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Biophysical interpretation of quantitative phase image
Štrbková, Lenka ; Kozubek,, Michal (referee) ; Hoppe, Andreas (referee) ; Chmelík, Radim (advisor)
Práce se zabývá interpretací kvantitativního fázového zobrazení pomocí techniky koherencí řízené holografické mikroskopie. Vzhledem k tomu, že tato technika generuje velké množství kvantitativních fázových obrazů o nezanedbatelné velikosti, manuální analýza by byla časově náročná a neefektivní Za účelem urychlení analýzy obrazů získaných pomocí koherencí řízené holografické mikroskopie je v této práci navržena metodika automatizované interpretace kvantitativních fázových obrazů pomocí strojového učení s učitelem. Kvantitativní fázové obrazy umožňují extrakci parametrů charakterizujících distribuci suché hmoty v buňce a poskytují tak cennou informaci o buněčném chování. Cílem této práce je navrhnout metodologii pro automatizovanou klasifikaci buněk při využití této kvantitativní informace jak ze statických, tak z časosběrných kvantitativních fázových obrazů. Navržená metodika byla testována v experimentech s živými buňkami, jimiž byla vyhodnocena výkonnost klasifikace a významnost parametrů získaných z kvantitativních fázových obrazů.
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Fluorescence imaging techniques in multimodal holographic microscope
Vašíček, David ; Procházková, Jana (referee) ; Čolláková, Jana (advisor)
The diploma thesis deals with the registration of images taken with the multimodal holographic microscope (MHM). The summary covers the fluorescent and holographic microscopy, and the multimodal holographic microscope combining both these microscopy types. Every pair of the images needs to be aligned in order to gain new information by combining both image types. The thesis contains an algorithm that registers images by phase correlation as well as a process created in MATLAB in accordance with the algorithm. The most important procedure parameters’ influence on the registration success is described and the results are annotated.
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Coherence-controlled holographic microscope in cell's life cycle research
Křížová, Aneta ; Kozubek,, Michal (referee) ; Uhlířová, Hana (advisor)
The goal of this diploma thesis was using of a coherence-controlled holographic microscope in cell’s life research. A brief history of interference microscopy and it’s applications in biology is described. Also other microscopy techniques routinely used for transparent objects imaging are mentioned and the biology of cell’s life cycle briefly explained. Characteristics describing the shape of a cell were proposed and tested with respect to identification of particular phases of its life cycle. The method of dynamic phase differences was modified in order to distinguish the internal motion of cell’s mass from the movement of the whole cell. Selected characteristics were used to evaluate observations carried out with the holographic microscope and the possibilities of their further applications were depicted. In conclusion, obtained findings were summarized and modifications of microscope construction as well as data-processing software were suggested.
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The Three-Dimensional Digital Imaging Methods for X-ray Computed Tomography and Digital Holographic Microscopy
Kvasnica, Lukáš ; Číp, Ondřej (referee) ; Štarha, Pavel (referee) ; Chmelík, Radim (advisor)
This dissertation thesis deals with the methods for processing image data in X-ray microtomography and digital holographic microscopy. The work aims to achieve significant acceleration of algorithms for tomographic reconstruction and image reconstruction in holographic microscopy by means of optimization and the use of massively parallel GPU. In the field of microtomography, the new GPU (graphic processing unit) accelerated implementations of filtered back projection and back projection filtration of derived data are presented. Another presented algorithm is the orientation normalization technique and evaluation of 3D tomographic data. In the part related to holographic microscopy, the individual steps of the complete image processing procedure are described. This part introduces the new orignal technique of phase unwrapping and correction of image phase damaged by the occurrence of optical vortices in the wrapped image phase. The implementation of the methods for the compensation of the phase deformation and for tracking of cells is then described. In conclusion, there is briefly introduced the Q-PHASE software, which is the complete bundle of all the algorithms necessary for the holographic microscope control, and holographic image processing.
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Microscopy of Time Variable Biologic Objects
Uhlířová, Hana ; Kozubek, Michal (referee) ; Peychl,, Jan (referee) ; Chmelík, Radim (advisor)
The subject of the PhD thesis is the application of a transmission digital holographic microscope (DHM) which was designed and constructed in the Laboratory of optical microscopy at the IPE BUT for the research of live cells dynamics. First part of the work is concerned with theoretical description of the microscope imaging properties dependent on the coherence of illumination. It is supplemented with experiments of imaging of a model and a real biological specimen. The following part describes construction modifications and innovations of the microscope and its equipment that enabled the utilization of the microscope for live cells observations. In the experimental part the methodology of live cells preparation and DHM imaging was worked out. The methodology was verified by the observation of cell dynamics during an apoptosis induced by the cytostaticum cis-platinum. Further experiments examined the dynamics of live cells in standard conditions and during a deprivation stimulus. A novel method of holographically reconstructed phase, named \uva{dynamic phase differences}, was set up to evaluate quantitative changes of cell mass distribution during the experiments. Depending on the degree of malignancy and density of cell outgrowth, various schemes of cancer cells behaviour during a specific reaction were revealed using this method. For the quantitative analysis of the DHM phase imaging, a suitable statistical characteristic and an interpretation of the measured data were proposed. Both of them were successfully applied for the comparison of cell motility of two cell types: parental and progeny cell lines. On the basis of the proposed processing, hypotheses describing the reaction mechanism of tumour cells to stress life conditions were established. In the conclusions we summarize our findings and suggestions for the construction and the applications of a new generation of the transmission DHM.
