This master’s thesis deals with the design of methodology for measurement of refractive index and thickness of living cells by coherence-controlled holographic microscope. The theoretical part summarises the holographic microscopy and its development at IPE FME BUT in Brno. The thesis focuses on the multimodal holographic microscope, its description, the principle, the procedure of work and data processing. Confocal microscopy is also described, which serves to compare the acquired values with the proposed methodology. The last part of the theoretical part deals with the testing of statistical hypotheses, which is needed for the processing of measured data. Experiments were designed for the verification of methodology for determination of the refractive index and cell thickness. The experimental part of the thesis deals with the sample preparation and measurement. The procedure and results of the proposed experiments and their evaluation follows.

Diplomová práca sa zaoberá rozptylom elektromagnetického žiarenia na biologickej bunke použitím metódy konečných diferencií v časovej oblasti (FDTD), Bornovej aproximácie a Rytovovej aproximácie. Metóda FDTD dáva presné výsledky v širokej škále problémov. Je spravené porovnanie Bornovej aproximácie a Rytovovej aproximácie prostredníctvom FDTD metódy. Ďaľšia časť práce zahrnuje krátky popis koherenciou riadeného holografického mikroskopu CCHM. Záverečná časť sa venuje zobrazeniu rozptýleného poľa získaného z jednotlivých simulácií pomocou simulácie objektového ramena mikroskopu CCHM.

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.

The Coherence Controlled Holographic Microscope (CCHM) was developed at BUT Brno for a quantitative phase imaging of living cells. Nowadays it ocurres that its imaging properties are enhanced by the use of additional modules. In the present the microscope is equipped with the epifluorescence module, which allows observation of fluorescently marked living cells. This thesis is going to follow up on the development of this module and is going to extend its options by confocal imaging. The disadvantage of current multi-channel confocal microscopes is a mechanical rotation of the Nipkow discs, which causes undesired mechanical vibrations. That is why in this thesis it is replaced by Digital Micromirror Device. With its use was developed optical system of the whole confocal model, whose correct funcion was simulated in optical CAD. The experimentally verified prototype serves to test the imaging properties. On this basis is designed an application idea of the fluorescence confocal module, which will be possible to connect to the CCHM microscope.

This diploma thesis describes the design of the illumination system for a Coherence--Controlled Holographic Microscope (CCHM). The theoretical part mentions the history of microscopy, the principle of holography and individual types of interference microscopy. To get closer to the topic, individual light sources and an overview of current illumination systems are mentioned. The diploma thesis also describes the procedure of designing a fiber-optic illumination module for microscopy from optical design through design of construction to the last step, which is assembly and testing of the module.

The goal of this diploma thesis was using of a coherence-controlled holographic microscope in cell’s life research. A brief history of interference microscopy and it’s applications in biology is described. Also other microscopy techniques routinely used for transparent objects imaging are mentioned and the biology of cell’s life cycle briefly explained. Characteristics describing the shape of a cell were proposed and tested with respect to identification of particular phases of its life cycle. The method of dynamic phase differences was modified in order to distinguish the internal motion of cell’s mass from the movement of the whole cell. Selected characteristics were used to evaluate observations carried out with the holographic microscope and the possibilities of their further applications were depicted. In conclusion, obtained findings were summarized and modifications of microscope construction as well as data-processing software were suggested.

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.

Studium chování opticky zachycených částic nám umožňuje porozumět základním fyzikálním jevům plynoucím z interakce světla a hmoty. Předkládaná práce podává vysvětlení zesílení tažné síly působící na opticky svázané částice ve strukturovaném světelném poli, tzv. tažném svazku. Ukazujeme, že pohyb dvou opticky svázaných objektů v tažném svazku je silně závislý na jejich vzájemné vzdálenosti a prostorové orientaci, což rozšiřuje možnosti manipulace hmoty pomocí světla. Následně se práce zaměřuje na levitaci opticky zachycených částic ve vakuu. Představujeme novou metodologii na charakterizaci vlastností slabě nelinearního Duffingova oscilátoru reprezentovaného opticky levitující částicí. Metoda je založena na průměrování trajektorií s určitou počáteční pozicí ve fázovém prostoru sestávajícím z polohy a rychlosti částice a poskytuje informaci o parametrech oscilátoru přímo ze zaznamenaného pohybu. Náš inovativní postup je srovnán s běžně užívanou metodou založenou na analýze spektrální hustoty polohy částice a za využití numerických simulací ukazujeme její použitelnost i v nízkých tlacích, kde nelinearita hraje významnou roli.

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.

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.