Epoxy Embedding Research Articles

Use of Melinex film for flat embedding tissue sections in LR White.

Tissue slices can undergo distortions during processing into resin for light and electron microscopy as a result of differential shrinkage of the various tissue components, and this may necessitate removal of a considerable amount of material from the final resin-embedded tissue block to ensure production of complete sections of the sample. To mitigate this problem, a number of techniques have been devised that ensure the sample is held flat during the final curing/polymerisation of the resin. For embedding in acrylic resins, oxygen must be excluded as it inhibits polymerisation, and methods devised for epoxy resin embedding are generally unsuitable. The method describes the preparation and use of air-tight flat-embedding chambers prepared from Melinex film and provides an inexpensive, technically simpler, and versatile alternative to chambers formed from either Thermanox coverslips or Aclar films that have previously been advocated for such purposes. Lay description: Tissue slices can undergo distortions during processing into resin for light and electron microscopy as a result of differential shrinkage of the various tissue components. Such distortions may necessitate removal of a considerable amount of material to ensure production of complete sections of the sample. For embedding in acrylic resins, oxygen must be excluded as it inhibits polymerisation, and methods devised for epoxy resin flat-embedding are generally unsuitable. Air-tight flat-embedding chambers prepared from either Thermanox coverslips, or a combination of PTFE-coated glass slides, polycarbonate film gaskets, and Aclar film have been advocated for such purposes. Thermanox coverslips are expensive and limited in size to 22mm × 60mm, and the alternative method is technically complicated. Melinex film is commercially available as 210mm × 297mm sheets and is approximately 1/20th the price of Thermanox and less than half the price of Aclar film. The method describes the preparation and use of embedding chambers made from Melinex film, glass slides and double-sided adhesive tape as a technically simpler, inexpensive and versatile alternative to both Thermanox coverslips and the Aclar film method.

Potential of digital autoradiography for characterization of uranium materials

This study evaluates the capabilities of a commercial digital autoradiography set-up in characterizing the spatial distribution of radioactivity in U-bearing materials. Two different imaging plates (IPs) from various manufacturers, as well as three read-out granularities (25 µm, 50 µm, and 100 µm) of a high-resolution IP scanner, were tested. A custom software, developed using Python, was used for processing of the image raw data obtained from the IP scanner. The software extracted 25 intensity profiles from each autoradiographic image, allowing for a detailed analysis of the data. By applying various mathematical fitting functions, important metrics such as edge width, amplitude of the flat top, and central core width of the intensity profiles were determined. This provided valuable insights into the interaction between the U samples and IPs, particularly for samples of different geometry and thickness. Comparisons were made between samples embedded in epoxy resin or modelling clay and non-embedded U specimens. The results highlighted the superior performance of epoxy embedding in the characterization of U-bearing materials. The variation in signal intensity between runs for individual samples reached up to 20 %, showing consistent behavior across all samples in each measurement. This suggests that the inconsistency in signal response during repetitive analysis is likely due to the IP scanner rather than the IP itself. By using the analytical method of global spatial autocorrelation (Moran’s I), we were able to effectively detect enrichment inhomogeneity within low enriched UO2 pellets. The Moran’s I value quantified the levels of inhomogeneity, providing a numerical measure that was not visually apparent from the digital autoradiographic image.

Potential of diagnostic electron microscopy of calcification, pathological neovascularization and elastolysis in combination with phenotyping of cell populations in large arteries

