Fast Phase Retrieval Method And Imaging Technology Of Cells Based On Interference Microscopy

Cell is the basic unit of the organism structure and functions, which is closely related to genetics,disease, immunity and rehabilitation in life science. As for individual cell, the morphological characteristic as well as the internal composition, plays a crucial role in its health status, function and activity. The research of cells, especially the analysis of its internal structure and dynamic behavior depends on the technical level of the microscopy imaging. The traditional microscope could not meet the requirement of the scientific research yet. Therefore, it is very necessary to grope new ways that can realize, nondestructive, fast, precise and dynamic imaging of cells with its original state. It is an urgent need and long-time goal of the research on life science.Quantitative phase imaging technology has became a very favorable tool for the microscopic imaging of biological cells as it has advantages of freeing from dyeing, non-invasion, non-destruction and allowing quantitative measurement. Over the past decades, many advanced quantitative phase imaging technologies have been proposed in succession, which are applied to phase imaging, classification recognition, parameter measurement and dynamic behavior analysis of cells successfully. Most quantitative phase imaging technologies are based on the principle of optical interference, and usually include the processes of the interference pattern acquisition, phase retrieval and possible phase unwrapping. In these steps, the quality, speed, as well as accuracy of phase imaging are determined mainly by the phase retrieval technology. So, it is significant to develop the phase retrieval method and the associated imaging technology. There are still many works to be actively carried out in the phase retrieval technology after systematically reading the related references. For instance, the singular problem of the phase shift of π in the general phase-shifting interferometry, is the first one. Secondly, it is required to balance the fast recovery speed and the detector bandwidth. Thirdly, there are some restrictions on the application. Aiming at these problems, the systematic research on its basic theory and key application technology is carried out in this paper supported by several national and provincial projects, and the expected results are achieved. Main achievements are as follows:(1) The characteristics of various typical phase imaging technologies, the associated phase retrieval methods as well as the determination methods of phase shifts are analyzed systematically. The reason of the singular case of the phase shift of π is pointed out. When the certain condition is satisfied, i.e., the intensity of the object wave is as same as that of the reference wave, an algebraic phase retrieval method is proposed, which can solve the singularity problem. By numerically calculating both the sum and the difference between two phase-shifted interferograms, one can directly retrieve the phase information, and the simulation results verify this method.(2) There are still some problems, such as multi-restriction, strict requirement of detection accuracy of phase shift and insufficient retrieval rate, existed in the traditional phase retrieval methods of interference microscopy technologies. Based on the sensitivity characteristic and numerical calculation ability of the local differential operator, we first propose a new derivative method for phase retrieval, which is applicable to all carrier interferometries, and then some phase derivative methods are proposed by analyzing two-step phase-shifting interferometry, which are applicable to an interferometry with an arbitrary interference recording mode. These derivative methods are verified in solving the problems mentioned above effectively by the simulation and experimental results. In addition, we propose a method of phase gradient to roughly estimate the morphology information of cells. The phase gradient results of heterogeneous white blood cells suggest that the effectiveness and feasibility of the method. Thus, it can be applied to recognize the type of cells.(3) Aiming at the noise in interferometry microscopy technique, a method for determining the phase shift is proposed by employing a matrix 2-norm algorithm in three-step phase-shifting interferometry. This method is easy to implement, because only the matrix 2-norm of the intensity difference between each two interference patterns is required to calculated, and no other physical quantities need to be measured. As a result, the imaging requirement is reduced and the robustness of the image is improved. In view of the restriction that the fringe number is more than 1, existed in the matrix 2-norm algorithm above, we propose a similar matrix 1-norm algorithm to determine phase shifts, in which the background intensity is eliminated by two interferograms with the phase shift difference of n. The validity and accuracy of these matrix norm methods are verified by the simulation and optical experiments.(4) By analyzing the statistical characteristics of the diffraction phase field, an expanded measurement method for phase shift is proposed by using a statistical average arithmetic and the spatial frequency relationship between two interference waves, in two-step or more-step phase-shifting interferometry. The phase shift can be set at any value within a complete period of (0,2π) excluding the singular case of π, so the real and general phase-shifting interferometry can be realized, and the precision of the phase shifter and the imaging requirement are reduced. The simulation results of a homogeneous red blood cell (RBC) and the experimental result of a polystyrene bead, suggest the high performance of this method in any environment whether it is ideal or not.(5) According to the theoretical results mentioned above, the associated design and discussion of the key technology are carried out. Especially, two kinds of instantaneous two-step phase-shifting imaging methods are proposed. One is realized by a detector and two identical beam splitters with the structure of the lateral displacement. Another is realized by two identical detectors and several common beam splitters. Considering the stability of the phase imaging system, a two-step diffraction phase microscopic imaging method is put forward. This method is realized by the zero-order,+1 order and -1 order diffraction lights produced by a grating, in which an optical interference between zero-order and+1 order diffraction lights is first recorded, and then another optical interference between zero-order and -1 order diffraction lights is recorded. This imaging method not only remains the advantages of high stability and real-time measurement in the traditional diffraction phase micro technique, but also expands the application scope. In addition, the accuracy of the phase retrieval is improved as well.This work makes some breakthroughs in theory. What is more, it gets some supports from the experiments. It not only offers more choices for the fast imaging and real-time measurement of cells, but also provides a reference for more in-depth quantitative detection of cells.