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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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Thickness determination of a cathodoluminescence active nanoparticles by means of Quantitative STEM imaging
Skoupý, Radim ; Krzyžánek, Vladislav
Labeling of specimens by nanoscale probes is common approach of complex biological\nsystems exploration. Namely gold nanoparticles immuno-staining is well established method\nin electron microscopy. However, if more than two label sizes are used, the differentiation of\nindividual nanoparticles becomes difficult.\nThis can be overcome by cathodoluminescence (CL) active particles – nanophosphors where\nlabels recognition is done by wavelength of emitted light. This gives a great opportunity to\nuse advanced multi probe labeling within one sample.\nThere is a huge variety of nanophosphors: green fluorescent protein, quantum dots, ZnO\nnanoparticles, organic molecules, rare earth-doped nanophosphors etc. Therefore, in order\nto choose best type of nanophosphors for a given task, it is important to measure particles\nsize/thickness, as the CL intensity is proportional to the probe volume.
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Structure investigation of hydrogels using a cryo-SEM
Adámková, Kateřina ; Hrubanová, Kamila ; Samek, Ota ; Trudičová, M. ; Sedláček, P. ; Krzyžánek, Vladislav
Hydrogels can be characterized as elastic hydrophilic polymer chains connected in network\nwhich are able to swell notably when exposed to aqueous media by absorbing considerable\namounts of water. Besides being a constituent of living organisms, nowadays, there are\nvarious fields hydrated polymers (e.g. polyvinyl alcohol, collagen, and starch) can be utilized\n– in both biological and non-biological form. Classic examples of such applications are\nhuman health and cosmetics (contact lenses, wound healing dressings and artificial\nreplacement tissues – skin, arterial grafts, cornea and spinal disc replacement), pharmacy\n(drug delivery systems), bioengineering, food industry, agriculture etc. Also, hydrogels\ncan reversibly change their shape when being exposed to a temperature change.
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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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Principal component analysis of Raman spectroscopy data for determination of biofilm forming bacteria and yeasts
Šiler, Martin ; Samek, Ota ; Bernatová, Silvie ; Mlynariková, K. ; Ježek, Jan ; Šerý, Mojmír ; Krzyžánek, Vladislav ; Hrubanová, Kamila ; Holá, M. ; Růžička, F. ; Zemánek, Pavel
Many microorganisms (e.g., bacteria, yeast, and algae) are known to form a multi-layered structure composed of cells and extracellular matrix on various types of surfaces. Such a formation is known as the biofilm. Special attention is now paid to bacterial biofilms that are formed on the surface of medical implants, surgical fixations, and artificial tissue/vascular\nreplacements. Cells contained within such a biofilm are well protected against antibiotics and phagocytosis and, thus, effectively resist antimicrobial attack.\nA method for in vitro identification of individual bacterial cells as well as yeast colonies is presented. Figure 1 shows an an example of the biofilm formed by Staphylococcus epidermidis bacteria and Candida parapsilosis yeasts known for forming biofilms. The\npresented method is based on analysis of spectral “Raman fingerprints” obtained from the single cell or whole colony, see figure 2(top). Here, Raman spectra might be taken from the biofilm-forming cells without the influence of an extracellular matrix or directly form the bacterial/yeast colony.
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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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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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