Aim. To determine the potential of diagnostic electron microscopy of intraplaque processes (severity of lipid damage, fibrous cap thickness and condition, severity of pathological neovascularization, presence, nature and severity of calcification, ratio and distribution of various cell populations).Material and methods. The study objects were plaques removed during endar­terectomy from the human carotid artery and segments of the human internal mammary artery removed during coronary bypass surgery. Whole specimens were subjected to chemical fixation, staining with heavy metal salts, embedding in epoxy resin followed by layer-by-layer grinding, polishing, contrasting, visualization using back-scattered electron scanning electron microscopy and three-dimensional reconstruction with color mapping (modified EM-BSEM).Results. The use of a modified EM-BSEM made it possible to: 1) visualize the fibrous cap thickness and assess the extracellular matrix; 2) analyze the neointimal lipid distribution; 3) perform three-dimensional reconstruction and analyze the microenvironment of calcifications of various sizes; 4) visualize endothelial cells, defects in interendothelial contacts and the basement membrane of neointimal capillaries with their subsequent three-dimensional reconstruction; 5) perform an analysis of age-dependent defects in the basement membrane and internal elastic membrane of the internal mammary artery. The resolution of the obtained images was significantly superior to intravascular imaging methods (intravascular ultrasound and optical coherence tomography), allowing additional assessment of capillary fluidity, the degree of calcification encapsulation and the condition of elastic fibers. Three-dimensional reconstruction of calcifications, neointimal capillaries and elastic fibers made it possible to assess their spatial density and heterogeneity. Simultaneously with the identification and assessment of these histological structures, objective phenotyping of cell populations was performed, which made it possible to isolate macrophages and foam cells, vascular smooth muscle cells, fibroblasts and endothelial cells in atherosclerotic plaques and automatically identify them through color mapping determined by their electronic contrast distribution signatures.Conclusion. The modified EM-BSEM method allows for universal electron microscopic diagnosis of atherosclerotic and elastolytic lesions of large arteries with high information content about vascular remodeling and high accuracy. Electronic contrast distribution signatures unique for each cell population indicate the possibility of their automated phenotyping using specialized neural network algorithms.

Simulated Hydrostatic Pulpal Pressure Effect on Microleakage-An Initial Study.

This study's purpose was to evaluate the effect of simulated in vitro hydrostatic pulpal pressure (HPP) on microleakage. Extracted third molars (n=12) were sectioned 5 mm below the cementoenamel junction, pulp tissue removed, and the sectioned crowns mounted on a Plexiglas plate penetrated by an 18-gauge stainless steel tube. The mounted specimen mesial surface received a 2×4×6 mm Class V preparation followed by restoration with a strongly acidic, one-step dental adhesive and a flowable microfilled resin, following all manufacturers' instructions. Restorations were finished to contour, and tubing was attached to a 20-cm elevated, 0.2% rhodamine G reservoir to the specimen steel tube for 48 hours. Specimens then received a nail polish coating to within 1 mm of the restoration margins and were placed in 2% methylene blue (MB) dye for 24 hours, followed by rinsing, embedding in epoxy resin, and sectioning into 1 mm slices using a diamond saw. Controls were intact molars (n=12) processed as above but without HPP. Specimen slices were evaluated using laser confocal microscopy with images exported to ImageJ software with microleakage assessed as the MB linear penetration as a percentage of the total interfacial wall length. Mean values were evaluated with the Kruskal Wallis/Dunn test at a 95% confidence level. The control specimens demonstrated significantly greater (p<0.0001) MB penetration than experimental specimens with simulated HPP. Under this study's conditions, simulated HPP significantly decreased MB dye penetration. Studies accomplished without simulated HPP may overestimate microleakage results.

Quantitative evaluation of embedding resins for volume electron microscopy.

Optimal epoxy resin embedding is crucial for obtaining consistent serial sections from large tissue samples, especially for block faces spanning >1 mm2. We report a method to quantify non-uniformity in resin curing using block hardness measurements from block faces. We identify conditions that lead to non-uniform curing as well as a procedure to monitor the hardness of blocks for a wide range of common epoxy resins used for volume electron microscopy. We also assess cutting repeatability and uniformity by quantifying the transverse and sectional cutting forces during ultrathin sectioning using a sample-mounted force sensor. Our findings indicate that screening and optimizing resin formulations is required to achieve the best repeatability in terms of section thickness. Finally, we explore the encapsulation of irregularly shaped tissue samples in a gelatin matrix prior to epoxy resin embedding to yield more uniform sections.