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Mathematical Methods for Image Processing in Biological Observations
Zikmund, Tomáš ; doc. RNDr.Petr Matula, Ph.D. (referee) ; Krejčí, František (referee) ; Chmelík, Radim (advisor)
The dissertation deals with the image processing in digital holographic microscopy and X-ray computed tomography. The focus of the work lies in the proposal of data processing techniques to meet the needs of the biological experiments. Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The phase images are affected by the phase aberrations that make the analysis particularly difficult. Here, we present a novel algorithm for dynamical processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. High resolution X-ray computed tomography is increasingly used technique for the study of the small rodent bones micro-structure. In this part of the work, the trabecular and cortical bone morphology is assessed in the distal half of rat femur. We developed new method for mapping the cortical position and dimensions from a central longitudinal axis with one degree angular resolution. This method was used to examine differences between experimental groups. The bone position in tomographic slices is aligned before the mapping using the propound standardization procedure. The activity of remodelling process of the long bone is studied on the system of cortical canals.
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Simulation of imaging in Reflected-light Holographic Microscope with optical aberrations
Effenberger, Adam ; Kvasnica, Lukáš (referee) ; Týč, Matěj (advisor)
The aim of this work is the simulation of a moving cell on a noisy backgroud. Experience gained during this work was used for correction of artificially created data. Subsequently it was used for the correction of real experiment a data. Spyder, open source cross-platform IDE for scientific programming in the Python language, was used for it. The simulation was successful, as well as the correction of artificially created data. The correction of real experiment data was only partly suscessfull . It gives us enough room for further improvement.
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Automatization of method of surface structure measurement by means of reflection mode digital holographic microscope.
Vacula, Daniel ; Šerý, Mojmír (referee) ; Lovicar, Luděk (advisor)
Reflected-light digital holographic microscope developed at IPE FME BUT uses off-axis holography principle and low spatial and temporal coherence illumination. Microscope allows reconstruction of the image amplitude and the image phase, which can be handled in real time. The only limiting factors are imaging speed of the detector and computer performance when processing holograms. Reconstruction of image phase and amplitude allows high-resolution profilometric measurements in the vertical axis direction. This thesis deals with the automatization of profilometric measurement method proposed in [2]. Proposed method uses the combination of the image phase and the image amplitude for the measurement of specimens with surface structure the vertical size of which cause the uncertainty of the image phase by a factor of 2pí. Futher the thesis deals with the construction design of the illumination system of the microscope and its realization together with experimental verification of functionality of proposed method automatization.
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Control of an interference-microscope optical stage based on the image phase
Kvasnica, Lukáš ; Číp, Ondřej (referee) ; Chmelík, Radim (advisor)
Digital holographic microscopy is an interferometric imaging technique, the principle of which is the off-axis image plane holography. The principle of this technique enables to reconstruct both the image intensity and the image phase from the output interferencesignal. The reconstruction can be carried out on the basis of a single image plane hologram. This leads to the possibility of a realtime image reconstruction. The speed of the reconstruction depends on the detection and the computing process. The aim of this diploma thesis is to develop user software for the control of the detection camera and for the image plane hologram reconstruction. The effort was to achieve the highest number of image reconstructions per time unit, with the maximum utilization of the data transfer between the camera and the computer.The next aim of this thesis is the stabilization of the optical table position. The method of stabilization is based on the image phase information, which is used for the control loop feedback between reconstructed image phase and the piezoelectric actuator placed inside of the optical table. Experimental results, which prove the functionality of the stabilization, are presented.
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Biophysical interpretation of quantitative phase image
Štrbková, Lenka ; Kozubek,, Michal (referee) ; Hoppe, Andreas (referee) ; Chmelík, Radim (advisor)
Práce se zabývá interpretací kvantitativního fázového zobrazení pomocí techniky koherencí řízené holografické mikroskopie. Vzhledem k tomu, že tato technika generuje velké množství kvantitativních fázových obrazů o nezanedbatelné velikosti, manuální analýza by byla časově náročná a neefektivní Za účelem urychlení analýzy obrazů získaných pomocí koherencí řízené holografické mikroskopie je v této práci navržena metodika automatizované interpretace kvantitativních fázových obrazů pomocí strojového učení s učitelem. Kvantitativní fázové obrazy umožňují extrakci parametrů charakterizujících distribuci suché hmoty v buňce a poskytují tak cennou informaci o buněčném chování. Cílem této práce je navrhnout metodologii pro automatizovanou klasifikaci buněk při využití této kvantitativní informace jak ze statických, tak z časosběrných kvantitativních fázových obrazů. Navržená metodika byla testována v experimentech s živými buňkami, jimiž byla vyhodnocena výkonnost klasifikace a významnost parametrů získaných z kvantitativních fázových obrazů.
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