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The Three-Dimensional Digital Imaging Methods for X-ray Computed Tomography and Digital Holographic Microscopy
Kvasnica, Lukáš ; Číp, Ondřej (referee) ; Štarha, Pavel (referee) ; Chmelík, Radim (advisor)
This dissertation thesis deals with the methods for processing image data in X-ray microtomography and digital holographic microscopy. The work aims to achieve significant acceleration of algorithms for tomographic reconstruction and image reconstruction in holographic microscopy by means of optimization and the use of massively parallel GPU. In the field of microtomography, the new GPU (graphic processing unit) accelerated implementations of filtered back projection and back projection filtration of derived data are presented. Another presented algorithm is the orientation normalization technique and evaluation of 3D tomographic data. In the part related to holographic microscopy, the individual steps of the complete image processing procedure are described. This part introduces the new orignal technique of phase unwrapping and correction of image phase damaged by the occurrence of optical vortices in the wrapped image phase. The implementation of the methods for the compensation of the phase deformation and for tracking of cells is then described. In conclusion, there is briefly introduced the Q-PHASE software, which is the complete bundle of all the algorithms necessary for the holographic microscope control, and holographic image processing.
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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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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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Automated Procedures for Coherence Controlled Holographic Microscope
Dostál, Zbyněk ; Štarha, Pavel (referee) ; Jákl, Petr (referee) ; Chmelík, Radim (advisor)
Coherence-Controlled Holographic Microscope (CCHM) and a Fluorescence Holographic Microscope (FHM) were developed particularly for quantitative phase imaging and measurement of live cell dynamics, which used to be a subject of digital holographic microscopy (DHM). CCHM and FHM in low-coherence mode extend capabilities of DHM in the study of living cells. However, this advantage following from the use of low coherence is accompanied by increased sensitivity of the system to its correct alignment. Therefore, the introduction of an automatic self-correcting system is inevitable. Accordingly, in the thesis, the theory of a suitable control system is derived and the design of an automated alignment system for both microscopes is proposed and experimentally proved. The holographic signal was identified as a significant variable for guiding the alignment procedures. On this basis the original basic realignment algorithms were proposed, which encompasses the processes for initial and advanced alignment as well as for long-term maintenance of the microscope aligned state. Automated procedures were implemented in both microscopes unique set of robotic mechanisms designed and built within the frame of the thesis work. All of the procedures described in the thesis were in real experimentally proved at real microscopes in the experimental biophotonics laboratory. In addition, the control software, which contains the needed automated procedures, was developed for FHM.
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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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Superresolution in holographic microscopy
Říha, René ; Komrska, Jiří (referee) ; Chmelík, Radim (advisor)
An approach that uses a two dimensional phase grating in certain distance behind a specimen to enhance the resolution in digital holographic microscopy is proposed. By theoretical considerations and with simulations the properties of new the arrangement are explored and the method of numerical reconstruction is described. The proposed approach is verified by electromagnetic simulation. Technical aspects and potential difficulties aren't involved.
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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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A method for the visualization of high phase gradients in a microscopic image
Druckmüllerová, Hana ; Martišek, Dalibor (referee) ; Chmelík, Radim (advisor)
Holografická mikroskopie je nekonvenční mikroskopická technika, vhodná zvláště pro vzorky s malou optickou hustotou, která umožňuje zviditelnit index lomu pozorovaných objektů. Na Ústavu fyzikálního inženýrství Fakulty strojního inženýrství VUT v Brně byl sestrojen unikátní transmisní digitální holografický mikroskop (TDHM). Pořízené snímky (hologramy) jsou zpracovány metodou založenou na Fourierově ransformaci, čímž je zrekonstruována intenzita a fáze světelné vlny procházející pozorovaným objektem. Fáze popisuje index lomu a tloušťku pozorovaného objektu. V místech, kde se mění index lomu nebo tloušťka, dochází i ke změně fáze. Úkolem této bakalářské práce bylo najít metodu pro zviditelnění míst s vysokým gradientem fáze. Podařilo se vytvořit metodu, která nevyžaduje navazování fáze, a proto je vhodná pro libovolné obrazy pořízené TDHM. Tato metoda byla implementována do počítačového programu Gradient3D, který kromě výpočtu gradientu ve dvou a třech rozměrech umožňuje i vytváření barevných obrazů, jejichž složkami jsou kombinace intenzity, fáze a gradientu. Program též umožňuje odstranění falešných gradientů v místech s nízkou intenzitou, kde je hodnota fáze nespolehlivá. Program byl testován na několika souborech hologramů pořízených TDHM při pozorování biologických vzorků.
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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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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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