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Minimal immunogen vaccines are being developed to focus antibody responses against otherwise challenging targets, including human immunodeficiency virus (HIV), but multimerization of the minimal peptide immunogen on a carrier platform is required for activity. Star copolymers comprising multiple hydrophilic polymer chains ("arms") radiating from a central dendrimer unit ("core") were recently reported to be an effective platform for arraying minimal immunogens for inducing antibody responses in mice and primates. However, the impact of different parameters of the star copolymer (e.g., minimal immunogen density and hydrodynamic size) on antibody responses and the optimal synthetic route for controlling those parameters remains to be fully explored. We synthesized a library of star copolymers composed of poly[ N -(2-hydroxypropyl)methacrylamide] hydrophilic arms extending from poly(amidoamine) dendrimer cores with the aim of identifying the optimal composition for use as minimal immunogen vaccines. Our results show that the length of the polymer arms has a crucial impact on the star copolymer hydrodynamic size and is precisely tunable over a range of 20-50 nm diameter, while the dendrimer generation affects the maximum number of arms (and therefore minimal immunogens) that can be attached to the surface of the dendrimer. In addition, high-resolution images of selected star copolymer taken by a custom-modified environmental scanning electron microscope enabled the acquisition of high-resolution images, providing new insights into the star copolymer structure. Finally, in vivo studies assessing a star copolymer vaccine comprising an HIV minimal immunogen showed the criticality of polymer arm length in promoting antibody responses and highlighting the importance of composition tunability to yield the desired biological effect.

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., (© 2024. The Author(s).)

A combination of experimental measurement preparations using pressure and temperature sensors in conjunction with the theory of one-dimensional isentropic flow and mathematical physics analyses is presented as a tool for analysis in this paper. Furthermore, the subsequent development of a nozzle for use in environmental electron microscopy between the specimen chamber and the differentially pumped chamber is described. Based on experimental measurements, an analysis of the impact of the nozzle shaping located behind the aperture on the character of the supersonic flow and the resulting dispersion of the electron beam passing through the differential pumped chamber is carried out on the determined pressure ratio using a combination of theory and mathematical physics analyses. The results show that nozzle shapes causing under-expanded gas outflow from the aperture to the nozzle have a worse impact on the dispersion of the primary electron beam. This is due to the flow velocity control. The controlled reduction in the static pressure curve on the primary electron beam path thus causes a significantly higher course of electron dispersion values than variants with shapes causing over-expanded gas outflow.

This paper describes the methodology of combining experimental measurements with mathematical-physics analyses in the investigation of flow in the aperture and nozzle. The aperture and nozzle separate the differentially pumped chamber from the specimen chamber in an environmental scanning electron microscope (ESEM). Experimental measurements are provided by temperature and pressure sensors that meet the demanding conditions of cryogenic temperature zones and low pressures. This aperture maintains the required pressure difference between the chambers. Since it separates the large pressure gradient, critical flow occurs on it and supersonic gas flow with the characteristic properties of critical flow in the state variables occurs behind it. As a primary electron beam passes through the differential pumped chamber and the given aperture, the aperture is equipped with a nozzle. The shape of the nozzle strongly influences the character of the supersonic flow. The course of state variables is also strongly influenced by this shape; thus, it affects the number of collisions the primary beam's electrons have with gas molecules, and so the resulting image. This paper describes experimental measurements made using sensors under laboratory conditions in a specially created experimental chamber. Then, validation using mathematical-physical analysis in the Ansys Fluent system is described.

