Heavy Metal Staining Research Articles

Electron holography of polymer blends

The characterization of polymer blend morphology by TEM often requires objectionable heavy metal staining to enhance contrast, enabling phase differentiation of materials with otherwise similar electron scattering properties. In some cases, the presence of a higher atomic number species (O, N, Cl, F, etc.) in the molecular framework of one phase provides sufficient contrast without staining. In the present example the only inherent contrast mechanism for a blend of polystyrene/polyethylene/ethylene-styrene copolymer (PS/PE/E-S) should be diffraction contrast due to the crystalline PE. Electron holography was investigated as an additional method of characterizing polymer blends.Electron holography was first described by Gabor in 1948 but did not become a practical tool until recently, due to the availability of field emission gun (FEG) TEMs equipped with an electron bi-prism. Electron holography is a wave front reconstruction process that involves hologram formation and image reconstruction. An interference pattern (hologram) between a reference electron beam (passing through vacuum) and a beam passing through the specimen contains both the amplitude and phase of the transmitted electron wave.

Simultaneous high-resolution SEM and STEM of organic compounds and biological macromolecules

A simple spray mounting preparation of hydrocarbon based macromolecules onto coated grids or Si chips was employed for secondary electron (HRSEM) or in tandem with scanning transmission electron (STEM) imaging. The grid specimens were staged in the lens of a Topcon DS-130 SEM equipped with a Schottky field emission (SFE) source. A 1 nm probe, created at 25 kV, elicited low radiation damage to the molecules on the formvar support. Molecules on grids were stabilized with heavy metal stains and contrast enhanced by a 1 nm fine-grain Cr film. The strategy was to generate sufficient SE-I electrons for above-lens detection simultaneous with the collection of transmitted electrons by the below specimen STEM detector.The bromine salt of a synthetic amphiphile, N-tetracosanyl-tetraethylenepentaammonium pentabromide was produced as a preliminary micelle solution of [10mM]. After vigorous sonication the solution was diluted to [1mM] with dH2O, filtered through 0.65μm polycarbonate membrane, and mixed vol./vol. with 2% uranyl acetate (freshly prepared and filtered through 0.2μm PC membrane).

Microwave-stimulated heavy metal staining of polymer blends for TEM

Microwave methods for fixation and staining of biological samples for microscopy have been under development for a decade. While microwave ovens are routinely used in the synthetic polymer field for sample digestion and ashing, potential microwave staining techniques for TEM of polymers have yet to be thoroughly investigated. Preliminary experiments have demonstrated that copper grids do not cause arcing problems in the microwave, and that most types of polymer sections can be safely and conveniently stained in an industrial microwave oven. The staining agent used was ruthenium tetroxide vapor generated from ruthenium trichloride. Improvements in the speed and selectivity of staining are attributed in part, but not entirely, to the increased reaction rates at higher temperatures compared to standard room temperature vapor staining techniques. Stringent waste disposal regulations make microwave staining an attractive method from the industrial hygiene point of view, with a minimum of toxic waste generation compared to staining with RuO4/CCl4 solutions.

Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the SOS monosomes and the large (608) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the “native” eukaryotic ribosome 3-D model.

Computerized oceanic particle characterization using heavy metal staining, SEM, EDXS and image analysis

Heterogeneous particles collected from seawater were analysed for physical dimensions and elemental composition using a computer-controlled image and chemical analysis system based on a scanning electron microscope and energy dispersive X-ray spectrometer. Staining the particles with the heavy metal ruthenium largely overcame the difficulties of forming an adequate image of organic particles that usually limits such automated analyses. Ruthenium staining provides an image comparable to that achieved with metal coatings, improves the contrast of organic particles, and provides a more uniform background grey level. In addition, this treatment simplifies sample preparation and improves the X-ray count rate compared with metal-coated specimens. The major X-ray energy peak of ruthenium interferes only with that of chlorine, which is usually removed during the sample preparation procedures. In its present configuration, the system can perform analyses of particles from 0.5 to 100 μm in one pass for abundance, size, shape, and elemental composition at a rate of about 500 particles per hour. A generalized particle classification scheme based on elemental proportions and ratios is presented for suspended marine particles. The scheme is applied to samples from three depths in the photic zone for a station in the Sargasso Sea. As illustrative data, particle abundance, volumes, and size-volume distributions are presented vs chemical class for the major types of particles found, and a bulk refractive index is calculated.