High Temperature Corrosion Behavior of 15-15Ti Cladding Tube Material in Contact with Liquid Lead, Outside, and Cs2MoO4, Inside

Lead-Cooled Fast Reactors are one of the emerging new technologies connected to the Generation IV reactor designs. Even though the reactor features are extensive and beneficial, some technical and regulatory difficulties remain that hinder the deployment. One of them is the corrosion behavior of fuel claddings in high burn-up and elevated temperatures, as fuel claddings are a part of accident barriers. In this study, 15-15Ti austenitic steel was the investigated cladding material.As this cladding would be used in a tube form, corrosion attack from both sides, inside and outside is studied in contact to their respective environments. From the outside of the tube, the proposed coolant liquid lead is the corrosive substance. At the inside of the tube, the major corrosive species in our test setup is, for simplicity reasons, Cs2MoO4. However, this choice has been made from some considerations regarding the fuel evolution upon burn-up.In a high burn-up situation, the generated fission products would start to accumulate to the gap between the fuel pellet and the cladding tube. These accumulated products are the so-called “Joint-Oxide Gain” (JOG) phases, and they are the corrosive substances in question when discussing the corrosion attack from the inside of a cladding tube. Cs2MoO4 has been identified as one of the main components of JOG phases and thus will be focused on in this study.A new capsule was designed to investigate the dual-atmosphere corrosion of cladding tubes. Unexposed capsule parts (a) and the basic schematic of the assembled capsule (b) can be seen in the Figure below. The cladding tube material itself is used as the container for JOG as it is filled with Cs2MoO4 and sealed on both ends. This assembly is then placed inside a bigger capsule, which is filled with lead powder to submerge the cladding tube assembly. All assemblies were done in Ar-filled glovebox. The capsules were then exposed to 600-1000 oC for 52 to 168 hours, which was followed by cutting and epoxy embedding to investigate the cross-sections. Figure 1

Qualitative Evaluation of Modified Polyvinyl Alcohol Gel Used as A Preservative Medium Compared to Traditional Modified Kaiserling Iii Solution in Pathology Museum

Pathology and anatomy museums preserve body tissues and organs traditionally in modified Kaiserling III solution and display in transparent glass jars, which serve as valuable teaching materials for students. Other techniques tried in preserving specimens are embedding in molten wax, embedding in epoxy resin, and plastination techniques. Modified Carbopol-940 gel has been tried recently as a preservative mounting medium in pathology museum. However, the use of modified Polyvinyl alcohol (PVA) gel as a preservative medium is not mentioned in the literature. Gel preservative medium can prevent leakage, toxic vapors, and irritation of eyes and skin. Aims: Qualitative evaluation of modified Polyvinyl alcohol gel as a preservative medium for specimens in pathology museums compared to modified Kaiserling III solution over one year. Objectives: 1) To qualitatively evaluate physical integrity of mounted tissue specimens stored in modified Kaiserling III solution and modified Polyvinyl alcohol gel for one year. 2) To qualitatively evaluate physical, chemical and microbiological parameters of modified Kaiserling III solution and modified Polyvinyl alcohol gel for one year. Materials and methods: In this qualitative cross-sectional study, six different organs, two sets each, were preserved in jars containing modified Kaiserling III solution and modified Polyvinyl alcohol gel. The media were evaluated for stability by physical tests, chemical test, and microbiological test, before and after the study. The specimens were assessed for physical disintegration by observing for tissue shredding, and evaluated for histopathological appearances, before and after the study. Results showed mild variability in stability of the preservative media but good tissue integrity of the specimens over a period of one year. &#x0D;

Applications of EMbedding and backscattered scanning electron microscopy for morphological and functional assessment of the thymus forcedly removed in children with congenital heart defects during the first weeks of life