Freezing and lyophilization have been utilized for decades to stabilize pharmaceutical and food products. Freezing a solution that contains dissolved salt and/or organic matter produces pure primary ice crystal grains separated by freeze-concentrated solutions (FCS). The microscopic size of the primary ice crystals depends on the cooling conditions and the concentration of the solutes. It is generally accepted that primary ice crystals size influences the rate of sublimation and also can impact physico-chemical behaviour of the species in the FCS. This article, however, presents a case where the secondary ice formed inside the FCS plays a critical role. We microscoped the structures of ice-cast FCS with an environmental scanning electron microscope and applied the aggregation-sensitive spectroscopic probe methylene blue to determine how the microstructure affects the molecular arrangement. We show that slow cooling at -50 °C produces large salt crystals with a small specific surface, resulting in a high degree of molecular aggregation within the FCS. In contrast, fast liquid nitrogen cooling yields an ultrafine structure of salt crystals having a large specific surface area and, therefore, inducing smaller aggregation. The study highlights a critical role of secondary ice in solute aggregation and introduces methylene blue as a molecular probe to investigate freezing behaviour of aqueous systems with crystalline solute., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bioactive moieties designed to bind to cell membrane receptors benefit from coupling with polymeric carriers that have enhanced affinity to the cell membrane. When bound to the cell surface, such carriers create a "2D solution" of a ligand with a significantly increased concentration near a membrane-bound receptor compared to a freely water-soluble ligand. Bifunctional polymeric carriers based on amphiphilic triblock copolymers were synthesized from 2-pent-4-ynyl oxazoline, 2-nonyl oxazoline and 2-ethyl oxazoline. Their self-assembly and interactions with plasma proteins and HEK 293 cells were studied in detail. The affinity of these triblock copolymers to HEK 293 cell membranes and organ tissues was tunable by the overall hydrophobicity of the polymer molecule, which is determined by the length of the hydrophobic and hydrophilic blocks. The circulation time and biodistribution of three representative triblock copolymers were monitored after intravenous administration to C57BL/6 albino mice. A prolonged circulation time was observed for polymers with longer hydrophobic blocks, despite their molecular weight being below the renal threshold., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

In protein analysis, fast techniques applicable for preliminary tests of the protein structural changes are sought. We show that using constant current chronopotentiometric stripping peak H, small amounts of oligomeric, denatured and aggregated bovine serum albumin (BSA) can be easily distinguished from native form. Different behavior of native, denatured, and aggregated BSA could be explained by combination of their different adsorption at charged surface and accessibility of electroactive amino acid residues. Ability to discriminate between individual forms allows to use chronopotentiometric stripping for study of processes responsible for structural changes, such as freezing treatment., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Dioecious plants possess diverse sex determination systems and unique mechanisms of reproductive organ development; however, little is known about how sex-linked genes shape the expression of regulatory cascades that lead to developmental differences between sexes. In Silene latifolia, a dioecious plant with stable dimorphism in floral traits, early experiments suggested that female-regulator genes act on the factors that determine the boundaries of the flower whorls. To identify these regulators, we sequenced the transcriptome of male flowers with fully developed gynoecia, induced by rapid demethylation in the parental generation. Eight candidates were found to have a positive role in gynoecium promotion, floral organ size, and whorl boundary, and affect the expression of class B MADS-box flower genes. To complement our transcriptome analysis, we closely examined the floral organs in their native state using field emission environmental scanning electron microscopy, and examined the differences between females and androhermaphrodites in their placenta and ovule organization. Our results reveal the regulatory pathways potentially involved in sex-specific flower development in the classical model of dioecy, S. latifolia. These pathways include previously hypothesized and unknown female-regulator genes that act on the factors that determine the flower boundaries, and a negative regulator of anther development, SUPERMAN-like (SlSUP)., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Frozen aqueous solutions are an important subject of study in numerous scientific branches including the pharmaceutical and food industry, atmospheric chemistry, biology, and medicine. Here, we present an advanced environmental scanning electron microscope methodology for research of ice samples at environmentally relevant subzero temperatures, thus under conditions in which it is extremely challenging to maintain the thermodynamic equilibrium of the specimen. The methodology opens possibilities to observe intact ice samples at close to natural conditions. Based on the results of ANSYS software simulations of the surface temperature of a frozen sample, and knowledge of the partial pressure of water vapor in the gas mixture near the sample, we monitored static ice samples over several minutes. We also discuss possible artifacts that can arise from unwanted surface ice formation on, or ice sublimation from, the sample, as a consequence of shifting conditions away from thermodynamic equilibrium in the specimen chamber. To demonstrate the applicability of the methodology, we characterized how the true morphology of ice spheres containing salt changed upon aging and the morphology of ice spheres containing bovine serum albumin. After combining static observations with the dynamic process of ice sublimation from the sample, we can attain images with nanometer resolution.

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.

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 ( Nt PIN3b-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.

