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.

• Klíčová slova
• advanced environmental scanning electron microscopy, extracellular matrix skeletons, pancreas decellularization, splenic vein perfusion, transplantation into the omentum,
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• mikroanalýza elektronovou sondou metody   MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• rostliny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The discovery and exploration of cryptic species have been profoundly expedited thanks to developments in molecular biology and phylogenetics. In this study, we apply a reverse taxonomy approach to the Brachionus calyciflorus species complex, a commonly studied freshwater monogonont rotifer. By combining phylogenetic, morphometric and morphological analyses, we confirm the existence of four cryptic species that have been recently suggested by a molecular study. Based on these results and according to an exhaustive review of the taxonomic literature, we name each of these four species and provide their taxonomic description alongside a diagnostic key.

• MeSH
• DNA helmintů genetika   MeSH
• druhová specificita MeSH
• ekosystém MeSH
• fylogeneze MeSH
• mikroskopie elektronová rastrovací MeSH
• molekulární biologie MeSH
• sekvenční analýza DNA MeSH
• sladká voda MeSH
• vířníci anatomie a histologie   klasifikace   genetika   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Nizozemsko MeSH
• Názvy látek
• DNA helmintů MeSH