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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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New Generation of a Coherence-Controlled Holographic Microscope
Slabý, Tomáš ; Novák,, Jiří (referee) ; Jákl, Petr (referee) ; Chmelík, Radim (advisor)
This doctoral thesis deals with design of a new generation of coherence-controlled holographic microscope (CCHM). The microscope is based on off-axis holographic configuration using diffraction grating and allows the use of temporally and spatially incoherent illumination. In the theoretical section a new optical configuration of the microscope is proposed and conditions for different parameters of the microscope and its optical components are derived. The influence of different sources of noise on phase detection sensitivity is studied. In the next section design of experimental setup is described and automatable adjustment procedure is proposed. Last section describes experimental verification of the most important optical parameters of the experimental setup. When compared to previous generation of CCHM, the newly proposed configuration uses infinity-corrected objectives and common microscope condensers, allows more space for the specimens, eliminates the limitation of spectral transmittance and significantly simplifies the adjustment procedure so that automation of this procedure is possible.
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Observation of Influence of Thin Layers on Cell Behaviour by Multimodal Holographic Microscope
Vengh, Martin ; Křížová, Aneta (referee) ; Štrbková, Lenka (advisor)
Surface treatment of materials for the cell-surface interaction and imitating intrinsic environment of cells is still subject of matter for their potentional usage in the bioengineering. One of the options to modify the surface of material is exhibiting them to effects of plasma of electrical discharge in steam of cyclopropylamine. In this type of plasma, the low pressure plasma polymerization takes place, which creates thin amine-rich layer. There is an assumption that this type of layer is appropriate for cell adhesion and proliferation. As a superior technique for a label-free monitoring of the cell-surface interaction, multimodal holographic microscope (MHM) Q-Phase was exploited to determine the biocompatibility of materials. MHM enables observation in quantitative phase imaging, where the phase is directly proportional to the cell dry mass. This gives opportunity to define various features able to determine the biocompatibility of materials. According to the results amine-rich films enhanced the conditions for the cell adhesion and proliferation.
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Analysis of changes in the phenotype of tumour cells induced by migrastatics in quantitative phase imaging
Kolínková, Veronika ; Netíková,, Irena Štenglová (referee) ; Veselý, Pavel (advisor)
This thesis focuses on the observation of living cells using the non-invasive methods of quantitative phase imaging (hiQPI). The imaging is enabled by a coherence-controlled holographic microscope (CCHM) developed in the Laboratory of Experimental Biophotonics at the VUT. Using this imaging technology, morphological changes of A549 and MCF7 after application of potential migrastatic drugs tumor cells are evaluated in the experimental part of the thesis. Migrastatics are defined as already approved drugs that could prevent the migration of cancer cells from the primary tumor and thus prevent metastasis. The RAC-GM (Rapid Assessment of Cell Growth and Migration in Vitro) method was chosen to assess their effect on tumor cells.
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Deep-learning methods for tumor cell segmentation
Špaček, Michal ; Kolář, Radim (referee) ; Gumulec, Jaromír (advisor)
Automatic segmentation of images, especially microscopic images of cells, opens up new opportunities in cancer research or other practical applications. Recent advances in deep learning have enabled efficient cell segmentation, but automatic segmentation of subcellular regions is still challenging. This work describes the implementation of the U-net neural network for segmentation of cells and subcellular regions without labeling in the pictures of adhering prostate cancer cells, specifically PC-3 and 22Rv1. Using the best-performing approach of all tested, it was possible to distinguish between objects and background with average Jaccard coefficients of 0.71, 0.64 and 0.46 for whole cells, nuclei and nucleoli. Another point was the separation of individual objects, i. e. cells, in the image using the Watershed method. The separation of individual cells resulted in SEG value of 0.41 and AP metric of 0.44.
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Design of the imaging part of the coherence-controlled holographic microscope
Dvořák, Vladislav ; Dostál, Zbyněk (referee) ; Špaček, Matěj (advisor)
The Experimental Biophotonics Research group at Brno University of Technology has been involved in the development of Coherence controlled holographic microscope (CCHM). For the purposes of further development in this field, it is necessary to build an open laboratory set-up of the microscope, which has only been available in a commercial design. The objective of this thesis is to design and assemble its imaging unit. The first part of the thesis is dedicated to a general overview of microscopic methods, with an emphasis on interference microscopy and principle of CCHM microscope. In the second part, different optical set-up concepts are discussed and compared. Then, the optical components are selected and the unknown parameters of these components are experimentally measured. With the use of measured parameters, the optical tracing and mechanical design are introduced. The imaging unit has been assembled based on the suggested design and experimentally verified.