Pyrrhocoroidea represents an important group of true bugs (Insecta: Hemiptera: Heteroptera) which includes fire bugs, cotton stainers and other taxa widely used in experimental studies or known as pests. However, the morphology and phylogeny of Pyrrhocoroidea have been only poorly studied so far. Here, structures of the external scent efferent system of the metathoracic scent glands are examined in 64 out of 71 currently valid genera of Pyrrhocoroidea and scanning electron micrographs are provided for most taxa. Several characters are revealed which define each of the three higher taxa within Pyrrhocoroidea: Larginae (small auriculate peritreme lacking manubrium and median furrow; metathoracic spiracle never surrounded by evaporatorium), Physopeltinae (large, widely open ostiole; large peritremal disc with manubrium [new term], lacking median furrow; mace-like mycoid filter processes of equal shape and size on both anterior and posterior margins of metathoracic spiracle), and Pyrrhocoridae (elongate auriculate peritreme with deep median furrow). Within Pyrrhocoridae, three main types (A, B and C) of the external scent efferent system are distinguished, differring in the amount of reductions. The findings are interpreted in the context of phylogenetic hypotheses available for Pyrrhocoroidea and their close relatives, Coreoidea and Lygaeoidea. An updated identification key to the families and subfamilies of Pyrrhocoroidea applicable for both sexes is provided., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Main Conclusion: 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.

The paper discusses the real-time monitoring of the changing sample morphology during the entire lyophilization (freeze-drying) and vacuum-drying processes of model biopharmaceutical solutions by using an environmental scanning electron microscope (ESEM); the device's micromanipulators were used to study the interior of the samples in-situ without exposing the samples to atmospheric water vapor. The individual collapse temperatures (T c ) of the formulations, pure bovine serum albumin (BSA) and BSA/sucrose mixtures, ranged from -5 to -29 °C. We evaluated the impact of the freezing method (spontaneous freezing, controlled ice nucleation, and spray freezing) on the morphologies of the lyophiles at the constant drying temperature of -20 °C. The formulations with T c above -20 °C resulted in the lyophiles' morphologies significantly dependent on the freezing method. We interpret the observations as an interplay of the freezing rates and directionalities, both of which markedly influence the morphologies of the frozen formulations, and, subsequently, the drying process and the mechanical stability of the freeze-dried cake. The formulation with T c below -20 °C yielded a collapsed cake with features independent of the freezing method. The vacuum-drying produced a material with a smooth and pore-free surface, where deep cracks developed at the end of the process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We present a complex analysis and optimisation of dynamic conditions in the environmental scanning electron microscope (ESEM) to allow in-situ observation of extremely delicate wet bio-polymeric spherical particles in their native state. According to the results of gas flow and heat transfer simulations, we were able to develop an improved procedure leading to thermodynamic equilibrium between the sample and chamber environment. To quantify and hence minimise the extent of any sample deformation during specimen chamber pumping, a strength-stress analysis is used. Monte Carlo simulations of beam-gas, -water, and -sample interactions describe beam scattering, absorbed energy, interaction volume and the emission of signal electrons in the ESEM. Finally, we discuss sample damage as a result of drying and the production of beam-induced free radicals. Based on all experimental and simulation results we introduce a Delicate Sample Observation Strategy for the ESEM. We show how this strategy can be applied to the characterization of polyelectrolyte complex spherical particles containing immobilized recombinant cells E. coli overexpressing cyclohexanone monooxygenase, used as a model biocatalyst. We present the first native-state electron microscopy images of the viscous core of a halved polyelectrolyte complex capsule containing living cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The freezing of ionic aqueous solutions is common in both nature and human-conducted cryopreservation. The cooling rate and the dimensions constraining the solution are known to fundamentally influence the physicochemical characteristics of the sample, including the extent of vitrification, morphology, and distribution of ions. The presence of some salts in an aqueous solution often suppresses the ice crystallization, allowing bulk vitrification during relatively slow cooling. Such a process, however, does not occur in NaCl solutions, previously observed to vitrify only under hyperquenching and/or in sub-micrometric confinements. This work demonstrates that, at freezing rates of ≥100 K min -1 , crystallized ice I h expels the freeze-concentrated solution onto the surfaces of the crystals, forming lamellae and veins to produce glass, besides eutectic crystallization. The vitrification covers (6.8% ± 0.6%) and (17.9% ± 1.5%) of the total eutectic content in 0.06M and 3.4 mM solutions, respectively. The vitrified solution shows a glass-to-liquid transition succeeded by cold crystallization of NaCl · 2H 2 O during heating via differential scanning calorimetry. We establish that ice crystallization is accompanied by increased basicity in freeze-concentrated solutions, reflecting preferential incorporation of chloride anions over sodium cations into the ice. After the sample is heated above the glass transition temperature, the acidity gradually returns towards the original value. The morphology of the samples is visualized with an environmental scanning electron microscope. Generally, the method of vitrifying the freeze-concentrated solution in between the ice I h crystals via fast cooling can be considered a facile route towards information on vitrified solutions.

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.