Thymectomy in a number of clinical situations is an unavoidable step of cardiac surgical treatment of congenital heart defects, but the issue of its effects on development of immunity in early ontogenesis remains open. There is still debates among scientists about the risk of immune deficiency states in children who underwent thymectomy at an early age. One may see completely different opinions, both in domestic and foreign publications. A tool for resolving this issue may be provided by morpho-functional studies of the removed thymic tissue by means of modern scanning electron microscopy techniques. The objective of our study was to assess the possibilities of EMbedding and backscattered scanning electron microscopy for morphological and functional evaluation of the thymus, which was forcedly removed in the infant patients with congenital heart defects over the first weeks of life. The thymus of a newborn infant (postnatal day 27) with congenital heart disease: ventricular septal defect was examined using EMbedding and backscattered scanning electron microscopy imaging after embedding in epoxy resin. The mass of thymus gland was 15.7 g, the dimensions of thymus were: transverse, 3.4 cm; longitudinal, 4.1 cm; thickness, 1.7 cm; volume, 12.4 cm3. The study showed ability of EMbedding and backscattered scanning electron microscopy in morphological and functional assessment of thymus gland considered the central organ of the immune system. Step-bystep visualization from low to high magnifications, from tissues to cells and intracellular structures, as well as layer-by-layer examination of thymic cortex, medulla, interlobular septa and vessels, allows you to effectively assess the functionality of the thymus. This research method is sufficient for scientific research of the forcedly removed thymus since it enables us to visualize its microanatomy, allowing cell phenotyping at different layers of the thymus, studying intercellular interactions of thymocytes with reticulo-epithelial cells, subtle features of Hassall’s bodies and, finally, the process of T lymphocytes’ release from thymus gland.

A Short Historical Review of Water-Soluble Aminoplastic Resin Use for Lipid Ultrastructure Research

Abstract In 1982, a water-soluble aminoplastic resin embedding method for electron microscopy (EM) was developed to study lipids. It involves a silylation technique for processing cured resins by converting them from hydrophilic to hydrophobic status, facilitating ultra-thin sectioning. The technique offers better preservation of lipovitellin material in frog (Rana amurensis) yolk platelets, and it shows membrane ultrastructure better than traditional epoxy embedding methods. An early (1982) pseudo-colored TEM image processing technique applied to the micrographs obtained by EM of silylated aminoplastic thin sections revealed the lipovitellin molecules, in relation to the biosynthesis of new membranes in the first cleavage of the egg, with remarkable clarity and explains the lipovitellin molecules in relation to the biosynthesis of new membranes. Because the original article was written in Chinese and published in China before its open policy, many scientists may not be aware of the publication. Thus, in this short review, the essential components of the procedure are presented in English to help guide investigators who might be interested in the use of water-soluble embedding methods for lipid research.

Qualitative Evaluation of Carbopol-940 Gel Used as a Preservative Medium Compared to Traditional Modified Kaiserling III Solution in Pathology Museum

Introduction: Body organs and tissue specimens have been preserved in human and veterinary anatomy, pathology museums for years in traditional modified Kaiserling III solution. Wax embedding, epoxy-resin embedding and Plastination techniques have also been tried. However, the use of Carbopol-940 gel as a preservative medium is not mentioned in the literature. Gel preservative medium can prevent leakage, toxic vapors, and irritation of eyes and skin. Aims: Qualitative evaluation of carbopol-940 gel as a preservative medium for specimens in pathology museums compared to modified Kaiserling III solution over one year. Objectives: 1) To qualitatively evaluate physical integrity of mounted tissue specimens stored in Kaiserling III solution and carbopol-940 gel for one year. 2) To qualitatively evaluate physical, chemical and microbiological parameters of Kaiserling III solution and carbopol-940 gel for one year. Materials and methods: In this qualitative cross-sectional study, different organs were mounted in jars containing modified Kaiserling III solution and carbopol-940 gel. The media were monitored for stability by physical tests, chemical test, and microbiological test, before and after the study. The organs were monitored for physical disintegration by observing for tissue shredding. Specimens were histologically assessed before and after the study for tissue integrity. Results showed good stability of the preservative media and good tissue integrity of the specimens over a period of one year.

