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Microscopy of Time Variable Biologic Objects
Uhlířová, Hana ; Kozubek, Michal (referee) ; Peychl,, Jan (referee) ; Chmelík, Radim (advisor)
The subject of the PhD thesis is the application of a transmission digital holographic microscope (DHM) which was designed and constructed in the Laboratory of optical microscopy at the IPE BUT for the research of live cells dynamics. First part of the work is concerned with theoretical description of the microscope imaging properties dependent on the coherence of illumination. It is supplemented with experiments of imaging of a model and a real biological specimen. The following part describes construction modifications and innovations of the microscope and its equipment that enabled the utilization of the microscope for live cells observations. In the experimental part the methodology of live cells preparation and DHM imaging was worked out. The methodology was verified by the observation of cell dynamics during an apoptosis induced by the cytostaticum cis-platinum. Further experiments examined the dynamics of live cells in standard conditions and during a deprivation stimulus. A novel method of holographically reconstructed phase, named \uva{dynamic phase differences}, was set up to evaluate quantitative changes of cell mass distribution during the experiments. Depending on the degree of malignancy and density of cell outgrowth, various schemes of cancer cells behaviour during a specific reaction were revealed using this method. For the quantitative analysis of the DHM phase imaging, a suitable statistical characteristic and an interpretation of the measured data were proposed. Both of them were successfully applied for the comparison of cell motility of two cell types: parental and progeny cell lines. On the basis of the proposed processing, hypotheses describing the reaction mechanism of tumour cells to stress life conditions were established. In the conclusions we summarize our findings and suggestions for the construction and the applications of a new generation of the transmission DHM.
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Fresnel Incoherent Correlation Holography (FINCH)
Bouchal, Petr ; Zemánek, Pavel (referee) ; Chmelík, Radim (advisor)
This master’s thesis develops a novel method of digital holography, from recent studies known as Fresnel Incoherent Correlation Holography (FINCH). The method enables the reconstruction of the correlation records of three-dimensional objects, captured under quasi-monochromatic, incoherent illumination. The experimental system is based on an action of a Spatial Light Modulator, driven by computer generated holograms to create mutually correlated beams. Both optical and digital parts of the experiment can be carried out using procedures of classical holography, diffractive optics and digital holography. As an important theoretical result of the master’s thesis, a new computational model was proposed, which allows to describe the experiment completely with respect to its two basic phases. The proposed model allows to understood the method intuitively and can be used additionally for analysis and interpretation of the imaging parameters and the system optimalization. The theoretical part of the master’s thesis also presents a detailed description of the correlation imaging based on an appropriate reconstruction process. Computational models were developed for both monochromatic and quasi-monochromatic illumination. In experimental part, all theoretical results were verified. The imaging parameters were examined using standard resolution target tests and appropriate biological samples. As an original experimental result, spiral modification of the system resulting in a vortex imaging was proposed and realized. Here, a selective edge enhancement of three-dimensional objects is possible, resulting in a significant extension of possible applications of the method.
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Theoretical description of imaging by a digital holographic microscope
Slabá, Michala ; Komrska, Jiří (referee) ; Chmelík, Radim (advisor)
The diploma thesis deals with theory of imaging in a transmitted-light digital holographic microscope using partially coherent illumination. The influence of spatial and temporal coherence state on optical sectioning property is solved. The coherent transfer function is calculated. From this function imaging characteristics for a two-dimensional scattering object are derived depending on its defocus. Two different designs of microscopes developed in the Laboratory of optical microscopy in IPE FME BUT are considered.
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Programmable illuminating system for an optical microscope
Lošťák, Martin ; Křupka, Ivan (referee) ; Chmelík, Radim (advisor)
A programmable illuminating system (PIS) uses a commercial multimedia projector together with a suitable optical relay system in order to illuminate specimens under microscope with transmitted light. The theoretical part of the diploma thesis describes some methods used in the optical transmission microscopy. All of these methods employ physical masks placed in the condenser front focal plane. In the case of the traditional methods the masks are used to enhance contrast (e.g. dark-field illumination) and resolution (oblique illumination). One of the methods (a condenser with rotating aperture) provides the information about the three-dimensionality of the specimen. The next part of the thesis contains the theory and the basic classification of the illuminating systems used in the optical transmission microscopy. An optical and mechanical design of the optical relay system used for PIS is introduced. The experimental part shows the results made with two different PIS arrangements. It was shown on two different specimens that the PIS provides the same illumination as the classical methods. It was also proved that the PIS can simulate the rotating aperture in the condenser front focal plane and thus to give the information about the three-dimensionality of the specimen. Some new static and dynamic illuminating methods were introduced.
