Nejvíce citovaný článek - PubMed ID 28826005
High-efficiency detector of secondary and backscattered electrons for low-dose imaging in the ESEM
This study explores the innovative approach in the development of freeze-dried hydrogel films, leveraging the unique properties of gum Karaya (GK), poly-(vinyl alcohol) (PVA), poly-(ethylene glycol) (PEG), and glycerol with a coating of octenidine dihydrochloride (OCT). These innovative hydrogel films exhibit at a certain glycerol concentration a sandwich-like structure, achieved through a tailored freeze-drying process, which enhances transparency and mechanical stability. OCT provides superior antibacterial performance, effectively combating multidrug-resistant bacteria with a controlled and gradual release mechanism, surpassing conventional OCT solutions that require frequent reapplication for infected wound treatment without the creation of bacterial resistance. Advanced environmental scanning electron microscopy (A-ESEM) reveals the complex microstructure of the hydrogel, highlighting the dense surface layer and interconnected porous bulk. Variations in glycerol concentrations proved to significantly impact hydrogels' properties. Increasing the glycerol concentration decreases the pore size (around 4.5 μm) while enhancing the polymer network density and flexibility. However, low concentration increases the pore size (7.8-15.6 μm), impacting enhanced swelling behavior and hydrolytic stability. OCT's rapid antibacterial action, releasing over 30% within the first hour and maintaining prolonged activity for up to 2 weeks, emphasizes the material's potential for diverse applications. Hydrogels' remarkable transparency, porosity, structural stability, and antibacterial efficacy against both Gram-positive and Gram-negative strains suggest promising uses as transparent dressings, biomedical devices, and infection-resistant surfaces.
- Publikační typ
- časopisecké články MeSH
In this work, we focus on the low-temperature behavior of concentrated aqueous solutions of cesium chloride and discover two hydrates of CsCl. We employ four different methods, namely, (i) simple cooling at rates between 0.5 and 80 K s-1, (ii) simple cooling followed by pressurization, (iii) hyperquenching at 106 to 107 K s-1, and (iv) hyperquenching followed by pressurization. Depending on the method, different types of phase behaviors are observed, which encompass crystallization involving freeze-concentration, pressure-induced amorphization, full vitrification, and polyamorphic transformation. The CsCl hydrates discovered in our work cold-crystallize above 150 K upon heating after ultrafast vitrification (routes iii and iv) and show melting temperatures below the eutectic temperature of 251 K. We determine the composition of these hydrates to be CsCl·5H2O and CsCl·6H2O and find evidence for their existence in ESEM, calorimetry, and X-ray diffraction. The dominant and less metastable hydrate is the hexahydrate, where the pentahydrate appears as a minority species. We also reveal the birthplace for the CsCl hydrates, namely, the freeze-concentrated solution (FCS) formed upon cold-crystallization of the fully glassy solution (from iii and iv). The spongy FCS produced upon cooling of the liquid (from i and ii) is incapable of crystallizing CsCl-hydrates. By contrast, the FCS produced upon heating the glassy solution (from iii and iv) shows tiny, fine features that are capable of crystallizing CsCl-hydrates. Our findings contradict the current knowledge that alkali chlorides only have hydrates for the smaller cations Li+ and Na+, but not for the larger cations K+, Rb+, and Cs+ and pave the way for future determination of CsCl-hydrate crystal structures. The pathway to metastable crystalline materials outlined here might be more generally applicable and found in nature, e.g., in comets or on interstellar dust grains, when glassy aqueous solutions crystallize upon heating.
- Publikační typ
- časopisecké články MeSH
The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM utilises machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator. A modified electron microscope was used, equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. A transparent ultra-thin film of ionic liquid is used to increase thermal and electrical conductivity of the samples and to minimize sample damage by free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not observed before by any microscopic technique. The ability to resolve nano-structural details of chromosomes using A-ESEM is validated by measuring gold nanoparticles with achievable resolution in the lower nanometre units.