Three‐dimensional reconstruction of bovine intradural spinal root myelin by electron microscope tomography

Electron microscope tomography was used to reconstruct three-dimensionally the configuration of heavy metal staining in bovine intradural spinal root myelin. Samples were fixed with glutaraldehyde, exposed to osmium tetroxide, embedded, thin sectioned, and finally stained with uranyl-acetate and lead citrate. Reconstructions up to 4.2 nm resolution showed a non-uniform distribution of stain in the planes of individual cytoplasmic appositions (major dense lines). In each reconstructed major dense line the stain was distributed in striated, well-defined structures. Those structures appear to be nearly parallel between neighboring major dense lines. The distribution of stain in the Schmidt-Lanterman cleft did not resemble the distribution of stain in the major dense line; however, weak striations were present. Evidence that the striated structures are not an artifact due to image calculation is discussed.

High‐angle annular dark‐field STEM imaging of immunogold labels

AbstractHigh‐angle annular dark‐field (HAADF) STEM imaging is a sensitive and efficient technique for detecting immunogold labels. Larger (5–15 nm) gold labels can be distinguished clearly from the heavy metal stain on tissue sections, but for smaller (<5 nm) labels the distinction is less clear. It is not possible to differentiate between ultra‐small (<1–3 nm) labels and stain, but the HAADF image shows sufficient contrast so specimens can remain unstained. On a TEM/STEM equipped with a LaB6 filament the smallest labels that are detectable are 1–2 nm. Efficient detection of ultra‐small labels requires a field emission microscope.

Simplified heavy metal staining techniques demonstrated with Fast Plant leaf tissue

Fast Plant (Brassica rapa, Cruciferae) leaf tissue fixed in glutaraldehyde-acrolein and post-fixed in osmium, was examined for response to several easily-prepared heavy metal stains. Lead and uranium, separately and in combination, gave typical results across the spectrum of cell organelles. As a single stain following osmium, bismuth produced images seemingly equivalent to lead and uranium. Phosphotungstic acid produced very good membrane delineation but produced a washed-out background image similar to that from lead staining. Carbohydrate compounds were especially responsive to ruthenium; the cytoplasm and the matrix of all organelles were also stained very well. The procedures were no more demanding than traditional staining methods and may be easily used in research and teaching. Fast Plant materials are a reliable, quick and easy source of living material.

Molecular architectural changes in hydrated macroporous styrene-divinylbenzene resin sorbents revealed by transmission electron microscopy using image analysis

A range of hydrophobic sorbent polymers based on crosslinked polystyrene have been prepared with high nominal crosslink ratios of 55–100%, and with varying levels of toluene as a porogen (comonomer/porogen volume ratios of 0.5 1 , 1 1 , 2 1 and 3 1 ). Contrast enhanced, energy filtered transmission electron micrographs, from dry and freeze-dried polymer embedded in Lowicryl resin and from frozen-hydrated sections of these sorbents, have been obtained in order to allow a direct visual comparison of these materials to be made without using heavy metal stains. In addition, ‘real-time’ electron microscopical images were captured via a video output to generate segmented pixel arrays in which grey level values were used to produce quantitative data relating to the polymers' pore structure. This was achieved by applying unsophisticated computer-assisted image analysis techniques. Despite the hydrophobic character of the sorbents, the results show clearly that these matrices expand significantly on hydration and that major changes in the pore sizes and their distribution take place. The relative changes observed within the matrix of sorbents examined are discussed in terms of the currently accepted structural models of pore structure. The techniques described offer the prospect of characterizing polymer sorbents in their hydrated and therefore applied state, and hence lay the foundation for a more rational design of sorbents in the future.

