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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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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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Freeze-fracture technique and artefacts caused by processing conditions
Vaškovicová, Naděžda ; Valigurová, A. ; Hodová, I. ; Melicherová, J. ; Krzyžánek, Vladislav
Freeze-fracture technique is a method used to visualise membrane surfaces of cell organelles. This method is based on cryo-fixation that stabilizes samples. The sample is rapidly frozen in nitrogen, and cut in the chamber under a vacuum and low temperature. Glycerol is used as a cryoprotectant preserving the fine structure of cells in their native stage. Although, cryoprotectants serve as a substitute for water and protect against ice crystal production, they could also affect the form of fracture through biological membranes. Figure 1 shows structures in a sample frozen in the presence of 25% glycerol. The temperature of the apparatus was not low enough during the process of fracturing and etching the sample. The structure of cells seems to be deformed due to melting glycerol. In contrast, figure 2 shows a replica with fine structure of frozen and proper good form of fracturing. The cells used for this study were human leukemic cells (HL-60). Another artefact is shown in figure 3A, compare with 3B. Each sample has to be fractured with a specific speed of cut. The force used for fracturing the membranes has to be set to optimal conditions, which depend on a hardness of sample and a coherence of drops. Low speed and unstable coherence of drops resulted in sample fragmentation. High speed of cut could cause cross-section of cellular structures, similar to ultrathin sections. Figure 3A shows fragmentation of nuclear membrane. This sample was not fractured, it was fragmented due to unstable coherence of drop. This overview shows how a combination of different conditions including the physical properties of the sample, cryoprotectants used and temperature could affect the form of fractures and hence significantly affect interpretation of morphological structures.
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A cryo high-vacuum shuttle for correlative cryogenic investigations
Tacke, S. ; Krzyžánek, Vladislav ; Reichelt, R. ; Klingauf, J.
The preservation of the native state is the key element in sample preparation. In the case of hydrated objects, embedding in vitreous (amorphous) ice and subsequent examination under cryogenic (cryo) conditions are the means of choice. Over the last years, cryogenic techniques such as cryo-electron microscopy (cryo-EM) or soft X-ray cryo-microscopy have become increasingly popular, as they provide a direct, unaltered view on the specimen. However, to provide a snapshot of the pristine architecture of the specimen, cryo techniques require constant cooling below the recrystallization temperature of 138°K and avoidance of any contamination. This has been proven to be particularly challenging in the case of correlative cryo investigations, since these methods include several transfer steps due to their extensive post-processing and complex workflow. In the past, several transfer concepts were introduced and they are now commercially available. However, these systems are limited either by not offering a high-vacuum environment or constraining the applications to a restricted workflow.
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Main Activites of the Institute of Scientific Instruments
Müllerová, Ilona ; Radlička, Tomáš ; Mika, Filip ; Krzyžánek, Vladislav ; Neděla, Vilém ; Sobota, Jaroslav ; Zobač, Martin ; Kolařík, Vladimír ; Starčuk jr., Zenon ; Srnka, Aleš ; Jurák, Pavel ; Zemánek, Pavel ; Číp, Ondřej ; Lazar, Josef ; Mrňa, Libor
Institute of Scientific Instruments (ISI) was established in 1957 to develop diverse instrumental equipment for other institutes of the Academy of Sciences. ISI has long experience in research and development of electron microscopes, nuclear magnetic resonance equipment, coherent optics and related techniques. Nowadays the effort concentrates on scientific research in the field of methodology of physical properties of matter, in particular in the field of electron optics, electron microscopy and spectroscopy, microscopy for biomedicine, environmental electron microscopy, thin layers, electron and laser beam welding, electron beam lithography using Gaussian and shaped electron beam, nuclear magnetic resonance and spectroscopy, cryogenics and superconductivity, measurement and processing of biosignals in medicine, non-invasive cardiology, applications of focused laser beam (optical tweezers, long-range optical delivery of micro- and nano-objects) and lasers for measurement and metrology. ISI works both independently and in cooperation with universities, other research and professional institutions and with private companies at national and international level.
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