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 μM and 10-50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ultraviolet-B (UV-B) radiation is a key environmental signal which initiates diverse responses that affect the metabolism, development, and viability of plants. In keeping with our previous studies, we concentrated primarily on how UV-B radiation affects Norway spruce [ Picea abies (L.) Karst.] somatic embryo maturation and how phenolics and polyamines (PAs) are linked to the defense response invoked by UV-B irradiation. We treated clusters of Norway spruce embryogenic culture (EC) with UV-B during the five stages of embryo maturation (early, cylindrical, precotyledonary, cotyledonary, and mature embryos). For the first time, we take an advantage of the unique environmental scanning electron microscope AQUASEM II to characterize somatic embryos in their native state. The severity of the irradiation effect on embryonal cell viability was shown to be dependent on the intensity of radiation as well as the stage of embryo development, and might be related to the formation of protoderm. The response of early embryos was characterized by an increase in malondialdehyde (MDA), a marked decrease in PA contents and a decline in phenolics. The reduced ability to activate the defense system seems to be responsible not only for the severe cell damage and decrease in viability but also for the inhibition of embryo development. The significant reduction in spermidine (Spd), which has been reported to be crucial for the somatic embryo development of several coniferous species, may be causally linked to the limited development of embryos. The pronounced decrease in cell wall-bound ferulic acid might correspond to failure of somatic embryos to reach more advanced stages of development. Embryos at later stages of development showed stress defense responses that were more efficient against UV-B exposure.

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., Competing Interests: The authors have declared that no competing interests exist.

Biogenesis of the plant secondary cell wall involves many important aspects, such as phenolic compound deposition and often silica encrustation. Previously, we demonstrated the importance of the exocyst subunit EXO70H4 for biogenesis of the trichome secondary cell wall, namely for deposition of the autofluorescent and callose-rich cell wall layer. Here, we reveal that EXO70H4-driven cell wall biogenesis is constitutively active in the mature trichome, but also can be activated elsewhere upon pathogen attack, giving this study a broader significance with an overlap into phytopathology. To address the specificity of EXO70H4 among the EXO70 family, we complemented the exo70H4-1 mutant by 18 different Arabidopsis ( Arabidopsis thaliana ) EXO70 paralogs subcloned under the EXO70H4 promoter. Only EXO70H4 had the capacity to rescue the exo70H4 - 1 trichome phenotype. Callose deposition phenotype of exo70H4-1 mutant is caused by impaired secretion of PMR4, a callose synthase responsible for the synthesis of callose in the trichome. PMR4 colocalizes with EXO70H4 on plasma membrane microdomains that do not develop in the exo70H4-1 mutant. Using energy-dispersive x-ray microanalysis, we show that both EXO70H4- and PMR4-dependent callose deposition in the trichome are essential for cell wall silicification., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

A new Combined System for high-efficiency detection of Secondary and Backscattered Electrons (CSSBE) in the ESEM consists of three detectors: an ionisation SE detector, an improved scintillation BSE detector, and a new Ionisation Secondary Electron Detector with an electrostatic Separator (ISEDS). The ISEDS optimizes conditions for electron-gas ionisation phenomena in the ESEM to achieve a strongly amplified signal from the secondary electrons with a minimal contribution from backscattered and beam electrons. For this purpose, it is originally equipped with an electrostatic separator, which focuses signal electrons towards a detection electrode and controls the concentration of positive ions above the sample. The working principle of the ISEDS is explained by simulations of signal electron trajectories in gas using the EOD program with our Monte Carlo module. The ability to detect the signal electrons in a selected range of energies is described with Geant4 Monte Carlo simulations of electron-solid interactions and proven by experimental results. High-efficiency detection of the ISEDS is demonstrated by imaging a low atomic number sample under a reduced beam energy of 5 keV, very low beam currents of up to 0.2 pA, and gas pressure of hundreds of Pa., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the study of lime as the basic component of historical mortars and plasters, four lime putties prepared from various kinds of lime of various granulometry and by various ways of preparation were evaluated. The rheological properties and micro-morphologic changes, growing of calcite crystals, and rate of carbonation were monitored. The lime putty prepared from lump lime achieves the best rheological properties, yield stress 214.7 Pa and plastic viscosity 2.6 Pa·s. The suitability of this lime putty was checked by testing the development of calcium hydroxide and calcite crystals using scanning electron microscopy and environmental scanning electron microscopy. The disordered crystals of calcium hydroxide exhibit better carbonation resulting in the large crystals of calcite; therefore, the mortar prepared from the lump lime has the highest flexural strength and compressive strength 0.8/2.5 MPa, its carbonation is the fastest and exhibits the longest durability. Also, thanks to the micro-morphological characterization of samples in their native state by means of environmental scanning electron microscopy, the new way of lime putty preparation by mixing was proven. The preparation consists in the mechanical crash of the lime particles immediately after hydration. This enables the properties of putty prepared from lump lime to be nearly reached.

