Advanced environmental scanning electron microscopy reveals natural surface nano-morphology of condensed mitotic chromosomes in their native state.

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.

Tuning polymer-blood and polymer-cytoplasm membrane interactions by manipulating the architecture of poly(2-oxazoline) triblock copolymers.

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.

Decellularized Pancreatic Tail as Matrix for Pancreatic Islet Transplantation into the Greater Omentum in Rats.

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.

Chronopotentiometric sensing of native, oligomeric, denatured and aggregated serum albumin at charged surfaces.

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.

Chemical genetics in Silene latifolia elucidate regulatory pathways involved in gynoecium development.

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).

ESEM Methodology for the Study of Ice Samples at Environmentally Relevant Subzero Temperatures: "Subzero ESEM".

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.

Correlative Light-Environmental Scanning Electron Microscopy of Plasma Membrane Efflux Carriers of Plant Hormone Auxin.

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.

External structures of the metathoracic scent gland efferent system in the true bug superfamily Pyrrhocoroidea (Hemiptera: Heteroptera: Pentatomomorpha).

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.

Silicon triggers sorghum root enzyme activities and inhibits the root cell colonization by Alternaria alternata.

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.

Dynamical in-situ observation of the lyophilization and vacuum-drying processes of a model biopharmaceutical system by an environmental scanning electron microscope.

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 (Tc) 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 Tc 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 Tc 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.

Simulation-based optimisation of thermodynamic conditions in the ESEM for dynamical in-situ study of spherical polyelectrolyte complex particles in their native state.

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.

In-situ preparation of plant samples in ESEM for energy dispersive x-ray microanalysis and repetitive observation in SEM and ESEM.

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.

Effects of copper and arsenic stress on the development of Norway spruce somatic embryos and their visualization with the environmental scanning electron microscope.

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.

Reverse taxonomy applied to the Brachionus calyciflorus cryptic species complex: Morphometric analysis confirms species delimitations revealed by molecular phylogenetic analysis and allows the (re)description of four species.

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.

Exocyst Subunit EXO70H4 Has a Specific Role in Callose Synthase Secretion and Silica Accumulation.

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.

High-efficiency detector of secondary and backscattered electrons for low-dose imaging in the ESEM.

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.

Effect of the preparation of lime putties on their properties.

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.

The low-temperature method for study of coniferous tissues in the environmental scanning electron microscope.

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.

Physical and bioengineering properties of polyvinyl alcohol lens-shaped particles versus spherical polyelectrolyte complex microcapsules as immobilisation matrices for a whole-cell Baeyer-Villiger monooxygenase.

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.

In situ study of live specimens in an environmental scanning electron microscope.

In this paper we introduce new methodology for the observation of living biological samples in an environmental scanning electron microscope (ESEM). The methodology is based on an unconventional initiation procedure for ESEM chamber pumping, free from purge-flood cycles, and on the ability to control thermodynamic processes close to the sample. The gradual and gentle change of the working environment from air to water vapor enables the study of not only living samples in dynamic in situ experiments and their manifestation of life (sample walking) but also its experimentally stimulated physiological reactions. Moreover, Monte Carlo simulations of primary electron beam energy losses in a water layer on the sample surface were studied; consequently, the influence of the water thickness on radiation, temperature, or chemical damage of the sample was considered.