Development of three-dimension refractive index microscopy for characterization and quantification of cells
Date Issued
2014
Date
2014
Author(s)
Hsu, Wei- Chen
Abstract
This dissertation mainly describes the construction, development and applications of three-dimensional (3D) refractive index (RI) microscopy.
Quantification of 3D RI with sub-cellular resolution was first achieved by a tomographic phase microscope (TPM) based on a Mach-Zehnder configuration. A simple piezoelectric actuator was used to generate phase shifts between sample and reference beams of the proposed TPM system. Two-dimensional phase images under a set of illumination angles were recorded for reconstruction of 3D RI tomograms based on optical diffraction tomography (ODT). 3D RI tomograms of various cell lines were quantified for demonstrating the feasibility of the TPM system on biological applications. However, the proposed TPM system based on phase shifting interferometry required multi-shot interferograms to obtain one phase image, resulting in limited spread and extension of bio-applications.
For speeding up acquisition efficiency of one RI tomogram and enhancing resistance of vibration, another common-path tomographic phase microscopy (cTPM) was developed as a novel technique for measuring 3D RI distribution. A diffraction grating was utilized to generate a reference beam that traversed a blank region of the sample in a common-path off-axis interferometry setup. In the cTPM system, 3D RI tomogram was reconstructed from single-shot phase images at multiple illumination angles implemented with ODT algorithm. The cTPM system inherently displayed high ability in resisting environmental vibration, leading to stable extraction of more accurate phase information than the TPM based on a Mach-Zehnder configuration. Besides, the cTPM system reduced the acquisition time of one RI tomogram due to the feature of single-shot phase imaging. The cTPM system was practically performed on mapping 3D RI distribution of HeLa cells.
In order to show extensive potentiality in bio-applications, the cTPM system was equipped with two light sources, a 473 nm diode laser and a 532 nm diode-pumped solid-state laser. The two-wavelength cTPM system became a powerful tool for stain-free visualization of 3D hemoglobin concentrations in single red blood cells (RBCs). In addition, cellular volume and morphology of individual RBCs could be also obtained in the two-wavelength cTPM system. This technique showed promising capabilities of charactering RBCs in blood specimens from patients with various blood deficiencies.
Subjects
三維影像
折射率
干涉儀
共光路
量化血色素
Type
thesis
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