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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-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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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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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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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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Analysis of motility in leukemia cells using incoherent holographic quantitative phase imaging
Smrčková, Zuzana ; Škarková, Aneta (referee) ; Zicha, Daniel (advisor)
This diploma thesis deals with the issue of motility analysis in leukemia cells. An accurate description of the cell movement and the detection of differences in motility under experimental conditions can be obtained by quantitative analysis of cell motility using time-lapse recording. The first part of this work describes various types of tumor cell migraton. The second part focuses on methods of analysis of cell motility in tissue culture using time-lapse recording, which include image acquisition and processing. Part of this chapter describes a coherence-controlled holographic microscope, which was used in the practical part and for which an insert was designed to ensure the exact and stable position of the individual chambers. The last part is focused on the research of leukemic cell motility, which is concluded by a discussion of the obtained results. The appendix contains a published study included acknowledgement to the author of this diploma thesis for participation in the project.
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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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Biophysical interpretation of quantitative phase imaging of live cells generated by coherence-controlled holographic microscopy
Šuráňová, Markéta ; Rösel,, Daniel (referee) ; Vomastek, Tomáš (referee) ; Veselý, Pavel (advisor)
The dissertation thesis deals with the biophysical interpretation of quantitative phase imaging (QPI – Quantitative Phase Imaging) obtained using coherence-controlled holographic microscopy (CCHM – Coherence-Controlled Holographic Microscopy) in the Q-PHASE microscope, Telight, Brno). The theoretical part of this thesis deals with the characteristics of quantitative phase imaging, which provides non-invasive information on the activity of living cells in vitro. The main part of the work consists in elaborating a concept and verifying it of a new methodology (PAMP – Primary Assessment of Migrastatic Potential) for the first critical evaluation of drugs for expected anti-migratory/metastatic potential. The result of this method is considered the first sorting evaluation when considering specific migrastatic agents for future complex oncological treatment. PAMP evaluates the speed of cell migration, the growth of tumor cells and controls the risk of appearance of invasive phenotypes. Furthermore, the correlation microscopy method between the Q-PHASE microscope and the laser scanning confocal microscope (LSCM) is proposed to evaluate cell behavior and the occurrence of focal adhesions after drug application. The quantitative phase image obtained using the Q-PHASE microscope is compared with the quantitative phase image from the HoloMonitor (PHI AB, Sweden), on which the PAMP method has been positively verified.
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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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