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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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COHERENCE-CONTROLLED HOLOGRAPHIC MICROSCOPE
Kolman, Pavel ; Křupka, Ivan (referee) ; Kozubek, Michal (referee) ; Chmelík, Radim (advisor)
ransmitted-light coherence-controlled holographic microscope (CCHM) based on an off-axis achromatic and space-invariant interferometer with a diffractive beamsplitter has been designed, constructed and tested. It is capable to image objects illuminated by light sources of arbitrary degree of temporal and spatial coherence. Off-axis image-plane hologram is recorded and the image complex amplitude (intensity and phase) is reconstructed numerically using fast Fourier transform algorithms. Phase image represents the optical path difference between the object and the reference arms caused by presence of an object. Therefore, it is a quantitative phase contrast image. Intensity image is confocal-like. Optical sectioning effect induced by an extended, spatial incoherent light source is equivalent to a conventional confocal image. CCHM is therefore capable to image objects under a diffusive layer or immersed in a turbid media. Spatial and temporal incoherence of illumination makes the optical sectioning effect stronger compared to a confocal imaging process. Object wave reconstruction from the only one recorded interference pattern ensures high resistance to vibrations and medium or ambience fluctuations. The frame rate is not limited by any component of the optical setup. Only the detector and computer speeds limit the frame rate. CCHM therefore allows observation of rapidly varying phenomena. CCHM makes the ex-post numerical refocusing possible within the coherence volume. Coherence degree of the light source in CCHM can be adapted to the object and to the required image properties. More coherent illumination provides wider range of numerical refocusing. On the other hand, a lower degree of coherence makes the optical sectioning stronger, i.e. the optical sections are thiner, it reduces coherence-noise and it makes it possible to separate the ballistic light. In addition to the ballistic light separation, CCHM enables us to separate the diffused light. Multi-colour-light
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Optical tweezers for coherence-controlled holographic microscope
Straka, Branislav ; Jákl, Petr (referee) ; Dostál, Zbyněk (advisor)
In the master's thesis, there has been described and explained the principle of operation of the second generation coherence controlled holographic microscope (CCHM2) designed at the Brno University of Technology. There has also been listed theoretical description of the operation of the optical trap, together with the calculation of the forces acting on it, ways of measuring the stiffness of the optical trap and the principle of~creating a time-shared optical traps. The optical tweezers forming a separate module connectable to CCHM2 was designed. Simulation and optimization of parameters of the optical system, mechanical design, manufacturing documentation, current source to power the laser diode which allows to control the diode output power by the controller card connected to the PC was designed. The galvano-optics mirror angle is controlled by the PC card too. The optical tweezer has been designed, manufactured and tested in conjunction with the CCHM2.
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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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Components for laser micromanipulation in a coherence-controlled digital holographic microscope
Straka, Branislav ; Antoš, Martin (referee) ; Dostál, Zbyněk (advisor)
In the Bachelor's thesis, there was described the general principle of operation of the optical trap, along with various options for creating optical tweezers. The function and the main parameters of holographic microscope with the off-axis achromatic and spatially invariant interferometer were described, too. Two variations of laser tweezers connectable to coherence-controlled second generation holographic microscope were described in detail, calculated and designed at IPE VUT. The optimization of the two variations was performed in the software ZEMAX. A 3D model in the Autodesk Inventor PROFESSIONAL 2010 was created for the chosen design.
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Identification of changes in the behaviour of tumour cells in low concentrations of migrastatics by coherence-controlled holographic microscope
Zábranská, Magdaléna ; Netíková,, Irena Štenglová (referee) ; Veselý, Pavel (advisor)
The subject of the diploma thesis was using of Coherence-Controlled Holographic Microscope in the research of the effect of migrastatics at different concentrations on tumor cells and the interpretation of quantitative phase imaging data. The theoretical introduction is divided into two parts. The first part is devoted to CCHM, which briefly describes the history of holographic microscopy, the principle and a detailed description of the main parts of the microscope. The second part is an introduction to the issue of cancer treatment and the introduction of migrastatic drugs. The experimental part of the work is the analysis of the influence of migrastatics on two tumor lines (H1299 and HT1080) from the measured time-lapse records taken by a holographic microscope. The effect of drugs was evaluated using characteristics describing morphological and dynamic cell changes in the SophiQ software. The data were then graphically and statistically processed to compare and evaluate the effect of migrastatics at low levels. In conclusion of the work, the findings were summarized and selected characteristics were proposed to analyze the impact of migrastatics.
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Flow-chambers for microscopy of living cells
Čolláková, Jana ; Ježek, Jan (referee) ; Antoš, Martin (advisor)
The perfusion chamber for long term observing of live cells by the means the Coherence-Controlled Holographic Microscope (CCHM) was designed. CCHM was built and designed at the Laboratory of the optical microscopy at the Institute of Physical Engineering, Brno University of Technology. CCHM can quantitatively evaluate dynamical changes inside live cells thanks to the quantitative information about phase shift in each pixel of the image. In order to demonstrate advantages of CCHM experimentally, it is important to keep the live cells in the good conditions. This is made by adding the fresh cultivation medium for studied cells directly in the microscope. In contrast to the stationary chamber the perfusion chamber allows both the cultivation medium exchange and the application of biological reagents without the necessity of removing the chamber from the microscope. Therefore we can study the vital signs of cells before and after the application of reagents. An original perfusion system with accessories compatible with CCHM was designed. The design is based on the previously published perfusion system solutions that are referred to in this thesis. The flow characteristics and medium exchange process was discussed and a modification of the internal geometry, based on numerical simulations, was introduced. The applicability of this perfusion chamber has been proven for the CCHM and even for different types of microscopes. The reactions of tumor and epithelial cells during the change of the environment from the cultivation medium to the physiologically solution were studied.
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