- MeSH
- chromozomy ultrastruktura MeSH
- kovové nanočástice chemie MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací * metody MeSH
- mitóza MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- zlato MeSH
The article describes the combination of experimental measurements with mathematical-physics analyses in flow investigation in the chambers of the scintillator detector, which is a part of the environmental scanning electron microscope. The chambers are divided with apertures by small openings that keep the desirable pressure differences between three chambers: The specimen chamber, the differentially pumped intermediate chamber, and the scintillator chamber. There are conflicting demands on these apertures. On the one hand, the diameter of the apertures must be as big as possible so that they incur minimal losses of the passing secondary electrons. On the other hand, it is possible to magnify the apertures only to a certain extent so the rotary and turbomolecular vacuum pump can maintain the required operating pressures in separate chambers. The article describes the combination of experimental measurement using an absolute pressure sensor and mathematical physics analysis to map all the specifics of the emerging critical supersonic flow in apertures between the chambers. Based on the experiments and their tuned analyses, the most effective variant of combining the sizes of each aperture concerning different operating pressures in the detector is determined. The situation is made more difficult by the described fact that each aperture separates a different pressure gradient, so the gas flow through each aperture has its own characteristics with a different type of critical flow, and they influence each other, thereby influencing the final passage of secondary electrons detected by the scintillator and thus affecting the resulting displayed image.
- Klíčová slova
- Ansys Fluent, ESEM, aperture, critical flow, one-dimensional flow theory, pressure sensor, scintillation detector,
- Publikační typ
- časopisecké články MeSH
This paper describes the combination of experimental measurements with mathematical-physical analysis during the investigation of flow in an aperture at low pressures in a prepared experimental chamber. In the first step, experimental measurements of the pressure in the specimen chamber and at its outlet were taken during the pumping of the chamber. This process converted the atmospheric pressure into the operating pressure typical for the current AQUASEM II environmental electron microscope at the ISI of the CAS in Brno. Based on these results, a mathematical-physical model was tuned in the Ansys Fluent system and subsequently used for mathematical-physical analysis in a slip flow regime on a nozzle wall at low pressure. These analyses will be used to fine-tune the experimental chamber. Once the chamber is operational, it will be possible to compare the results obtained from the experimental measurements of the nozzle wall pressure, static pressure, total pressure and temperature from the nozzle axis region in supersonic flow with the results obtained from the mathematical-physical analyses. Based on the above comparative analyses, we will be able to determine the realistic slip flow at the nozzle wall under different conditions at the continuum mechanics boundary.
- Klíčová slova
- Ansys Fluent, low pressure, nozzle, shear stress, slip flow,
- Publikační typ
- časopisecké články MeSH
Infusing pancreatic islets into the portal vein currently represents the preferred approach for islet transplantation, despite considerable loss of islet mass almost immediately after implantation. Therefore, approaches that obviate direct intravascular placement are urgently needed. A promising candidate for extrahepatic placement is the omentum. We aimed to develop an extracellular matrix skeleton from the native pancreas that could provide a microenvironment for islet survival in an omental flap. To that end, we compared different decellularization approaches, including perfusion through the pancreatic duct, gastric artery, portal vein, and a novel method through the splenic vein. Decellularized skeletons were compared for size, residual DNA content, protein composition, histology, electron microscopy, and MR imaging after repopulation with isolated islets. Compared to the other approaches, pancreatic perfusion via the splenic vein provided smaller extracellular matrix skeletons, which facilitated transplantation into the omentum, without compromising other requirements, such as the complete depletion of cellular components and the preservation of pancreatic extracellular proteins. Repeated MR imaging of iron-oxide-labeled pancreatic islets showed that islets maintained their position in vivo for 49 days. Advanced environmental scanning electron microscopy demonstrated that islets remained integrated with the pancreatic skeleton. This novel approach represents a proof-of-concept for long-term transplantation experiments.
