|
| |
|
| |
|
|
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.
|
|
|
Determination of thickness refinement using STEM detector segments
Skoupý, Radim ; Krzyžánek, Vladislav
Quantitative STEM imaging together with Monte Carlo simulations of electron scattering in solids can bring interesting results about properties of many thin samples. It is possible to determine thickness of a sample, to calculate mass of particles and measure mass per length/area. Appropriate calibration is one of the crucial parts of the method. Even a small error or inaccuracy in detector response to electron beam either blanked or full brings significant error into thickness determination. This problem can be overcome by parallel STEM imaging in more segments of the detector. Comparing more segments gives a possibility to use a signal from different segments for different thicknesses of a sample. Accuracy of individual parts of the detector depends on the captured signal quantity. It is desirable to use such a STEM detector segment that provides the greatest signal change to a unit of thickness. To demonstrate the usage, we used a sample of Latex nanospheres placed on thin carbon lacey film, diameter of the nanospheres was around 600 nm in order to compare the results from different detector segments. Thanks to the known thickness of the sample (calculated from its geometrical shape), it is possible to estimate the optimal acquisition settings and post processing steps with the known and the true state of the sample.
|
|
|
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.
|
|
|
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.
|
|
| |
|
|
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.
|
|
|
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.
|
|
|
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.
|
host ::
RSS.