Fluid Flow in the Gas Diffusion Layer Using Computational Fluid Dynamics and Microscopy Techniques

The utilization of a proton exchange membrane fuel cell (PEMFC) as an energy provider using hydrogen as a fuel has increased drastically. The reasons are the current limitation of fossil fuel-based devices, pollutants-free, high efficiency, and zero-carbon emission. The life-cycle assessment results of this type of fuel cell have also indicated the lowest contribution to global warming, human health, and resource scarcity in comparison to other types of fuel cells. In this regard, improving the performance of PEMFC is of importance.As an important component of the PEMFC, the gas diffusion layer (GDL) transports the gas reactants to the catalyst layer with the least electrical resistance. The GDL is often made of carbon fibers and should have a surface with good electrical contact and hydrophobic properties to facilitate the water removal. Remaining water in the GDL will result in difficulties during the cold-start and enhances the degradation of this layer. It is believed that the water removal of the GDL has a direct relationship with the capillary pressure, which is strongly linked to the GDL’s microstructure and wettability. However, further investigations can be done once a comprehensive simulation model is developed to monitor the changes in the GDL liquid removal by the effective parameters.In this regard, the Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) has been used to perform three-dimensional cross-section imaging of the GDL. The GDL samples are provided by Freudenberg in which the Microporous layer (MPL) is impregnated into the GDL. The average porosity of this sample is in the range of 75% while having the in-plane gas permeability of 2.4 under a compressive stress of 1 MPa. The utilized resin for FIB-SEM imaging is a mixture of Epoxy embedding medium (diglycidyl ether of bisphenol – A) with two different hardeners DDSA (2-Dodecenylsuccinic anhydride) and MNA (Methylnadic anhydride), which will be mixed with DPM – 30 [2,4,6 – Tris(dimethylaminomethyl)phenol] as the accelerator, all provided by Sigma Aldrich. After the preparation of the resin, 0.4 gr of Cobalt (II) acetylacetonate nanoparticles (supported by Sigma Aldrich) were added to 7.14 ml of the resin to improve the contrast of the images. The samples were impregnated with the resin under vacuum to be pressurized (3 MPa) for 20 minutes, and afterward, heated in an oven at 60℃ for 12 hours. The surfaces for analyses were cut by a diamond wire, polished by abrasive plates down to 0.1 and gold coated (20nm).FIB-SEM acquisition consisted of the polishing of cross-sections with a focused ion beam (LMIS Ga+ source) at 30 keV and 1 nA (Zeiss Crossbeam 540), followed by imaging with an electron beam at acceleration voltages of 1.0, 3.0 kV. Milling and imaging were performed at a working distance of 5.2 mm and stage tilt of 54°, i.e., the coincidence point of the electron and ion beams.Once the three-dimensional cross-sections of the GDL are obtained, the segmentation and reconstruction will be made to provide the needed geometry for fluid flow simulation and to calculate the microstructural properties. Fig. 1 shows the generated structure of the GDL after the segmentation and reconstruction of the cross-section images. The geometry will be then used to characterize the effective parameters of the thermal/water management of the PEMFC. This study can also be a valid reference for future computational fluid dynamic analyses in the GDL using the numerical modeling with the conservative equations or the Lattice Boltzmann modeling (LBM) with the kinetic and particle distribution equations. Keywords : Proton exchange membrane fuel cell (PEMFC); Gas Diffusion Layer (GDL); Focused Ion Beam- Scanning Electron Microscopy (FIB-SEM); Three-dimensional simulation; Lattice Boltzmann method (LBM) Figure 1

Application of ultramicrotomy and infrared imaging to the forensic examination of automotive paint

AbstractIn several previously published studies, Lavine and coworkers have demonstrated that infrared (IR) spectra from all layers of an intact multilayered automotive paint chip can be collected in a single analysis by scanning across each layer of a cross sectioned paint chip using a Fourier transform IR imaging microscope. Applying alternating least squares to the spectral data, the IR spectrum of each layer of an original equipment manufacturer paint chip can be extracted from a line map of the spectral image. To further develop this imaging technique for automotive paint analysis, the capability to cross section “small” paint chips (1 mm or less) using an ultramicrotome has been incorporated into our current imaging methodology. An ultramicrotome does not require epoxy or other embedding media for the paint chip and will simplify the analysis. However, extracting the IR spectra for each layer of an original equipment manufacturer paint chip by alternating least squares can be problematic for thin peels (less than one micron thickness), necessitating the use of target testing factor analysis to determine whether a specific layer is present in the line map and modified alternating least squares to recover the IR spectrum of the layer. Using a new sample preparation technique and the appropriate multivariate curve resolution methods, high quality IR spectra of the layers of a modern automotive paint system can be obtained from paint fragments that are smaller than what is practical to analyze by conventional Fourier transform IR spectroscopy.

