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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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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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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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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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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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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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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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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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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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