Somatic embryogenesis (SE) is an important biotechnological technique used for the propagation of many pine species in vitro. However, in bog pine, one of the most endangered tree species in the Czech Republic, limitations were observed, which negatively influenced the development and further germination of somatic embryos. Although initiation frequency was very low-0.95 %, all obtained cell lines were subjected to maturation. The best responding cell line (BC1) was used and subjected to six different variants of the maturation media. The media on which the highest number of early-precotyledonary/cotyledonary somatic embryos was formed was supplemented with 121 μM abscisic acid (ABA) and with 6 % maltose. In the end of maturation experiments, different abnormalities in formation of somatic embryos were observed. For visualization and identification of abnormalities in meristem development during proliferation and maturation processes, the environmental scanning electron microscope was used. In comparison to the classical light microscope, the non-commercial environmental scanning electron microscope AQUASEM II has been found as a very useful tool for the quick recognition of apical meristem disruption and abnormal development. To our knowledge, this is the first report discussing somatic embryogenesis in bog pine. Based on this observation, the cultivation procedure could be enhanced and the method for SE of bog pine optimized.

Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.

This article describes the surface structure of Norway spruce early somatic embryos (ESEs) as a typical culture with asynchronous development. The microstructure of extracellular matrix covering ESEs were observed using the environmental scanning electron microscope as a primary tool and using the scanning electron microscope with cryo attachment and laser electron microscope as a complementary tool allowing our results to be proven independently. The fresh samples were observed in conditions of the air environment of the environmental scanning electron microscope (ESEM) with the pressure from 550Pa to 690Pa and the low temperature of the sample from -18°C to -22°C. The samples were studied using two different types of detector to allow studying either the thin surface structure or material composition. The scanning electron microscope with cryo attachment was used for imaging frozen extracellular matrix microstructure with higher resolution. The combination of both electron microscopy methods was suitable for observation of "native" plant samples, allowing correct evaluation of our results, free of error and artifacts., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The use of non-standard low-temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low-temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low-temperature method. An influence of the low-temperature method on sample stability was also studied. The results indicate that high-moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long-time observation at different operation parameters. We finally show high applicability of the low-temperature method on different types of conifers and Oxalis acetosella., (© 2014 Wiley Periodicals, Inc.)

Direct comparison of key physical and chemical-engineering properties of two representative matrices for multipurpose immobilisations was performed for the first time. Polyvinyl alcohol lens-shaped particles LentiKats® and polyelectrolyte complex microcapsules were characterised by advanced techniques with respect to the size distribution of the particles, their inner morphology as revealed by fluorescent probe staining, mechanical resistance, size-exclusion properties, determination of effective diffusion coefficient and environmental scanning electron microscope imaging. While spherical polyelectrolyte complex microcapsules composed of a rigid semipermeable membrane and a liquid core are almost uniform in shape and size (diameter of 0.82 mm; RSD = 5.6 %), lens-shaped LentiKats® are characterised by wider size distribution (diameter of 3.65 mm; RSD = 10.3 % and height of 0.341 mm; RSD = 32.3 %) and showed the same porous structure throughout their whole volume at the mesoscopic (micrometre) level. Despite differences in their inner structure and surface properties, the pore diameter of ∼ 2.75 nm for regular polyelectrolyte complex microcapsules and ∼ 1.89 nm for LentiKats® were similar. These results were used for mathematical modelling, which provided the estimates of the effective diffusion coefficient of sucrose. This value was 1.67 × 10(-10) m(2) s(-1) for polyelectrolyte complex microcapsules and 0.36 × 10(-10) m(2) s(-1) for LentiKats®. Recombinant cells Escherichia coli-overexpressing enzyme cyclopentanone monooxygenase were immobilised in polyelectrolyte complex microcapsules and LentiKats® for comparison of their operational stability using model Baeyer-Villiger oxidation of (±)-cis-bicyclo [3.2.0] hept-2-en-6-one to regioisomeric lactones as important chiral synthons for potential pharmaceuticals. Both immobilisation matrices rendered high operational stability for whole-cell biocatalyst with no reduction in the biooxidation rate over 18 repeated reaction cycles.