Study On Phase Contrast Imaging For Micro Objects By Digital Holographic Microscopy

With the rapid development of the photoelectrical device and the computer, with the success of the recording configuration and the reconstruction algorithm, digital holography is developed rapidly based on the traditional optical holography, and becomes more and more popular recently. Up to now, the topic about the digital holography is never than active. The methodology and experiment are unceasingly expanding, expecially its application has been widened to lots of fields. Digital holography replaces the traditional recording material with the photoelectrical device, and implements its reconstruction process in the computer. Besides whold-field, no-touch, no-destruction, being access to the full information including the amplitude and phase, which are the common ones with the traditional optical holography, digital holography has lots of unique advantages, such as simple reconstruction in the computer, easy processing, quantitative measurement, easy storage, and so on. In this thesis, the topic is Study on Phase Contrast Imaging for Micro Objects by Digital Holographic Microscopy. It is analyzed from the view of theory and experiment, such as the scheme of the recording configuration, the realization of the reconstruction algorithm, the imaging for different kinds of samples. Following, the obtained production will be depicted one by one:1. Study of the reconstruction algorithm in digital holography. Three algorithms for diffraction propagation, named Convolution Method, Angular Spectrum Method, and Fresnel Transform Method, are analyzed in the aspects of the reconstructed quality, the efficience, the relationship with the reconstruction distance. It is indicated that both the Convolution Method and the Angular Spectrum Method present an optimal reconstruction distance, only in this case the reconstructed image has the good quality and resolution. In the case of distance bigger than the optimal one, the hologram or its spectrum is needed certain compensation to guarantee the whold field without any overlapping. While Fresnel Transform Method needs the Fourier transform only once, and has no-worse-than reconstruction quality and resolution compared with the other two methods, so it is determined as the best reconstruction algorithm. With regard to the phase aberration, the two-step-method is adopted to correct it. And the least square phase unwrapping algorithm is applied to obtain the unwrapped phase image. All of the theoretical knowledge will play an essential role when processing the experiment.2. Study for the recording configuration in the digital holography. First of all, the lensless Fourier transform digital holography is chosen because of its simple configuration and reconstruction algorithm. After collocating the proper experimental parameters, the phase contrast images and quatitative measurement are obtained for the USAF test target, the micro-lens array, the biological teaching plate, and the living biological cell. The achieved resolution is about 3.1m, which is adequate for the employed samples. In order to observe more details in the biological samples, the off-axis Fresnel digital holography with pre-magnification is established to improve the lateral resolution. The morphology of a single biological cell could be seen clearly with the sub-micron resolution. All the important experimental results offer a new prospect for the measurement of the micro-object. Especially the successful observation of the dynamic variation for the biological cell, it is expected to be applied to obtain the dynamic properties of the cell, the interaction between cells, and the response of the cell to the medicine, which could be useful to the early medicinal determination and the medicine design.3. Study for the autofocusing problem in the reconstruction of the digital hologram. Here, it is focused on the pure phase object. With regard to the lensless Fourier transform digital holography and the off-axis Fresnel digital holography with pre-magnification, the problem is discussed, which is about how to obtain the in-focus reconstructed image. Through applying the criterion function to the reconstructed image, the position of extremum is related with the in-focus reconstruction distance. Usually, the criterion function is applied to the reconstructed amplitude image, and the in-focus reconstruction distance is related with the minimum of the criterion function for the pure phase object. Here, the criterion function is directly applied to the reconstructed phase image, and the result is not the same with when applied to the reconstructed amplitude image. The method presented here is obviously more accurate, because the phase image is the one we are more concerned with. In experiment, firstly the standard phase grating is used to verify our idea. Then, the biological cells offer more evidence.4. Study for the elimination of the twin image in the in-line digital holography. The phase retrieval algorithm is chosen to solve the problem, and the absorption of the object is used as a constraint. After combined with the phase flipping method, with regard to the simple-shape object, it has the better efficience and quality. With regard to the complex-shape object, it can help to find the support of the object. Then the Fienup algorithm is further applied to retrieve the information of the object. The approach combining the Phase Flipping Method and the Fienup Algorithm is named Hybrid Phase Retrieval Approach. The simulation and experimental results verify the approach with good vadility and accuracy. It is reasonable to believe that it will become an effective approach to eliminate the twin image in the in-line digital holography.