Study On Phase Contrast Imaging And The Refractive Index Measurement For Biological Cells By Digital Holographic Microscopy

In recent years, with the development of digital image processing technology,computer technology and photoelectric detection technology, the researches aboutdigital holographic technology are carried out widely and rapidly and has become oneof the most indispensable and important research direction in the microscopic imaging.Microscopy has been a key technology in biomedical research. The traditionalqualitative two-dimensional observation approaches with low resolution are unable tomeet the multifaceted demands of the current biological microscopic technology, so itis necessary that developing the real-time non-destructive and non-contact approachesto image the transparent biological cells with high resolution. The several advantagesof digital holography make it that be mixed fully with microscopic technology andbecome a new opportunity for three-dimensional microscopic imaging. The digitalholography record holograms with photoelectric detection devices such asCharge-coupled Device (CCD), instead of the traditional recording materials,therefore the subsequent multi-step processes of chemical treatment can be avoidedeffectively. Then, the reconstruction processes are operated on the computer, which isgiving full play to the advantages of digital image processing technology andcharacterized by the real-time measurement and quantitative analysis.In the life sciences and medical field, the demands of quantitative and dynamicmeasurement for the living cells is increasing, so the methods have been unable tomeet them, such as the ordinary optical phase contrast microscope, interferencemicroscope, etc. The most important reason is that these methods can only play a rolein the qualitative detection for cells and cannot provide the information of the truethickness about biological cells. Compared with the ordinary electronic microscopeand traditional optical microscope, the digital holographic microscopy imagingtechnology can not only measure reliably and non-destructively the quantitativeparameters of biological cells such as phase, thickness and refractive index withoutdyeing, but also be combined with digital image processing technology and realize thereal-time three-dimensional imaging and parameters analysis. These advantages isexpected to be applied in public health care and bio-medical field, then this approachcan contribute to the early detection of epidemic disease and early diagnosis ofserious diseases.This thesis systematically analyzes the research status of the phase contrastimaging for biological cells at home and abroad. And on the base of the digitalholography imaging theories, the digital holography is applies in the detection andanalysis of biological cells and the measurement of the refractive index. The main contents are expounded as follows:1. Based on the pre-magnification off-axis image digital holographic theory, aninverted setup of digital holographic microscopy is designed and built in accordancewith the need of observing the living cells. This structure do not need the step of thenumerical diffraction propagation by computer and greatly simplifies the numericalreconstruction. Through the determination, the actual transverse resolution in theamplitude image and phase image are measured by using the USAF resolution mask,which are0.87μm and0.977μm. First of all, the quantitative phase contrast imagesof three kinds of the living cells in the natural state are achieved. These living cells arerespectively Hippocampal Neuron Cells, neurosphere and Hela cells. Then, dynamicchanges of the Hela cells are detected, which are added different concentrations oforganic solvents and the basic parameters of these cells morphology are also obtained.The results show that the digital holography can be used for quantitative detection ofmorphology and dynamic changes of the living cells and it provides a reliable way fortesting and analyzing the functions and behavior of cells in the bio-medical field.2. Based on the principle of the refractive index measurement withdual-wavelength digital holographic microscopy, an inverted imaging device isdesigned and built, which is composed of532nm laser and633nm laser. And theactual transverse resolution in the amplitude image and phase image are1.10μm and1.55μm. Next, the cover glass in the edible oil is used as the object to do a feasibilitystudy about the refractive index measurement with dual-wavelength digitalholography, whose refractive index of is known. Then, the phase images of thetree-ring cells with dual-wavelength are obtained. After calculations, the refractiveindex distribution is measured. Thus, it can be seen that the dual-wavelength digitalholography can satisfy the demands of the refractive index measurement of biologicalcells and has high and practical values.