Preparation Of Ultrathin Sections Research Articles

CHANGES IN THE ULTRASTRUCTURAL ELEMENTS OF PERIODONTAL NEUROTROPHY UNDER CONDITIONS OF ACUTE SIMPLE COAGULATION DYSTROPHY IN THE EXPERIMENT.

Aim: To determine the role of damage to the ultrastructural elements of the periodontal nervous system in the pathogenesis of dystrophic periodontal disease. Materials and Methods: The basis of the experimental part of the study was the preparation of ultrathin sections from blocks of gum tissue of white rats, which were prepared using the UMTP-3M device. The study and analysis of biopsy samples was carried out with the help of an electron microscope UEMV-100K. Results: With the help of transmission electron microscopy, it was found that from the first minutes after the injection of hemolysate of isogenic erythrocytes into the rats, aggregates of erythrocytes, clumps of blood plasma, clusters of fibrin monomer masses, bundles of fibrin fibers, platelet and homogeneous were present in the connective tissue of the gums, and in particular in the lumens of hemocapillaries microthrombi, which confirms damage to the ultrastructures of the periodontium, which lead to the development of a pathological process, which is described when simple coagulation dystrophy is reproduced. Conclusions: Coagulative damage to the ultrastructural elements of the periodontal nervous system is one of the important factors in the pathogenesis of dystrophic periodontal damage. Under these conditions, trophic disturbances occur, similar to those that occur when the integrity of the nerve is disturbed - neurotrophic mechanism of dystrophy.

Dissection and ultramicroscopic observation of an apical pollen tube of Pyrus.

The pollen tube is ideal for studying cell polar growth, and observing the ultrastructure of the pollen tube tip using transmission electron microscopy (TEM) is the primary method for studying pollen tube growth. The preparation of ultrathin sections of the pollen tube tip sample is important for its successful microscopic observation. The direction of pollen tube growth in vitro is irregular, and it is difficult to dissect the tip of the pollen tube during ultrathin sectioning. Here, we used two methods to efficiently obtain an ultrathin section of the pollen tube tip of Pyrus. In the first method, laser micro-cutting was used to obtain the pollen tube tip, followed by ultrathin sectioning. In the other method, the pollen tubes were cultured in the same growth direction, followed by ultrathin sectioning. Ultrathin sections, which were observed via TEM, showed typical characteristics of the pollen tube tip, such as dense vesicles, numerous mitochondria, and secretory vesicles of the Golgi. We concluded that these two methods are effective in pollen tube tip sample preparation for ultrathin sectioning and provide the foundation for observing the ultrastructure of pollen tube tips.

Ultrastructural features of dentinoenamel junction revealed by focused gallium ion beam milling.

To take full advantage of focused ion beam (FIB) in preparation of ultrathin sections of biological tissues, we have used a cryo-milling process. In this study, extracted human teeth were scanned by optical coherence tomography to inspect the samples for intactness and to determine the area of interest. Then, the selected area of interest was cross-sectioned for examination under a confocal laser scanning microscope to determine the target location of the dentinoenamel junction (DEJ) that was later milled by cryo-FIB at preset parameters, followed by transmission electron microscope examination of the final sliced specimens for ultrastructural characterization. The proposed technique was able to outline the DEJ and to identify the different tooth layers in a single section, without artefacts or tissue damage. The DEJ was outlined as fine longitudinal projections intermingling between the solid electron-dense enamel and intricate electron-lucent hollow dentin. In conclusion, this study has shown the great potential of cryo-FIB in handling different biological tissues having different physical properties, with great precision and accuracy and minimum artefacts.

The Technology in Sample Preparation for Transmission Electron Microscopy and Ultrastructure Observation of Lung Tissue in Rats with Experimental Silicosis

ts hard to get ideal ultrathin sections because of the adamant SiO2 dust in silicosis, after perfusion fixation methods and strict control of the cutting speed, improving the success rate of the Silicosis tissue TEM sample preparation of ultrathin sections,so we can more clearly and accurately observed ultrastructural changes of silicosis,and it also can offer morphological basis for research the silicosis organizations function histological changes.

