Phase-Contrast Imaging In Digital Holographic Microscopy For Biological Samples

Digital holography is the technology of the combination of the traditional opticalholography, computer technology and photoelectric detection technology withadvantages of fast, real-time, full field, non-contact and quantitative phase-contrastimaging. It is especially suitable for the imaging and measurement of biologicaltissues in vivo state. With the development of commercial digital holographicmicroscope, the research of digital holography began to move from basic theory toexploring its potential applications. At present, the research work in digitalholography is active at home and abroad both in basic theory and algorithm and theexpansion of application fields.In this thesis, the topic is mainly about the application of digital holographicmicroscopy phase contrast imaging method in the imaging of biological samples. Inthe fields of key algorithms, coherent noise suppression methods, optical setups andimaging of biological samples in digital holographic microscopy, a series oftheoretical analysis and experimental results are given. They are depicted one by oneas follows:1. The numerical reconstruction algorithms in digital holography are studied. Inthe numerical reconstruction of digital holography, there are three traditionalnumerical propagation algorithms: the angular spectrum algorithm, the convolutionalgorithm and the Fresnel algorithm. Using the traditional numerical propagationalgorithms, the pixel size, the number of pixels and the position of the reconstructedimage are fixed. So that it is not suitable for a number of occasions, such as colordigital holography, particle field measurements. To solve this problem, we proposeddirect integration Fresnel algorithm and direct integration Rayleigh-Sommerfeldalgorithm, with which the pixel size, the number of pixels, and the position of thereconstructed images are adjustable. It makes the numerical reconstruction moreflexible. Using the proposed algorithms, we adjusted the pixel size, the number ofpixels and the center position of the reconstructed experimental results. Based on this,we achieved a step-by-step reconstruction.2. The phase unwrapping algorithms are studied. In digital holography, thequality of the reconstructed phase map depends on the phase unwrapping algorithm.With a unified mathematical model, a comparative study on the phase unwrappingalgorithms was done. On the basis of an off-axis Fresnel digital holographicexperiment, we concluded that the phase unwrapping algorithm based on least squaresfitting is appropriate in digital holography.3. The phase aberration compensation algorithms are studied. In the phasecontrast imaging of digital holographic microscopy, phase aberrations always exist, which prevent to obtain the correct phase information. Some phase aberrationcompensation methods require manual intervention, while some others requireaccurate system parameters. In digital holographic biological phase-contrast imaging,we proposed a surface fitting algorithm based on an improved mathematical modelthat can remove the main phase aberrations automatically. The improvedmathematical model contains not only the first order and quadratic items, but alsosome cross ones, which can fit the phase aberration more accurately. It can beresolved by a non-iterative algorithm, which greatly simplifies the surface fittingprocess and eases the computational load.4. A coherent noise suppression method based on the multiple obliqueilluminations is studied. The method based on the multiple oblique illuminations cansuppress the coherent noise and obtain high-quality image. However, the systems arealways complicated. We proposed an improved coherent noise suppression methodbased on the multiple oblique illuminations, which doesn't demand a large anduniform light spot. Based on the proposed method we built up a compactfiber-coupled lensless Fourier transform digital holographic system. Multiple digitalholograms with different illumination angles can be obtained automatically, whichleads to suppression of the coherent noise by averaging.5. A method for imaging through scattering media is studied. It is an importantarea of research to image through scattering or turbid media with visible light, and haspotential application values in biological imaging. Based on the analysis of thecharacteristic of photon passing though scattering media, we propose ashort-coherence digital holographic imaging method which utilize multi-step speckleillumination method to suppress the coherent noise. With a low-power shortcoherence laser diode, we achieved to image through3mm thick fresh chicken breast.6. Based on the off-axis Fresnel digital holography with pre-magnification, acompact fiber-coupled inverted digital holographic microscopy system is built.Combined with our study on algorithms, we obtained phase-contrast imaging of livingcells adherent growing in the culture dish.7. The physical compensation of phase aberrations in digital holographicmicroscopy is studied. In digital holography, it is necessary to ease computationalload as far as possible for long term real-time imaging and observation. Theoreticalanalysis shows that the spherical phase aberration could be compensated physicallywith proper optical setups. Subsequently, we built a phase aberration free digitalholographic microscope, and achieve long term observation of living cells with a highprecision.