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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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The study of the different percentage performance of nanoparticles on the properties of epoxy resin
Hudec, Jiří ; Neděla, Vilém ; Polsterová, H.
This paper deals with the study of impact of different percentage filling of nanoparticles on the electrical properties of epoxy resin, which has very good mechanical and electrical properties. The sample is the blended mixture which is evacuated, subjected to ultrasound and then cured. It is expected that the formation of lumps should be minimised due to the influence of microwaves. Nanoparticles should be equally distributed in epoxide volume for this case. Unfortunately, this assumption was not proven. The mixture contains an epoxy resin CY228, hardener HY918, softener DY045 and accelerator DY062. Nanoparticles of alumina (Al2O3), sulfur dioxide (SiO2), titanium dioxide (TiO2) and tungsten oxide (WO3) from Sigma Aldrich Company were used as a filler. There were made samples for each filler with 0.25, 0.5, 1, 2 weight percent for our experiment and were determined values of the dissipation factor tg.delta., permittivity .epsilon.r and resistivity .rho.v by measuring.
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Imaging of carbon nanostructures by low energy STEM below 5 keV
Pokorná, Zuzana ; Knápek, Alexandr ; Jašek, O. ; Prášek, J. ; Majzlíková, P.
Our work deals with the imaging of nanostructures composed of light biogenic elements, such as carbon nanotubes, by low energy scanning transmission electron microscopy (STEM). Compared to imaging at the voltages commonly used for TEM and STEM, low energy electrons seem very promising in terms of specimen damage that is caused by a number of elastic andn inelastic collisions. In carbonaceous materials, the most problematic is probably the knock-on damage, where the structure can be impaired by carbon atom displacement. To avoid this problem with structures composed of light elements, a reduction in beam voltage going down to 5 keV has recently been proposed. The range below 5 keV has not been explored yet for this purpose, although electron scattering in matter is lower for these energies, which allows achieving a higher spatial resolution. We aim to demonstrate that additional reduction of incident electron energy may yield interesting contrast features.
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Using environmental scanning electron microscopy (ESEM) as a non-invasive method to studying fixed parasites
Mašová, Šárka ; Neděla, Vilém ; Tihlaříková, Eva
Scanning electron microscopy (SEM) is popular and for taxonomy of parasites very important and not substituted method in many times. However sometimes taxonomists have only one specimen and cannot use classical SEM, because their sample (poor conductor) have to be fixed, dehydrated and coated before it can be observed. This method condemns samples for destroying and do not allow other using of it, e.g. for molecular study or depositing as type material in museum. Moreover, the specimen preparation is often long and slow. Environmental scanning electron microscopy (ESEM) brings two main advantages: elimination of speed of sample preparation and non-invasivity.
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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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The Study of Ice Impurities Using the Environmental Scanning Electron Microscopy at Higher Pressures and Temperatures
Neděla, Vilém ; Runštuk, Jiří ; Klán, P. ; Heger, D.
Natural ice and snow accumulate and concentrate significant amounts of impurities that can be stored or chemically transformed, and eventually released to the environment. The location of impurities and their interactions with the water molecules of ice have not yet been sufficiently clarified. The aim of this work is to observe an uranyl-salt brine layer on the ice surface using a back scattered electron detection and the ice surface morphology using a secondary electron detection under equilibrium conditions in a specimen chamber of environmental scanning electron microscope (ESEM). Our specially modified ESEM AQUASEM II equipped with the YAG:Ce3+ backscattered electron detector, an ionization detector of secondary electrons, a special hydration system and a Peltier cooled stage was used. The pressures between 400-700 Pa, 50% water-vapor saturation, and the temperatures above 250 K were utilized in our experiments. At these conditions, the phenomena of etching and subsequent stripping of impurities are largely suppressed.
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Characterization of .beta.-phase in Al-Mg-Si alloys by SLEEM and STLEEM techniques
Ligas, A. ; Hida, S. ; Matsuda, K. ; Mikmeková, Šárka
Knowledge of the distribution and morphology of the Mg2Si precipitates (i.e. .beta.-phase) in Al-Mg-Si alloys are very important for many practical reasons and the scanning electron microscopy (SEM) technique is widely used for their visualization. Unfortunately, in the standard SEM images these precipitates are barely visible and finding them can be very difficult. Using the cathode lens (CL) mode in the SEM (so called SLEEM) these difficulties have been overcome and a very high contrast between the hexagonal-shaped .beta.-phase and the matrix has been obtained. Moreover, it has been found that the SLEEM images offer the possibility to distinguish between the hexagonal-shaped and the conventional .beta.-phase based on their different brightness, not only on their shape, which can be in some cases difficult or even impossible. Mg2Si precipitates have been also characterized by means of the scanning transmission low energy electron microscopy (STLEEM) method based on the using of a STEM detector in the SEM operated in the CL mode.
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Microstructural characterization of metallic materials using advanced SEM techniques
Piňos, Jakub ; Konvalina, Ivo ; Kasl, J. ; Jandová, D. ; Mikmeková, Šárka
The development of advanced materials is inseparably connected with detailed knowledge of the relationship between microstructure and mechanical properties. Traditional high-voltage scanning electron microscopy (SEM) is one of the most commonly used techniques for microstructure analysis, though it may be insufficient particularly for the characterization of advanced materials exhibiting a complex microstructure. The benefits of using slow electrons have been described in several articles. Experiments have been performed with a XHR SEM Magellan 400L (FEI Company) equipped with two detectors for secondary electrons (SE), an Everhart Thornley detector and an in-lens TLD detector, and solid-state BSE detector (CBS) located below the pole piece. This microscope can also be operated in the beam deceleration (BD) mode. The field of the BD not only decelerates the primary electrons, but also accelerates the emitted (signal) electrons towards the detector. Furthermore, high-angle backscattered electrons (BSE) are also collimated towards the optical axis and are detected. These electrons carry, first and foremost, crystal orientation contrast. SE and low-angle BSE can be detected by the TLD detector located inside the objective lens. Angle-resolved detection of BSE is performed using a CBS detector divided into four concentric segments.
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