Morphological studies on machined implants of commercially pure titanium and titanium alloy (Ti6Al4V) in the rabbit

The aim of this study was to evaluate the bone response to commercially pure titanium grade I and titanium alloy grade V (90% Ti, 6% Al, and 4% V, depicted Ti6Al4V) after 8 weeks in rabbit tibia. Interference microscopy and scanning electron microscopy were used for surface analyses. Transmission electron microscopy (TEM) was used for evaluation of surface crystallinity and chemistry after preparation of ultrathin sections using focused ion beam (FIB) microscopy. Three different embedding resins commonly used for histological preparation were evaluated with respect to adaptation to a turned implant surface. Epoxy Agar 100 resin and acrylic Technovit 7200 resin showed low separation while acrylic LR White resin showed large separation at the interface. The retrieved specimens were embedded in acrylic Technovit 7200 resin after fixation and dehydration. The histological evaluation revealed osseointegration for both c.p. titanium grade I and Ti6Al4V alloy, but no quantitative differences in bone contact and bone area were detected. Because a separation of implant and tissue occurred in the interface between implant and bone embedded in acrylic Technovit 7200 resin, additional factors related to implant surface properties and technical procedures are likely to influence the possibilities to prepare ultrathin sections by FIB.

A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis.

The epidermis of higher plants generates the cuticle layer that covers the outer surface of each plant. The cuticle plays a crucial role in plant development, and some mutants with defective cuticle exhibit morphological abnormalities, such as the fusion of organs. The way in which the cuticle forms and its contribution to morphogenesis are poorly understood. Conventional detection of the cuticle by transmission electron microscopy (TEM) requires laborious procedures, which include fixation, staining with osmium, and preparation of ultra-thin sections. It is also difficult to survey entire surfaces of expanded leaves because of the limited size of specimens that can be examined. Thus, TEM is unsuitable for large-scale screening for mutants with defective cuticle. We describe here a rapid and inexpensive method, designated the toluidine-blue (TB) test, for detection of cuticular defects in whole leaves. We demonstrated the validity of the TB test using mutants of Arabidopsis thaliana, including abnormal leaf shape1 (ale1), fiddlehead (fdh), and five eceriferum (cer) mutants, in which the structure and/or function of the cuticle is abnormal. Genetic screening for mutants using the TB test allowed us to identify seven loci. The cuticle-defective regions of leaves of the mutants revealed five intrinsic patterns of surface defects (classes I through V), suggesting that formation of functional cuticle on leaves involves various spatially regulated factors.

Ultrastructure of Achilles tendon from rats after treatment with fleroxacin.

Quinolone therapy can be associated with tendon disorders (tendinitis, ruptures), but little is known about possible ultrastructural changes in tendons after exposure to these antimicrobials. We studied the Achilles tendons from fleroxacin-treated adult rats by electron microscopy. Wistar rats were treated orally with single oral doses of 0, 30, 100, 300 or 600 mg fleroxacin/kg body weight (n = 6 per group). The animals were killed 4 weeks after treatment. Achilles tendon samples were collected and tangential sections were made from the distal part of the tendon. Subsequently, tendons were cut crosswise for preparation of ultrathin sections. Samples were fixed by using glutaraldehyde, osmium tetroxide, tannic acid and finally contrasted with uranyl acetate/lead citrate before they were examined by transmission electron microscopy. The rats did not show any general effects such as behavioural changes or body weight changes which could be attributed to the treatment. However, we were able to detect pathological changes even at the lowest dose level (30 mg/kg), which increased in incidence and severity with increasing doses. Tenocytes exhibited degenerative changes such as multiple vacuoles and large vesicles in the cytoplasm that resulted from swelling and dilatation of cell organelles (mitochondria, endoplasmic reticulum). The nucleus became dense and the chromatin had clumped to form rough plaques. The cells detached from the extracellular matrix. Other important findings were a general decrease of the fibril diameter and an increase in the distance between the collagenous fibrils. The finding that these rather low single dose of a fluoroquinolone induce ultrastructural changes in Achilles tendons from rats, which were not associated with clinical symptoms and which were still present 4 weeks after treatment, is of concern. Further toxicological as well as clinical studies are needed to characterize the conditions under which quinolone-induced tendon lesions develop.

The ultrastructure of the periodontal ligament cells-titanium interface with vitro

Several studies have shown that a peridontal ligament can form on titanium dental implants which were placed in contact with the periodontal ligament of the retained roots. However, the following question is still critical: how do the periodontal ligament cells adhere to the implant surface? Little is known about the ultrastructure of the periodontal ligament cells-titanium interface. The objective of this study, therefore, was to gain insight into the morphology of the interface. Human periodontal ligament cells were cultured on thin titanium substrates obtained by electron beam evaporation of titanium. This technique allowed for the preparation of ultra-thin sections for transmission electron microscopy containing titanium substrate as well as the cells adhering to it. Transmission electron microscopy showed that periodontal ligament cells not only came into close contacts with the titanium surface, but also extended numerous cellular processes that contained microfilaments and an electron-dense plaque. In addition, collagen fibres were observed in some zones of the extracellular matrix contacts. The results provide evidence that periodontal ligament cells can develop attachments on titanium surface.

