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An appropriate method for assessing hydrogel pore sizes by cryo-sem
Adámková, Kateřina ; Trudicová, M. ; Hrubanová, Kamila ; Sedláček, P. ; Krzyžánek, Vladislav
The aim of our work was to examine and describe ultrastructure of the agarose hydrogel and any possible structural concentration dependencies, and to assess the distribution and size of pores of agarose hydrogel in dependence on its concentration. Four concentrations were prepared (0.5 %, 1.0 %, 2.0 % and 4.0 % of dry weight content) and cryo-SEM and turbidimetry methods were executed on wet (original) samples in order to image the ultrastructure and measure the pore sizes within. \nReasonable results were obtained for the wet samples as they were closer to their native state they are usually used for applications in. Cryo-SEM and turbidimetry provided comparable results of pore diameters and allowed to compare pore diameters dependant on the concentrations, moreover, it showed more detailed and realistic structure.
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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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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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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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Beam damage of embedding media sections and their investigations by SEM
Krzyžánek, Vladislav ; Novotná, V. ; Hrubanová, Kamila ; Nebesářová, J.
A scanning transmission electron microscope (STEM) is useful device combining features of scanning and transmission electron microscopes. The sample in form of the ultrathin section is scanned by the electron probe and the transmitted electrons are detected. Except the dedicated STEMs this mode can exist as options in both TEM and SEM. The STEM based on the SEM equipped by a transmission detector was used for presented experiments. Nowadays, such low voltage STEM is more often used, and in many cases replaces the typical TEM. Here, we report investigations of embedding media that are typically used for TEM preparation of biological samples. The STEM detector in SEM may be able to detect both bright-field and dark-fields images. It uses much lower acceleration voltages (30 kV and below) than conventional TEM or STEM. However, materials like biological samples, polymers including embedding media are electron beam sensitive. Two the most important beam damages are the mass loss and the contamination. Both types of damages depend on the used electron energy and the electron dose applied to the sample. The mass loss depends on the sample composition, and the contamination results from the poor vacuum in the specimen chamber of the SEM, cleanness of the sample surface, etc.
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Comparison of freeze fracture images of mixed bacterial/yeast biofilm in cryo-SEM with high pressure freezing fixation
Hrubanová, Kamila ; Nebesářová, Jana ; Růžička, F. ; Krzyžánek, Vladislav
Microscopic organisms include bacteria and yeasts have been studied in this project. Besides the planktonic way of living, microbes are able to adhere to surfaces or interfaces and to form organized communities, a so-called biofilm, which are embedded in a matrix of extracellular polymeric substances that they produce; visualization and quantification of this microscopic formation is the main goal of this study. In medicine the biofilm formation allows microorganisms to colonize the surface of implants and it also protects the microbial cells from attacks by the immunity system as well as from the effect of antibiotics. Therefore, the biofilm is considered to be important virulence factor in these microorganisms. The characteristic features of the biofilm infections, especially high resistance to antifungal agents, complicate therapy. Understanding of the biofilm structure can contribute to understanding the biofilmformation and basic biochemical mechanisms underlying this process. It may help to develop more efficient treatment strategy for biofilm infection.
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