Detailed anatomical study of the peripheral motor branches of the facial nerve in the swine model: A novel investigative approach for facial paralysis research

IntroductionLarge animal models aid in innovating surgical approaches and developing medical devices for the treatment of facial paralysis. However, there is a lack of information on facial nerve anatomy in swine. This study aimed to investigate the branching pattern and histologic characteristics of the swine facial nerve, thereby establishing the anatomical patterns of preclinical models in facial paralysis research. Material and methodsThe five peripheral motor branches of the facial nerve were dissected in 30 hemifaces of fresh swine cadavers. Starting at the stylomastoid foramen, the course, branching pattern, and diameter of each motor branch was recorded. Samples were taken at the start of each branch for epoxy embedding, toluidine blue staining, and histomorphologic analysis. ResultsThe dissections demonstrated consistent anatomy of the buccal and marginal mandibular branches in contrast to the temporal and zygomatic branches, which showed more variation in branching patterns. The buccal branch had the largest mean diameter of 1.34 mm (± 0.26 mm), whereas the marginal mandibular branch had the largest fascicular area of 0.558 mm2 (± 0.12 mm2) and highest axon count of 3636.35 (± 526.36). The zygomatic branch had both the smallest diameter of 0.74 mm (± 0.25 mm) and fascicular area of 0.187 mm2 (± 0.14 mm2). ConclusionsThe swine facial nerve is anatomically similar to the human facial nerve, making the pig a suitable large animal model. Detailed anatomical and histological information is crucial for developing preclinical models of novel facial nerve reconstruction approaches.

Evaluation of Marginal and Internal Fit of Ceramic Laminate Veneers Fabricated with Five Intraoral Scanners and Indirect Digitization.

The long-term success of ceramic laminate veneers (CLVs) is influenced by the marginal and internal fit of the restorations. However, studies comparing the fit of CLVs using different intraoral scanners or the indirect digitization technique are lacking. The purpose of this study was therefore to assess the marginal and internal fit of CAD/CAM-milled CLVs using different intraoral scanners and the indirect digitalization technique. An ivorine typodont maxillary left-central incisor was prepared; the tooth and the neighboring teeth were scanned and used as a template to print ninety 3D partial models. Thereafter, ceramic laminate veneers (CLVs) (N = 90) were milled from IPS-Emax CAD blocks and divided into six equal groups (15 specimens each) according to the type of intraoral scanner (IOS), as follows: Omnicam IOS, SC3600 IOS, Trios 3 IOS, Emerald IOS, I500 IOS. Fifteen further CLVs were fabricated using the conventional indirect digitalization technique. After cementation on the resin dies and embedding in clear epoxy resin, specimens were sectioned inciso-gingivally and mesio-distally. At the incisal and cervical positions, the marginal discrepancy was measured and evaluated in addition to the internal gap at six locations using SEM (200×). Differences between gap measurements among the six groups were determined using ANOVA. Games-Howell multiple comparisons for homogenous variances and LSD multiple comparisons for non-homogenous variances were used with 95% confidence intervals. The significance level was set at 0.05. The lowest mean absolute marginal gap at the incisal margins (AMGI) was recorded for Omnicam group (203.28 ± 80.14) µm, while the highest mean absolute marginal gap at the cervical margins (AMGC) was recorded for Omnicam group (147.16 ± 59.78) µm. The mean AMGC was reported to be significantly different between the conventional technique (146.75 ± 38.43) µm and Trios 3 (91.86 ± (35.51) µm; p = 0.001) and between Emerald (112.37 ± (50.31) µm; p = 0.042) and I500 (86.95 ± (41.55) µm; p < 0.001). The mean MGI was found to be significantly different between the conventional technique (114.11 ± (43.45) µm and I500 group (186.99 ± (73.84) µm) only (p = 0.035). However, no significant differences were found in the mean MGI between all types of IOSs. The means of AMG and MG were significantly different at incisal or cervical areas between the conventional technique and IOSs and within the scanner groups (p > 0.05). Marginal gaps were higher in the incisal region compared to the cervical region with both the indirect digitization technique and the IOSs. Ceramic laminate veneers (CLVs) fabricated using IOSs produced overall internal and marginal fit adaptation results comparable to CLVs fabricated from the indirect digitalization method, and both techniques produced clinically acceptable results.

