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Characterization of scanning transmission electron microscopy images of thin biological sections
Novotná, Veronika ; Nebesářová,, Jana (oponent) ; Krzyžánek, Vladislav (vedoucí práce)
This master´s thesis describes physical principles of the TEM, the SEM and the STEM and their suitability for observation of electron-sensitive samples (embedding media, biological specimens). The interaction between the incident electron beam and the sample is also discussed, as well as the sample preparation for the STEM (TEM). Furthermore, a description of microscopic image processing is included with a section about quantitative comparison of the STEM images. The thesis is focused on the mass loss of pure embedding media (Epon, Spurr, LR White) caused by the electron beam in the low voltage STEM. The samples of different thicknesses were investigated using different microscope settings (acceleration voltage, total dose, probe current, cleaning of the sample surface and specimen chamber) and the STEM imaging modes (bright-field, dark-field). Furthermore, biological samples of Euglena gracilis embedded in Epon and Spurr are examined. Collected images, created algorithms and obtained results are widely discussed.
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Scanning Electron Microscopy and its Applications for Sensitive Samples
Hrubanová, Kamila ; Nováček, Jiří (oponent) ; Schröfel,, Adam (oponent) ; Krzyžánek, Vladislav (vedoucí práce)
The presented dissertation thesis titled “Scanning electron microscopy and its applications for sensitive samples” describes an instrumental and methodological development in the field of scanning electron microscopy leading to an innovative solution that could be particularly applicable in microbiological research. A summary of the history and current state of electron microscopy (EM) as a scientific imaging and analytical technique is provided in the introductory chapters. The undeniable contribution of EM in the biological and medical sciences is evidenced by many cited scientific publications. This dissertation thesis contains innovations and improvements in specimen preparation and cryogenic scanning electron microscopy (cryo-SEM) produced at the Institute of Scientific Instruments of the CAS in Brno. In particular, the new constructions of special sample holders together with methodological development in the field of microbiological sample preparation resulted in finding optimal parameters for individual processes. In the experimental part there is showed a verification of methodological procedures in the study of hydrated and electron beam sensitive specimens. Subsequent comparison of different methodological approaches on a defined microbiological system contributes to extending the interpretation of the hitherto known results. Among the microbiological strains investigated were the biofilm positive bacteria Staphylococcus epidermidis and yeasts such as Candida albicans and Candida parapsilosis, which are considered to be clinically significant because they are often involved in serious infections and especially threaten immunocompromised patients. Furthermore, the effect of the biofilm formation of Bacillus subtilis on the biodeterioration and biodegradation of poly--caprolactone films was studied. The new development in low temperature cryo-SEM was employed in the research of microbes with biotechnological potential such as Cupriavidus necator and Sporobolomyces shibatanus.
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Quantitative Imaging in Scanning Electron Microscope
Skoupý, Radim ; Buršík, Jiří (oponent) ; Shimoni, Eyal (oponent) ; Krzyžánek, Vladislav (vedoucí práce)
This thesis deals with the possibilities of quantitative imaging in scanning (transmission) electron microscope (S|T|EM) together with its correlative applications. It starts with quantitative STEM (qSTEM) method description, where estimated local sample thickness can be related to irradiated dose and create a mass-loss study, which was applied on samples of ultrathin epoxy resin sections at variate conditions (age, temperature, staining, plasma cleaning, carbon covering, probe current). The possibilities of the detector calibration process, the necessary background of the Monte Carlo simulations of electron scattering and achievable accuracy of the method are discussed and demonstrated. The method is then extrapolated for the use of back-scattered electron (BSE) detector, where new detector calibration technique, based on primary beam deflection on electron mirror, was postulated, developed and tested on various thin coating layers with thicknesses in range from 1 to 25 nm. The use of BSE detector brings the opportunity to measure the thickness of not only the electron transparent samples as in case of qSTEM, but also thin layers on substrates – qBSE. Both above-mentioned methods (qSTEM and qBSE) are intensity-based. This brings complication in the need of proper calibration, where just a slight drift of base-signal level causes a significant change of the results. This insufficiency was overcome in case of qSTEM by using the most probable scattering angle (captured by pixelated STEM detector) instead of an integral image intensity captured by an annular segment of STEM detector. The advantage of this method is its applicability post-acquisition, where no special previous actions are needed before each imaging session. The disadvantage is the limited range of detectable thicknesses given by the peak creation in signal/scattering-angle dependency. In general, low thickness region is immeasurable as well as those too thick (usable thickness range for latex is 185 - 1,000 nm; given by detection geometry and pixel size). Moreover, multiple applications of conventional and commercially available quantitative techniques of cathodoluminescence (CL) and energy-dispersive X-ray spectroscopy (EDX) are presented in correlation with high-resolution images taken in secondary and transmitted electrons.
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Monte Carlo simulace elektronového rozptylu v rastrovacím prozařovacím elektronovém mikroskopu
Záchej, Samuel ; Hrubanová, Kamila (oponent) ; Krzyžánek, Vladislav (vedoucí práce)
Diplomová práca popisuje elektrónový rozptyl v STEM systémoch na objektoch rôzne-ho tvaru, akými sú hranol, guľa alebo dutá kapsula. Na kvantifikáciu viacnásobného rozptylu elektrónov v materiáloch sú využité Monte Carlo simulácie. Okrem teoretického rozboru elektrónového rozptylu a metodiky simulácií, obsahuje práca aj návrh a realizáciu algoritmu pre simulácie na zadaných objektoch. Práca zahŕňa overenie robustnosti simulácií na základe porovnania výsledkov so známymi signálmi pre daný objekt. Funkčnosť algoritmu bola overená experimentálnym meraním elektrónového rozptylu na vrstve uhlíka.
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Komplexní strukturní charakterizace semi-interpenetrovaných biopolymerních hydrogelů
Trudičová, Monika ; Krzyžánek, Vladislav (oponent) ; Sedláček, Petr (vedoucí práce)
Náplní této diplomové práce bylo otestovat využitelnost dostupných technik strukturní analýzy na vhodném modelovém hydrogelovém systému. Hlavním cílem bylo zhodnotit výhody a nevýhody vybraných technik strukturní analýzy a vliv změny kompozice daného hydrogelového systému na jeho vnitřní strukturu. Jako modelový systém byly zvoleny semi-interpenetrované hydrogely na bázi agarózy o různé koncentraci, tento typ materiálu byl vybrán pro jednoduchou a opakovatelnou přípravu a taktéž pro jeho aplikační potenciál. Technikami strukturní analýzy byly zvoleny elektronová mikroskopie (SEM, kryoSEM), rtuťová porozimetrie a turbidimetrie. Získané informace poslouží k prohloubení znalostí o vlivu kompozice hydrogelu na jeho strukturu a porovnání vybraných technik poslouží pro volbu vhodných strukturních analýz v budoucnu, které budou aplikovatelné i na jiné hydrogelové systémy.
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Novel embedding resins for 3D-SEM II
Šlouf, Miroslav ; Pavlova, Ewa ; Strachota, Beata ; Strachota, Adam ; Nebesářová, Jana ; Týč, Jiří ; Krzyžánek, Vladislav ; Skoupý, Radim
Research report for Thermo Fisher Scientific s. r. o., based on common project awarded by TA CR (program: National center of competence, TN01000008).
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