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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 (referee) ; Schröfel,, Adam (referee) ; Krzyžánek, Vladislav (advisor)
Předložená dizertační práce s názvem “Rastrovací elektronová mikroskopie a její aplikace pro senzitivní vzorky” pojednává o problematice rastrovací elektronové mikroskopie v kontextu instrumentálního a metodologického vývoje vedoucího k inovativnímu řešení, které je dobře aplikovatelné zejména v mikrobiologickém výzkumu. Součástí práce je rozprava o historii a současném stavu elektronové mikroskopie (EM) jakožto vědecké zobrazovací a analytické techniky, tato část se nachází v úvodních kapitolách. Nepopiratelný přínos EM v biologických a lékařských oborech je dokazován mnoha citovanými vědeckými publikacemi. Předložená dizertační práce přináší novinky z oblasti přípravy preparátů a kryogenní rastrovací elektronové mikroskopie (cryo-SEM) vyvinuté na pracovišti Ústavu přístrojové techniky AV ČR, v.v.i. v Brně. Jedná se především o návrhy a výrobu speciálních držáků vzorků a vývoj nových metodik v oblasti přípravy mikrobiologických preparátů vedoucích k nalezení optimálních parametrů jednotlivých procesů. V experimentální části se nachází ověření metodologických postupů při studiu hydratovaných a na elektronový svazek senzitivních preparátů. Následné srovnání různých přístupů na definovaném biologickém systému z oblasti mikrobiologie přispívá k rozšíření interpretace doposud známých výsledků. Mezi zkoumanými mikrobiologickými kmeny byly biofilm-pozitivní bakterie Staphylococcus epidermidis a kvasinky jako Candida albicans a Candida parapsilosis, jež jsou považovány za klinicky významné, protože se podílejí na vzniku závažných infekcí zejména u imunokompromitovaných pacientů. Dále byl studován vliv růstu biofilmu bakterie Bacillus subtilis na biodeteriorizaci a biodegradaci poly--kaprolaktonových fólií. Vývoj v oblasti cryo-SEM byl aplikován ve výzkumu mikrobů s biotechnologickým potenciálem, jako jsou např. Cupriavidus necator a 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 simulations of electron scattering in scanning transmission electron microscopy
Záchej, Samuel ; Hrubanová, Kamila (referee) ; Krzyžánek, Vladislav (advisor)
This thesis deals with an electron scattering in STEM microscopy on objects with dif-ferent shapes, such as cuboid, sphere and hollow capsule. Monte Carlo simulations are used for description of multiple electron scattering. Except the theoretical analysis of the electron scattering and simulation methods, the thesis contains design and realiza-tion of an algorithm simulating electron scattering in given objects. In addition, there is a design for robustness evaluation of the simulation, based on comparison between results and known signals for a given object. Reliability of the algorithm was verified by experimental measurements of the electron scattering on a carbon layer.
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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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