Micromechanics of non-embedded spruce wood: Novel polishing and indentation protocol

Since the start of nanoindentation in wood, cell walls have been supported by epoxy resin during the microtoming of surfaces. However, wood embedding in epoxy resin was initially developed to harvest thin sections destined for transmission electron microscopy. In nanoindentation, embedding can affect the identified mechanical properties and might be difficult for modified wood samples. For this reason, we propose a protocol that does not involve resin within the wood specimen, uses a more robust miller to achieve surfaces with root means squared roughness of around 35 nm, and can exclude invalid edge indents through a novel evaluation procedure based on fitting 2D histograms with a linear combination of bi-variate lognormal probability density functions. We examined twelve samples of Norway spruce (Picea abies) late wood and their counterparts soaked with a biological preservation pretreatment. A nanoindentation protocol on every sample led to a mean reduced modulus and mean hardness of around 12 % and 26 %, respectively, lower than those found in the literature for embedded Norway spruce. These results agree well with previous results found for Loblolly pine (Pinus taeda L.) when comparing embedded and non-embedded specimens.

Improvement of mechanical properties of oak-wood by bi-directional laminations – Efficacy of standard and pre-stressed glass fibre implants

This paper brings results of the bending testing of oak-wood laminated elements for structural use. The bi-directional laminations were reinforced with six types of implants made of glass fibre reinforced polymer (GFRP), some of which were pre-stressed before embedding in epoxy resin. Previously we reported on improving the mechanical properties of oak-wood laminated elements using carbon fibre implants (CFRP). Although it proved as an efficient improvement of the mechanical properties, it also raised the question of the cost effect to the final product. Therefore, within the larger study on investigating effects of different reinforcement systems, we tested glass fibres reinforced polymer (GFRP) as an alternative. Results showed that the introduction of implants improved the effective stiffness between 4 and 51 % and the ultimate load to failure up to 55 %. Furthermore, not only that insertion of the GFRP layers significantly contributed to the strength and stiffness improvement, but the ductility of the elements increased up to 32 %. Pre-stressing of the glass fibres reinforced polymer implants in tension before their installation contributed to the strength increase to a much greater extent than the increase in the number of layers. The best reinforcement effect is obtained if two layers of pre-stressed GFRP are used.

A combination of scanning electron microscopy and broad argon ion beam milling provides intact structure of secondary tissues in woody plants

The secondary tissues of woody plants consist of fragile cells and rigid cell walls. However, the structures are easily damaged during mechanical cross-sectioning for electron microscopy analysis. Broad argon ion beam (BIB) milling is commonly employed for scanning electron microscopy (SEM) of hard materials to generate a large and distortion-free cross-section. However, BIB milling has rarely been used in plant science. In the present study, SEM combined with BIB milling was validated as an accurate tool for structural observation of secondary woody tissues of two samples, living pine (Pinus densiflora) and high-density oak wood (Quercus phillyraeoides), and compared with classical microtome cross-sectioning. The BIB milling method does not require epoxy resin embedding because of prior chemical fixation and critical point drying of the sample, thus producing a three-dimensional image. The results showed that xylem structures were well-preserved in their natural state in the BIB-milled cross-section compared with the microtome cross-section. The observations using SEM combined with BIB milling were useful for wide-area imaging of both hard and soft plant tissues, which are difficult to observe with transmitted electron microscopy because it is difficult to obtain sections of such tissues, particularly those of fragile reaction woods.