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Control of an interference-microscope optical stage based on the image phase
Kvasnica, Lukáš ; Číp, Ondřej (referee) ; Chmelík, Radim (advisor)
Digital holographic microscopy is an interferometric imaging technique, the principle of which is the off-axis image plane holography. The principle of this technique enables to reconstruct both the image intensity and the image phase from the output interferencesignal. The reconstruction can be carried out on the basis of a single image plane hologram. This leads to the possibility of a realtime image reconstruction. The speed of the reconstruction depends on the detection and the computing process. The aim of this diploma thesis is to develop user software for the control of the detection camera and for the image plane hologram reconstruction. The effort was to achieve the highest number of image reconstructions per time unit, with the maximum utilization of the data transfer between the camera and the computer.The next aim of this thesis is the stabilization of the optical table position. The method of stabilization is based on the image phase information, which is used for the control loop feedback between reconstructed image phase and the piezoelectric actuator placed inside of the optical table. Experimental results, which prove the functionality of the stabilization, are presented.
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Coherence-controlled holographic microscope in cell's life cycle research
Bartoníček, Jan ; Chmelík, Radim (referee) ; Uhlířová, Hana (advisor)
The subject of the bachelor thesis is live-cell imaging in a transmitted-light holographic microscope which was designed at the Institute of Physical Engineering BUT and comparing this imaging method with the phase-contrast microscopy. The first part is dedicated to a basic description of used imaging techniques and a cell biology. A description of an experiment preparation follows. In the part dedicated to a data analysis the method of dynamic phase differences is described and the method of growth monitoring is proposed. Both methods were used for the analysis of experiments which are described in the last part of this work. Experiments were focused on acquiring time-lapse data of a cell’s cycle and particularly the mitosis.
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Fresnel Incoherent Correlation Holography (FINCH)
Bouchal, Petr ; Slabý, Tomáš (referee) ; Chmelík, Radim (advisor)
In the Bachelor's thesis, the recently proposed method known as Fresnel Incoherent Correlation Holography (FINCH) is examined both theoretically and experimentally. Its main advantage consists in a possibility to realize holographic reconstruction of 3D objects illuminated by incoherent light. In FINCH, the object recording is performed applying methods of optical holography and digital diffractive optics. The object reconstruction is realized numerically and utilizes principles of digital holography. In experiments, the modern optoelectronic devices known as Spatial Light Modulators are effectively used. The Bachelor's thesis includes a short review including description of the basic principles of FINCH but its own contribution consists in the mathematical description of the method and creation of the numerical simulation model in Matlab. The main result of the thesis is design and realization of experiments enabling verification of the method. In the Bachelor's thesis, results of two independent experiments realized with different types of Spatial Light Modulators HOLOEYE and HAMAMATSU are presented. An agreement of experimental results with theoretical predictions is very good. A short discussion of the obtained results, further research topics and FINCH applications is also included in the Bachelors's Thesis.
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Numerical refocusing in a digital holographic microscope with a partially coherent illumination
Slabá, Michala ; Komrska, Jiří (referee) ; Chmelík, Radim (advisor)
The bachelor thesis deals with issue of numerical refocusing in a holographic microscope with partially space coherent light. The numerical refocusing is a computation of the complex amplitude of an image wave in planes differing from the image plane. The calculation of the region where the numerical refocusing is usable is based on application of Rayleigh-Sommerfeld diffraction integral and the Fresnel approximation of a spherical wave. The description of coherence state and propagation of partially coherent light follows from statistical methods in optical coherence theory. In this thesis the thickness of field is calculated where the numerical refocusing is usable. The thickness depends on microscope parameters - the size of the light source and parameters of lenses in microscope. The result is applied to the microscope in the laboratory IPE FME BUT.
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Synthetic Holography
Tvarog, Drahoslav ; Chmelík, Radim (referee) ; Kotačka, Libor (advisor)
The thesis presents method of a creation of computer-generated holograms. The first part is devoted to an introduction of theoretical principles of a generation of synthetic holograms. The second part of the discourse deals with several practical methods of synthetic holograms creation. We present a computer code computing a discrete Fourier transform of an input image. The output of the code is an image matrix binarized in a specifically given way and printed on a transparent slide. When reconstructing the synthetic hologram we initially illuminate the slide with CGH by a plane wave and a reconstructed pattern is magnified through a camera objective to obtain a pattern observable by naked eyes. Finally, we analyze the quality of reconstructed pictures obtained from the synthetic holograms.
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