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Complementary analysis of procaryotic cells by electron microscopy and Raman spectroscopy
Ikrényiová, Terézia ; Sedláček, Petr (oponent) ; Hrubanová, Kamila (vedoucí práce)
This master thesis deals with conventional methods of bacterial cell analysis, polyhydroxyalkanoates, Raman spectroscopy and electron microscopy in the theoretical part. The production of polyhydroxybutyrate by selected thermophilic bacteria and their analysis by gas chromatography, cryogenic scanning electron microscopy and Raman spectroscopy is described in the experimental part. The chosen sample was analyzed by a transmission electron microscope. Comparing the results from previous mentioned methods it was found that the bacteria Schlegelella thermodepolymerans accumulated the highest amount of PHB. The lowest amount of PHB was obtained by bacteria Rubrobacter xylanophilus. The assumption that the PHB granules formed so-called needle-like plastic deformations during freeze-fracturing was affirmed by cryo-SEM photos analysis. Moreover, it was found that the bacterial cell characterization deduced from microscopic observation of samples corresponded to the description in the literature. TEM provided better resolution photos and in consequence the cells and PHB are more visible. The thesis is also focused on chemical fingerprint analysis of cells by Raman spectroscopy. Several biomolecules were identified by measured Raman spectra for the particular samples.
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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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Charakterizace mechanických vlastností semi-IPN hydrogelů na bázi poly(vinylalkoholu)
Přibyl, Jiří ; Pekař, Miloslav (oponent) ; Kalina, Michal (vedoucí práce)
Tato diplomová práce se zabývá problematikou mechanických vlastností semi-IPN hydrogelů na bázi poly(vinylalkoholu). Připravené poly(vinylalkohol) hydrogely byly modifikovány přídavkem alginátu sodného, dextranu, DEAE-dextranu, chitosanu a poly(ethylenglykol). Následně byly mechanické vlastnosti připravených hydrogelů studovány za využití oscilačních amplitudových reologických test, dynamické mechanické analýzy, bobtnacích experimentů a sušících charakteristik. Mikrokalorimetrie byla využita pro posouzení interakcí mezi poly(vinylalkoholem) a biopolymery a v neposlední řadě byla morfologie připravených hydrogelů vizualizována za pomocí kryo-SEM. Hlavní předpoklad pro semi-IPN hydrogely je fakt, že dochází k minimálnímu ovlivnění mechanických vlastností, ale vede k modifikaci vazebných míst obsažených ve struktuře hydrogelu, což má velký potenciál při transportních vlastnostech. Z experimentálních výsledků byly stanoveny vlivy jednotlivých biopolymerů na mechanické vlastnosti. Ze získaných výsledků bylo patrné, že přídavky biopolymerů nemají významný vliv na mechanické vlastnosti poly(vinylakoholových) hydrogelů, avšak v případě bobtnacích experimentů dochází k významnému vlivu počtu cyklů mražení/tání, iontové síly prostředí a použitého biopolymeru
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Complementary analysis of procaryotic cells by electron microscopy and Raman spectroscopy
Ikrényiová, Terézia ; Sedláček, Petr (oponent) ; Hrubanová, Kamila (vedoucí práce)
This master thesis deals with conventional methods of bacterial cell analysis, polyhydroxyalkanoates, Raman spectroscopy and electron microscopy in the theoretical part. The production of polyhydroxybutyrate by selected thermophilic bacteria and their analysis by gas chromatography, cryogenic scanning electron microscopy and Raman spectroscopy is described in the experimental part. The chosen sample was analyzed by a transmission electron microscope. Comparing the results from previous mentioned methods it was found that the bacteria Schlegelella thermodepolymerans accumulated the highest amount of PHB. The lowest amount of PHB was obtained by bacteria Rubrobacter xylanophilus. The assumption that the PHB granules formed so-called needle-like plastic deformations during freeze-fracturing was affirmed by cryo-SEM photos analysis. Moreover, it was found that the bacterial cell characterization deduced from microscopic observation of samples corresponded to the description in the literature. TEM provided better resolution photos and in consequence the cells and PHB are more visible. The thesis is also focused on chemical fingerprint analysis of cells by Raman spectroscopy. Several biomolecules were identified by measured Raman spectra for the particular samples.
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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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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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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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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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