Ultrastructure of neointima of native and artificial elements of the blood circulatory system

To compare the neointima structure in conduits for coronary bypass grafting, bioprosthetic heart valves, tissue-engineered vascular grafts, and metal stents. The objects of the study were the fragments of the human internal thoracic artery, experimental biodegradable vascular prostheses, leaflets of xenopericardial bioprostheses of heart valves, and fragments of stented vessels. Tissue samples were fixed in formalin and post-fixed in osmium tetroxide. After dehydration and epoxy resin embedding, the samples were ground and polished. Samples were counterstained with uranyl acetate and lead citrate and visualized by means of backscattered scanning electron microscopy. Neointimal pattern in all samples was similar. Neointima was comprised of endothelial cells, smooth muscle cells, fibroblasts, and the extracellular matrix. Endothelial cells showed significant diversity both between different elements of the circulatory system and within the same tissue, having either elongated or polygonal shape. Adhesion of leukocytes testified to the endothelial cell activation. In the absence of inflammation in the superficial layer of the neointima, the arrangement of smooth muscle cells and extracellular matrix fibers was parallel to the endothelium. Clusters of foam cells were frequently detected around the neointimal layers with solid inclusions (metal stents or calcium deposits). Thickening of the neointima was accompanied by the presence of capillaries and capillary-like structures. Neointima formation is a typical response to the damage inflicted to the elements of the circulatory system. Neointima underwent a constant remodeling characterized by an altered cellular composition, macrophage invasion, neovascularization, and calcification.

光 - 電子相関顕微鏡法とアレイトモグラフィー法を組み合わせて蛍光を放つオルガネラを電顕で捉える

Correlative light and electron microscopy (CLEM) is an analytical technique used to evaluate the correlation between fluorescence images of identical samples and regions of fluorescent-labeled organelles captured using light microscopy and electron microscopy. By utilizing various methods during sample preparation, the fluorescence images of fluorescent-labeled organelles are juxtaposed with ultra-structures of the same region obtained using scanning electron microscopy (SEM) or transmission electron microscopy. Unlike animal tissues and adherent cells, visualizing the organelles detected in plant samples via CLEM analysis is difficult because of the non-adherence of plant tissue and cultured cells to glass plates and the size of the cells. Therefore, we combine the serial section SEM method (Array tomography) based on ultra-thin sections and CLEM to identify fluorescent-labeled organelles in plant cells and reconstruct them in three dimensions (3D). In particular, as osmium (Os) epoxy-tolerant fluorescent proteins have recently been reported to be resistant to Os fixation and epoxy resin embedding in animal adherent cells, we applied these fluorescent tags to plant samples. In this paper, we introduce the sample preparation method for the CLEM analysis of organelles labeled with Os epoxy-tolerant fluorescent proteins in plant tissues, alongside the method for obtaining CLEM images and the combined 3D-CLEM analysis method comprising CLEM and array tomography.

A novel tight cylindrical mold for epoxy resin embedding allows enhanced microscopic analysis of microcores extracted from woody plants

A novel mold was devised to embed microcores extracted from stems of trees in epoxy resin, which has been widely used for optical and electron microscopic analysis of xylem formation. The embedding mold of a tight cylindrical shaped tube was designed to avoid displacement of microcores from the right position during the process of resin embedding. Microcores of a ring-porous hardwood species, Quercus crispula, with higher wood density and much larger differentiating vessel elements laid down on the boundary between the current xylem and the previous one, which generally cause difficulty in thin sectioning and breaks in sections, respectively, were embedded in the cylindrical molds full of epoxy resin. Locations of the three principal planes of wood anatomy could be determined in cylindrical resin-embedded microcores as follows: the transverse plane could be found on their side of cylinder, the radial one was vertical to the transverse, and the tangential ones were their circular ends of cylinder. The present embedding mold, therefore, can provide all three principal sections for microscopic wood anatomy from the side or ends of the same cylindrical microcore in principle. To confirm the usefulness of the resin-embedded microcores, we examined the differentiation of vessel elements during the period of earlywood formation on their transverse sections under microscopes, consequently could observe cell division in the cambial zone and sequential stages of vessel element differentiation, including cell expansion and deposition of the secondary cell wall. The present embedding mold for epoxy resin is simple but highly useful and innovative for a wide range of applications of microcores in microscopy for studies on tree-ring formation.