Novel TEM approaches to imaging of microstructures in carbonates; clues to growth mechanisms in calcite and dolomite

Relating microstructures imaged by transmission electron microscopy (TEM) to specific growth mechanisms requires imaging of both the surface microtopography on crystal faces and its corresponding microstructure in the bulk crystal. Pt-C replicas of as-grown surfaces are ideally suited to this task, as they provide a high-resolution, nearly three-dimensional image of surface topography that can be correlated with microstructures visible in sections at high angle to the as-grown surface. Ultramicrotomy enables the preparation of ultrathin sections more quickly than conventional ion-thinning and can be used to investigate chemical heterogeneities by analytical electron microscopy (AEM). We evaluate the potential of both techniques for the study of microstructures in calcite and dolomite. (1) TEM images were obtained from Pt-C replicas of synthetic calcite. The as-grown (101̅4) face of a Mg/Mndoped crystal growth showed growth hillocks aligned along preferred orientations. In Pb/Mn/Srdoped calcite, sections at high angle to as-grown faces showed a uniform microstructure in {101̅4} sectors. The {011̅2} sectors contained concentric zones that alternated from striated to uniform microstructures, suggesting periodic variations in growth rate and possibly in the growth mechanism. An oscillatory-zoned Mn-doped calcite showed fine-scale banding (30-150 mm) and periodic roughening of (101̅4) surfaces that suggest repetitive transitions between growth mechanisms. (2) In sedimentary dolomites, Pt-C replicas of surfaces cleaved parallel to a (101̅4) face showed a hillocky topography or smooth (101̅4) surfaces modified by non-equivalent facets. Surfaces produced by cleavage at high angle to a (101̅4) face exposed sectors with fine-scale banding crosscut by sharp boundaries, suggestive of closely spaced growth hillocks intercalated with non-equivalent subsectors. Ultrathin sections prepared by microtomy showed some disruption of the crystal structure but, in some dolomite samples, domains with distinctive microstructures similar in scale and shape to those seen in the Pt-C replica were identified. (3) The growth microstructures in {101̅4} sectors of dolomite present striking similarities to the larger scale compositional zoning patterns produced by growth spirals in calcite doped with trace elements. At this stage, Pt-C replicas and ultramicrotomy can distinguish among dolomites that are petrographically very similar.

The Progression from Optical Light Microscopy to Transmission Electron Microscopy in the Study of Soils

AbstractThe use of electron microscopy to study clay microfabrics in thin-sections is discussed. A technique is described to isolate undisturbed microparts of pedofeatures from thin-sections, which are subsequently used for TEM analysis. Re-embedding with a polyester resin of undisturbed, in situ, neoformed clay microfabrics, obtained by microdrilling and preparation of ultrathin sections by microtoming with a diamond knife are emphasized; these steps enable micromorphology, clay mineralogy, microchemical and HRTEM analysis to be performed on one unique microsample of clay fabrics, with conserved micro-organization. Two examples on clay neoformation are presented to demonstrate that this technique can successfully be applied to unravel the impact of mineral alteration and clay neoformation in undisturbed soil samples on a micro- and a nanometer scale.

Detection of internal Pt clusters in zeolite FAU using serial sectioning with diamond-knife ultramicrotomy and HRTEM

Zeolite Y or X (FAU) has a very open framework structure with 13 Å diameter cages. Small metal clusters fit well in such cages. In catalytic research it is essential to localize the metal clusters; are they in the zeolite cavities or on the zeolite surface? In this study we will describe a method, using high resolution transmission electron microscopy (HRTEM) imaging of ultrathin sections, that give evidence that platinum is located within the zeolite. It will be shown that clusters preferably aggregate in the larger cages of the frequently occurring {111}-twin planes (TP). Zeolite NaY (Si/Al=2.8) crystals were ion exchanged with Pt(NH3)4 Cl2 according to standard literature procedures. After ion exchange the specimen was oxidized in O2 at 383 K for three hours and finally reduced in H2 at 298 K for two hours.The zeolite crystals were embedded in epoxy resin (Spurr) for preparation of ultrathin sections. The composition of the resin was 15.0 g vinylcyclohexene dioxide (ERL 4206), 6.0 g polypropylene glycol diglycidyl ether (DER 736), 39.0 g nonenyl succinic anhydride (NSA), and 0.3 g dimethylamino ethanol (S-l).

