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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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Holographic module for a light microscopy
Škrabalová, Denisa ; Slabý, Tomáš (referee) ; Dostál, Zbyněk (advisor)
The new arrangement of the off-axis holographic module, which is using polarizationactive diffraction grating divides signal into reference and subject wave of an interferometer based on their polarization. However, current design of the module does not have a possibility to tune a length of the optical paths. Thus the inability to tune optical paths leads to a reduced quality of interference structure during observation of biological samples. The current module is only suitable for technical applicating due to this limitation. Possibility of tuning branches is key step in biological applications. Therefore a new computer-controlled module is created in order to enable use for biological samples.
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Development of Biophysical Interpretation of Quantitative Phase Image Data
Křížová, Aneta ; Jákl, Petr (referee) ; Vomastek, Tomáš (referee) ; Chmelík, Radim (advisor)
This doctoral thesis deals with biophysical interpretation of quantitative phase imaging (QPI) gained with coherence-controlled holographic microscope (CCHM). In the first part methods evaluating information from QPI such as analysis of shape and dynamical characteristics of segmented objects as well as evaluation of the phase information itself are described. In addition, a method of dynamic phase differences (DPD) is designed to allow more detailed monitoring of cell mass translocations. All of these methods are used in biological applications. In an extensive study of various types of cell death, QPI information is compared with flow cytometry data, and preferably a combination of QPI and fluorescence microscopy is used. The DPD method is used to study mass translocations inside the cell during osmotic events. The simplified DPD method is applied to investigate the mechanism of tumor cell movement in collagen gels.
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Novel cancer biomarkers derived from quantitative phase imaging of biopsy cells
Plišková, Diana ; Týč, Matěj (referee) ; Kolářová, Jana (advisor)
The main objective of this work is the development of novel cancer biomarkers usable in personalized treatments. To understand why this issue is important, a brief description of cancer, including statistical results over the past years, is provided. The work also describes individual methods of light microscopy that can be used in cell analysis and subsequent image processing consisting of segmentation, tracking, feature extraction and classification. In this work, the main cell features, such as cell motility and shape, are presented. These features can be potential biomarkers in the treatment of cancer.
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Coherence-gate assisted three-dimensional imaging by holographic microscope
Maršíková, Barbora ; Heintzmann, Rainer (referee) ; Chmelík, Radim (advisor)
Tato diplomová práce se zabývá výzkumem na téma vlivu prostorové koherence osvětlení. Účelem je určit schopnost osové lokalizace při zobrazení Koherencí řízeným holografickým mikroskopem (CCHM) v závislosti na různé prostorové koherenci světelného zdroje. Osová lokalizace je v tomto případě zkoumána jako kvalita rozlišení drobných detailů trojrozměrného vzorku, umístěných nad sebou. Teorie zobrazení holografickým mikroskopem a teorie rozptylu v nehomogenních prostředích je shrnuta v první části práce, v rozsahu nutném pro pochopení části praktické. Základní princip fungování mikroskopu a přesný popis jeho uspořádání je zde podrobně popsán. Proběhl mechanický návrh stavební úpravy mikroskopu tak, aby bylo možno využívat kondenzorovou optiku s vysokou numerickou aperturou a omezenými optickými vadami. Několik různých přístupů, které by mohly vést ke zlepšení zobrazovacích vlastností mikroskopu, bylo navrženo a vyzkoušeno a jsou zde popsány i s jejich výhodami a nevýhodami. Pro experimentální část práce byl vyroben modelový vzorek. Závislost osové lokalizace na prostorové koherenci osvětlení byla demonstrována pomocí simulace a následně ověřena experimentálně, pozorováním vyrobeného modelového vzorku. Experimentální výsledky potvrzují základní principy vycházející ze zmíněné teorie. Na závěr jsou navržena možná vylepšení, pro budoucí zpřesnění výsledků.
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Critical review of culture devices used for study of live cells in the microscope
Ukropcová, Iveta ; Štrbková, Lenka (referee) ; Dostál, Zbyněk (advisor)
Coherence-controlled holographic microscope (CCHM) is used mainly in live cell microscopy in vitro. The cells observed must be placed in a culture device which enables hologram registration. With using the quantitative phase imaging (QPI) the live cells are inspected. Conventional cultivation devices are usually not adapted to the QPI method. In this text requirements are specified for cultivation devices for CCHM. A critical review of commercially available cultivation devices is the crucial part of the thesis, as well as an assessment of whether these devices meet the specified requirements. This work also deals with the issue of microfluidic and its application to live cell imaging. In the last part of the text two hybrid cultivation devices optimized for CCHM are described, which allow microfluidic cellular experiments.
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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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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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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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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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