the feasibility and reliability of the 3-D RI distribution reconstruction by combining digital holographic microscopy with tomographic imaging technique.Based on the Fourier diffraction projection theorem, the theoretical model of digital holographic microscopy diffraction tomography was thoroughly studied. The diffraction tomography reconstruction formula under the way of image-plane digital holographic microscopic recording was deduced. Two kinds of diffraction tomography reconstructionalgorithm based on the Fourier diffraction projection theorem were studied, and the principle and computer implementation steps of the algorithms were described in detail respectively. The numerical simulation was performed to demonstrate the feasibility and reliability of the 3-D RI distribution reconstruction by combining digital holographic microscopy with diffraction tomographic imaging technique in the case of diffraction cannot be ignored. Furthermore, the reconstruction accuracy, calculation efficiency and anti-noise robustness of the two algorithms were compared and analyzed according to the simulation results.A single-mode fiber and a polarization maintaining fiber werer used to simulate the biological sample having cylindrical symmetry and non-cylindrical symmetry structure respectively, and a translucent micro worm was selected as the actual biological sample. The digital holographic microtomography system was employed for experiments. According to ideal cylindrically symmetric structure of the sample, only single image-plane digital hologram was recorded to obtain projection data required for tomographic reconstruction algorithms, and in order to overcome the reconstruction error caused by incomplete correction of the wavefront aberration, the corresponding appropriate method to effectively eliminate the distortion of reconstructed image was put forward. Experimental results verify that Fourier diffraction tomography reconstruction algorithm can get more accurate results than the tomography reconstruction algorithm based on the Fourier projection slice theorem in the case of diffraction cannot be ignored. It provides a new way for the non-destructive and quantitative measurement of 3-D RI distribution of small biological samples.