Growth and Dissolution Characteristics of Dolomite Studied in Ultrathin Section: A New TEM Approach

A sample preparation technique involving ultramicrotomy is presented for the preparation of ultrathin sections of single crystals of synthetic calcite and natural dolomite cements, This technique offers certain advantages over the conventional ion-thinning technique (Wenk et al., 1983) since more material can be prepared and surveyed more quickly by transmission electron microscopy (TEM). On the other hand, the high shear strain involved in ultramicrotomy induces a nearly pervasive microcleavage parallel to {10i4} and only fragments from the original crystal are recovered. However, individual crystal fragments remain largely coherent and the nearlypervasive microcleavage highlights differences in the physical properties among different carbonate phases. TEM observations of ultrathin sections from diagenetic dolomites show that the microcleavage is not necessarily uniform through single growth sectors and reveal a pronounced domain structure. The effect of intracrystalline compositional and structural heterogeneity on the reactivity of carbonate minerals can also studied qualitatively by exposing ultrathin sections to various solutions directly on a TEM grid.

In situ Hybridization at Electron Microscopic Level.

In the current study, we developed an improved method for ISH at the electron microscopic level, in which we employed pre-embedding hybridization using a BrdU labeled probe followed by post-embedding immunoglobuline gold colloid staining (IGS). Because metabolic labeling of BrdU provides an almost evenly labeled DNA probe with high labeling index, pre-embeddingly hybridized BrdU-labeled probe preserves its detectable antigenicity even after the preparation of ultrathin sections. We demonstrated precise cell identification, early stage of gene expression and alternative splicing patterns using this technique.

Effects of monomeric acrylic embedding media on the antigenicity of two epitopes of the MIC2-encoded Ewing's sarcoma cell membrane antigen.

Comparative electron microscopic studies of pre- and postembedding immunolabeling experiments have shown that the antigenicity of some epitopes is lost during acrylic resin embedding of the respective tissues. In the present investigation we have tested the sensitivities of two embedding-labile epitopes (HBA-71 and HBA-45) of the Ewing's sarcoma-associated MIC2-encoded E2 antigen to the effects of the different treatment steps, which are necessary for the preparation of ultrathin sections. The extent of antigenic retention was quantitated using flow cytometry and enzyme-linked immunosorbent assays (ELISA) of tumor cell lines, thymocytes and cell membrane extracts. Fixation, dehydration and high temperature treatment of MIC2-positive cells showed only minor effects on the reactivity with the HBA-71 and HBA-45 antibodies. However, exposure of the cells to the monomeric acrylic resins LR White (LRW), LR Gold (LRG) and Lowicryl K4M at 4 degrees C for 2-18 h resulted in a significant reduction of the HBA-71 and HBA-45 reactivities. In contrast, the antigenicity of both epitopes was maintained during treatment with the apolar Lowicryl HM20 embedding medium under these non-polymerizing conditions. The resins have no direct effect on the HBA-71/HBA-45 antigen, since it could be extracted in intact form from membranes of native, but not of fixed, tumour cells using LRW for membrane solubilization. These data indicate that the HBA-71/HBA-45 antigen remains in the cell membrane and is indirectly influenced by the extraction/modification of adjacent membrane constituents. The adverse effects of the polymerization process, in the case of embedding at low temperature in Lowicryl HM20, destroyed MIC2-antigenicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Method for ultrastructural studies of the intact tissue-metal interface

Samples were prepared for ultrastructural studies of the intact interface between metallic implants and tissue by transmission electron microscopy. The method is based on plastic embedding of implant and tissue and subsequent removal of the bulk metal by electrochemical dissolution (electropolishing), to facilitate preparation of ultrathin sections for transmission electron microscopy. Surface sensitive spectroscopy (Auger electron microscopy and X-ray photoemission spectroscopy) and transmission electron microscopy EDX results show that the method produces samples with an intact interface, containing the implant surface oxide and the adjacent tissue. Examples of application of the method on titanium, zirconium and aluminium implants in soft tissue are given.