Pumping in vacuum chambers is part of the field of environmental electron microscopy. These chambers are separated from each other by a small-diameter aperture that creates a critical flow in the supersonic flow regime. The distribution of pressure and shock waves in the path of the primary electron beam passing through the differentially pumped chamber has a large influence on the quality of the resulting microscope image. As part of this research, an experimental chamber was constructed to map supersonic flow at low pressures. The shape of this chamber was designed using mathematical-physical analyses, which served not only as a basis for the design of its geometry, but especially for the correct choice of absolute and differential pressure sensors with respect to the cryogenic temperature generated in the supersonic flow. The mathematical and physical analyses presented here map the nature of the supersonic flow with large gradients of state variables at low pressures at the continuum mechanics boundary near the region of free molecule motion in which the Environmental Electron Microscope and its differentially pumped chamber operate, which has a significant impact on the resulting sharpness of the final image obtained by the microscope. The results of this work map the flow in and behind the Laval nozzle in the experimental chamber and are the initial basis that enabled the optimization of the design of the chamber based on Prandtl's theory for the possibility of fitting it with pressure probes in such a way that they can map the flow in and behind the Laval nozzle.
- Klíčová slova
- BD sensor, ESEM, Prandtl’s theory, differentially pumped chamber, mach number, static pressure, static probe,
- Publikační typ
- časopisecké články MeSH
Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.
- Klíčová slova
- auxin carriers, correlative microscopy, nanodomains, plasma membrane,
- MeSH
- Arabidopsis genetika růst a vývoj MeSH
- buněčná membrána genetika metabolismus ultrastruktura MeSH
- konfokální mikroskopie MeSH
- kovové nanočástice chemie MeSH
- kyseliny indoloctové metabolismus MeSH
- mikroskopie elektronová rastrovací * MeSH
- počítačové zpracování obrazu MeSH
- protoplasty metabolismus ultrastruktura MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- tabák genetika metabolismus ultrastruktura MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
- zlato MeSH
Silicon inhibits the growth of Alternaria alternata into sorghum root cells by maintaining their integrity through stimulating biochemical defense reactions rather than by silica-based physical barrier creation. Although the ameliorating effect of silicon (Si) on plant resistance against fungal pathogens has been proven, the mechanism of its action needs to be better understood on a cellular level. The present study explores the effect of Si application in sorghum roots infected with fungus Alternaria alternata under controlled in vitro conditions. Detailed anatomical and cytological observations by both fluorescent and electron microscopy revealed that Si supplementation results in the inhibition of fungal hyphae growth into the protoplast of root cells. An approach of environmental scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy enabling spatial detection of Si even at low concentrations showed that there is no continual solid layer of silica in the root cell walls of the rhizodermis, mesodermis and exodermis physically blocking the fungal growth into the protoplasts. Additionally, biochemical evidence suggests that Si speeds up the onset of activities of phenylpropanoid pathway enzymes phenylalanine ammonia lyase, peroxidases and polyphenol oxidases involved in phenolic compounds production and deposition to plant cell walls. In conclusion, Si alleviates the negative impact of A. alternata infection by limiting hyphae penetration through sorghum root cell walls into protoplasts, thus maintaining their structural and functional integrity. This might occur by triggering plant biochemical defense responses rather than by creating compact Si layer deposits.
- Klíčová slova
- Cell integrity, Fungal infection, In vitro cultivation, Phenolics, Plant defense, Root anatomy,
- MeSH
- Alternaria * účinky léků MeSH
- fenylalaninamoniaklyasa MeSH
- kořeny rostlin * účinky léků enzymologie mikrobiologie MeSH
- křemík * farmakologie MeSH
- nemoci rostlin mikrobiologie MeSH
- Sorghum * účinky léků enzymologie mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fenylalaninamoniaklyasa MeSH
- křemík * MeSH
The Extended Low Temperature Method (ELTM) for the in-situ preparation of plant samples in an environmental scanning electron microscope enables carrying out repetitive topographical and material analysis at a higher resolution in the vacuum conditions of a scanning electron microscope or in the low gas pressure conditions of an environmental scanning electron microscope. The method does not require any chemical intervention and is thus suitable for imaging delicate structures rarely observable with common treatment methods. The method enables both sample stabilization as close to their native state as possible, as well as the transfer of the same sample from a low vacuum to an atmospheric condition for sample storage or later study. It is impossible for wet samples in the environmental scanning electron microscope. Our studies illustrate the high applicability of the ELTM for different types of plant tissue, from imaging of plant waxes at higher resolution, the morphological study of highly susceptible early somatic embryos to the elemental microanalysis of root cells. The method established here provides a very fast, universal and inexpensive solution for plant sample treatment usable in a commercial environmental scanning electron microscope equipped with a cooling Peltier stage.
