Imaging Theory And Experimental Demonstration Of Digital Holographic Microscopy

Digital holography is a new holographic technique, which uses a charged-coupled device(CCD) camera to replace the traditional holographic material for hologram recording and uses a computer to simulate the optical diffraction process of wave front for reconstructing the original object field, implementing a totally digitalized holographic process of recording, storing, transmitting and reconstructing. Because of its advantages, in recent years, digital holography has become one of the most interested research fields all over the world. It has wide application prospect in various aspects, including cell culture observation, microcircuit examination, granularity analysis, profilometry and shape microvariation measurement, transparence field measurement, etc.Through theory analysis, simulation computation and experiment demonstration, this paper addresses the basic issues of digital holography in application field, including the recording condition, the reconstruction algorithm, the resolution of the digital holographic imaging and the method to improve it, the reconstruction for three dimensional object and the depth of field, depth of focus of the holographic imaging system. The main content and the significative results are as follows:1. In this paper, the method of extremum-spatial-frequency is used to deduce the expression of the minimum recording distance and the offset of reference point source in different optical configuration, meeting the conditions of sampling and separation of the reconstructed image simultaneously. The results are compared with the related papers. With the off-axis Fresnel digital holography, the different results have been obtained, but rigorous and accurate in this paper. With other configuration, the same results have beeb obtained. Computer simulation indicates that if the off-axis Fresnel digital hologram is recorded in the condition given in this paper, the well-separated reconstructed image with high resolution can be achieved.2. Based on the point spread function of the digital holography without pre-magnification, the lateral resolution limitation of the reconstructed image is analyzed, and the shortages in the published papers are pointed out. An abnormal recording condition is proposed, that is reducing the recording distance and the off-set of reference point source further compared with the minimum theoretical value, letting the three orders of the reconstructed image separate with each other only for the target imaing region. This method can improve the imaing resolution, and the expression of corresponding recording condition is also given. With the lenless Fourier transform digital holography, the resolution of reconstructed image can be improved from 3.91μm to 2.76μm. And, the experimental results testify the feasibility and validity of the given method.3. The paper deduces the point spread function of the digital holography with pre-magnification firstly in detail, and analyzes the determing fator to the resolution limitation. It is pointed out that the matching requirement of imaging resolution between the microscopic objective and the CCD. The conclusion is significative to the recording of digital hologram with pre-magnification.4. The phase reconstruction algorithms of digital holograpy are studied. The term of phase aberration is decuced in detail to establish the phase mask with the lensless Fourier transform digital holography and the digital holography with pre-magnification. The auto-focusing methods in ditigal imaging are compared with each other, including the gray variance method, the sum of Fourier spectrum logarithm method, the Tenengrad operator method and the sum of Laplacian operator method. When use them to reconstruct the Fresnel digital hologram with pre-magnification, finding the accurate position of the reconstructed image plane, it is found that all the four methods are suitable for this configuration. Using the automatic procedure for aberration compensation to reconstruct phase image of the hologram, it is found that in the condition of strong noise, in order to get the accurate three-dimensional image, it is nessesary to combine the automatic aberration compensation procedure with the manual adjustment together.5. The issues of depth of field and depth of focus of the digital holographic imaging system are studied in detail. In several common digital holographic recording systems, the depth of focus is decuded and analyzed respectively using the reconstructed-image-intensity-criterion. The results of computer simulation and experiment indicate that the depth of focus accords with the theoretical prediction with the reconstructed-image–intensity-criterion. Then according to the 1/4 wave-imaging-difference limitation of the ideal imaging, the depth of focus of Fresnel digital holography is analyzed further from the off-focusing phase error. The difference between the two methods to evaluate the depth of focus is one third. It is indicated that the depth of focus of the digital holographic imaging system is not only related with the numerical aperture and the reference wavelength, but also related with the configuration and the offset of reference wave.