The Diamond Knife: A Necessary Tool in Light and Electron Microscopy

Since the introduction by H. Fernández-Morán in 1953 [1], the diamond knife has found wide acceptance by the majority of electron microscopists for the preparation of ultrathin sections of biological samples.Through the years the basic architecture of the knife itself remained unchanged: The cutting edge was formed by the intersection of two planes making an angle of approximately 45°. It is assumed that this angle was taken from the nominal 45° angle of the glass knives.Reasons for keeping such values were: By in large, 45° angle diamond knives gave satisfactory results for morphological studies. Factors, other than the sectioning artifacts, seemed to be more important for limiting image quality. In recent years electron microscopes have been developed to an extremely high performance, the resolution reaching atomic level. Due to all of these developments, ultramicrotomy became a needed tool not only for plastic embedded and frozen biological samples, but also in materials research for virtually all types of materials.

The Diamond Knife: A Necessary Tool in Light and Electron Microscopy

Since the introduction by H. Fernández-Morán in 1953 [1], the diamond knife has found wide acceptance by the majority of electron microscopists for the preparation of ultrathin sections of biological samples.Through the years the basic architecture of the knife itself remained unchanged: The cutting edge was formed by the intersection of two planes making an angle of approximately 45°. It is assumed that this angle was taken from the nominal 45° angle of the glass knives.Reasons for keeping such values were: By in large, 45° angle diamond knives gave satisfactory results for morphological studies. Factors, other than the sectioning artifacts, seemed to be more important for limiting image quality. In recent years electron microscopes have been developed to an extremely high performance, the resolution reaching atomic level. Due to all of these developments, ultramicrotomy became a needed tool not only for plastic embedded and frozen biological samples, but also in materials research for virtually all types of materials.

Specific use of electron microscopic techniques in the characterization of ABS polymers

The supermolecular structure of various types of ABS polymers was characterized by employing various preparation and electron-microscopic observation techniques. The results thus obtained were compared with the classical techniques of preparation of ultra-thin sections. The techniques were chosen so as to reveal the structure of the interior of the sample and the rubber particles (material contrast on cut surfaces, separation of particles by dissolving the matrix, fracture at low and high temperatures), the surface structure (surface etching) and the mechanism of the fracture process (fracture surface at room temperature). By treatment of the fracture surfaces (relaxation in vapours of the solvent of the matrix or OsO 4-staining), rubber particles on the fracture surface or adjacent to it can be revealed. The size distribution of rubber particles, determined by using one sample and most of the methods mentioned above, shows an agreement in the range of the most frequently occurring particles. Systematic deviations in the range of large particles (aggregates) are explained by differences between the individual preparation methods.

Ultrastructural study of epithelial cell attachment to implant materials.

Attachment of gingival tissues to the protruding post of an endosseous dental implant is of great importance for the prognosis of its clinical success. Little is known about the requirements an implant material must meet to secure a durable permucosal seal. The objective of this study, therefore, was to gain insight into the morphology of epithelial cell-implant interfaces for a number of well-known or potential implant materials. Guinea pig epithelial cells were cultured on gold, titanium, carbon, hydroxylapatite, carbonate apatite, and modified polystyrene substrates. The metallic and carbon substrates were obtained by vapor-phase deposition of thin films on polystyrene carriers. This technique allowed for the preparation of ultra-thin sections for transmission electron microscopy containing substrate as well as the cells adhering to it. The cultures on apatite were decalcified prior to sectioning. The results revealed tht as attachment structures, focal contacts, extracellular matrix contacts, and hemidesmosome-like contracts could be distinguished. Hemidesmosome-like contacts were only observed on apatite and polystyrene and not on the metallic or carbon surfaces. The results of this study suggest the existence of some, as yet unknown, property of the substrate that determines the nature and the structure of the contact site with epithelial cells.

The structure of ?-FeOOH�Cln (akaganeite) and its uptake of amino acids

Yellowish brown precipitates in samples of Red Sea hot brine have recently been identified as β-FeOOH·Cln or akaganeite. Synthetic β-FeOOH·Cln has been prepared by hydrolysis of FeCl3-solution for transmission electron microscope studies of the structure of this iron (III) oxide hydroxide. Extreme care has been taken to avoid destabilization of the crystalline compound during preparation of ultrathin sections for the uptake of micrographs. The results indicate that previously reported and debated structures in the size range of 40–60 A of the somatoidic akaganeite crystals are no attifacts but instead a superimposed structure that is sensitive to washing and drying procedures. This superimposed structure might be important for the uptake of amino acids by β-FeOOH·Cln and may perhaps also be suitable for catalytic effects such as prebiotic synthesis of polypeptides.