| Contrast-agent-free microscopy is highly desirable to study the dynamics and physiological activity of various structures in living cells. Refractive index is an intrinsic contrast source, but at the same time it is an important biochemical parameter that is proportional to the concentration of molecules. By measuring the refractive index quantitatively, the alteration of cells under chemicals or drugs as well as their normal physiological activities can be monitored in most native conditions.Tomographic phase microscopy (TPM) is a novel microscopy for quantitative measurement of3D refractive index map of a transparent biological sample based on quantitative phase microscopy (QPM). However, there are some technical issues for TPM used in the detection of circulating tumor cells (CTCs), such as measurement limit and image reconstruction algorithms.In this paper, we explored a three-dimensional quantitative phase microscopy system based on QPM and implemented it to image the simple structure and low scattering samples such as single polystyrene beads and single living cells, and an accurate refractive index estimation and high spatial resolution were described.Then the three-dimensional quantitative phase microscopy system was applied to measure the3D refractive index distribution of samples with complex structures, and its performance was evaluated. The samples included one-layer of polystyrene beads, two-layers of polystyrene beads and two-layers of HeLa cells.For the numerical simulation, we generate scattering fields by using the forward operator. As sample we used3D polystyrene bead phantom and modified3D Shepp-Logan phantom with negligible absorption, which provide the foundation for reconstruction algorithm study.The numerical reconstruction of the3-D refractive index map from the2-D phase images was done in various ways. In chapter four, we adopted a filtered back-projection algorithm which is based on the projection approximation. In chapter five,3D refractive index of the sample was reconstructed under the first Born approximation and the first Rytov approximation respectively. This thesis compared the performance of filtered back-projection algorithm and diffraction tomography with the first Born and Rytov approximation.In most practical applications of three-dimensional quantitative phase microscopy, the angular coverage of an incident beam is limited due to finite system numerical aperture. The refractive index map reconstructed from the restricted data set suffers from the missing cone artifact:elongation of the reconstructed shape along the optical axis and underestimation of the value of the refractive index. This thesis applied an iterative constraint algorithm to minimize the artifact introduced by the missing cone problem.Another contribution of this paper is developing a method to achieve an automatic image processing for the images with symmetric structure. |