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Analysis of Microscopic Images of Cancer Cells
Vičar, Tomáš ; Matula,, Petr (referee) ; Sladoje, Natasa (referee) ; Kolář, Radim (advisor)
Tato disertační práce je zaměřena na analýzu různých forem mikroskopických obrazových dat nádorových buněk (statické 2D snímky, statické 3D obrazy, 2D časosběrné zobrazování živých buněk). Hlavní pozornost je věnována datům získaným koherencí řízeným holografickým mikroskopem, který je relativně novou modalitou schopnou kotrastních záznamů živých buněk bez barvení (label-free) a poskytuje kvantitativní informaci (kvantitativní fázové zobrazení - QPI). V práci je popsán základní postup analýzy těchto snímků a jsou vytvářeny nové metody a zdokonalovány metody pro jednotlivé kroky této analýzy. Největší část práce je věnována segmentaci buněk, kde jsou shrnuty klasické metody i metody založené na hlubokém učení. Jsou také vyvinuty nové metody vhodné právě pro QPI data. Část práce je také věnována segmentaci 3D fluorescenční jader a detekci DNA zlomů pomocí hlubokého učení. Práce se zabývá i dalším zpracování v podobě sledování buněk, extrakce příznaků a následné analýze, kde je detekována buněčná smrt a jsou vytvořeny vhodné interpretovatelné příznaky pro klasifikaci buněčné smrti na apoptickou a lytickou. Celkově tato práce přispívá k rozvoji jednotlivých kroků analýzy obrazu nádorových buněk a odráží současný pokrok v oblasti analýzy obrazu, zejména přístupy hlubokého učení, což je také demonstrováno na několika výzkumných aplikacích.
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Measurement of the local phase of metasurfaces using digital holographic microscopy
Weiss, Vlastimil ; Bouchal, Petr (referee) ; Dvořák, Petr (advisor)
This bachelor's thesis consists of research studies of optical metasurfaces that are capable of modifying and governing incident radiation via the shift of a local phase. It also discusses experimental microscopic techniques with the ability to measure the distribution of the said phase. Experimental results of the phase distribution of the electromagnetic wave impinging on metal metasurface presented in this thesis are captured through quantitative in-line and off-axis digital holographic microscopy. These metasurfaces utilize both geometrical phase and localised surface plasmon resonance (LSPR). Measured results are in accordance with previous scientific studies. Finally, the successful outcome in the form of measurement of the geometrical phase introduced by the single building block is presented as well as the application of an analytical model for characterization of phase response generated by interaction with optical metasurfaces.
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Analysis of changes in the phenotype of tumour cells induced by migrastatics in quantitative phase imaging
Kolínková, Veronika ; Netíková,, Irena Štenglová (referee) ; Veselý, Pavel (advisor)
This thesis focuses on the observation of living cells using the non-invasive methods of quantitative phase imaging (hiQPI). The imaging is enabled by a coherence-controlled holographic microscope (CCHM) developed in the Laboratory of Experimental Biophotonics at the VUT. Using this imaging technology, morphological changes of A549 and MCF7 after application of potential migrastatic drugs tumor cells are evaluated in the experimental part of the thesis. Migrastatics are defined as already approved drugs that could prevent the migration of cancer cells from the primary tumor and thus prevent metastasis. The RAC-GM (Rapid Assessment of Cell Growth and Migration in Vitro) method was chosen to assess their effect on tumor cells.
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Cell And Sub-Cellular Segmentation In Quantitative Phase Imaging Using U-Net
Majerčík, Jakub ; Špaček, Michal
The ability to automatically segment images, especially microscopy images of cells, opensnew opportunities in cancer research or other practical applications. Recent advancements in deeplearning enabled for effective single-cell segmentation, however, automatic segmentation of subcellularregions is still challenging. This work describes an implementation of a U-net neural networkfor label-free segmentation of sub-cellular regions on images of adherent prostate cancer cells,specifically PC-3 and 22Rv1. Using the best performing approach, out of all that have been tested,we have managed to distinguish between objects and background with average dice coefficients of0.83, 0.78 and 0.63 for whole cells, nuclei and nucleoli respectively
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