Interrelationships of ultrastructure and function in the microvasculature of normal and ischaemic myocardium

This paper reviews various methods involving electron microscopy that have been used to investigate the ultrastructure of the vasculature of the normal and diseased heart. Whereas scanning electron microscopy is more commonly employed to record surface topography, it can be used to examine freeze-fracture planes within the myocardium and, using heavy-metal staining and back-scattered electron imaging, to examine large 2-mu-thick resin-embedded sections through the heart. The latter technique allows the comparison of structural alterations across the wall of the heart and thus accurate definition of the transmural progression of pathological processes. Transmission electron microscopy can then be used to provide more detailed information from precisely localised regions. Human myocardium can be usefully studied up to 12 hours post-mortem provided that suitable control material is included. Intravascular tracers including low-viscosity resin and nuclear track emulsion can be used to determine whether or not particular vessels allow flow at the time of fixation, and thus changes in the pattern of flow through the microvasculature due to ischaemia and reperfusion can be quantified and defined. Particular care is required in the fixation of ischaemic tissues because oxygen dissolved in the fixative can lead to the rapid formation of oxygen-free radicals on contact with the tissue. This produces artefactual reoxygenation damage characterised by membrane disruption and cell and organelle swelling, which has previously been attributed to ischaemic injury per se. Bubbling glutaraldehyde with nitrogen substantially reduces this artefact.

Characterization of the attachment matrix ofPseudomonas fragiattached to non‐porous surfaces

Microbial attachment to food‐contact surfaces is of interest to those involved with the production, processing and safety of the food supply. The attachment of Pseudomonas fragi to food contact surfaces is reviewed. Initial efforts in the author's laboratory focused on methods to study the attachment of this organism to stainless steel. Scanning electron microscopy was utilized to visualize adherence of P. fragt to stainless steel. Improved visualization was obtained by adding ruthenium red and sodium cacodylate buffer to the SEM fixation procedure. Heavy metal staining and transmission electron microscopy were used to tentatively identify the attachment matrix as a complex polysaccharide. Further evidence from the use of specific chemicals and enzymes is presented which confirms that the material involved in the adherence of P. fragi to stainless steel is a complex polysaccharide.

Thermal evolution of organic matter in source rocks — Experimental and scanning electron microscopic studies

Five major forms (lamellar, banded, crack-like, disseminated and segregated encrustation) of organic matter distribution in source rocks have been revealed under scanning electron microscope by using the heavy metal staining technique. The degree of organic impregnation is related to the amount of liquid hydrocarbons in the rocks, and from this relationship a rough estimation of organic matter can be made on the basis of electron microscopic observations. In conjunction with experimental studies it has been found that the distribution forms of organic matter are a function of its maturity in the process of thermal evolution and accordingly some microscopic criteria can be developed for the assessment of source rocks.

Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron microscopy

We describe the development and application of a novel approach to high-resolution ultrastructural analysis of cells and tissues. It is based on the preparation of ultrathin frozen sections of fixed tissues, rinsing of the sections, followed by their embedding on the grid in a layer of vitrified ice, and direct observation with a cryoelectron microscope. Examination of smooth muscle, kidney and heart tissues showed that although no heavy metal staining was used, high-contrast images are obtained. Fine details of cytoplasmic filaments and organelles, membranes and membrane-associated structures, as well as connective-tissue elements are all visible. The new method is suitable for immunolabeling, including high resolution localization of specific molecules within the cytoplasm.

The effect of embedding resins on ultramicrotomed polymer membrane morphology

A variety of microporous polymeric membranes are used in separation processes. Because the unique chemical and physical properties of these membranes affect performance, determining the membrane ultrastructure is essential to understanding how the structure and performance are related.TEM examination of ultramicrotomed membrane cross-sections is the most appropriate means of observing the thin active edge where the critical separation process occurs. Observation and interpretation of the image, however, is often difficult due to the low electron scattering of the polymer and alteration of the ultra-structure by the embedding resin. To solve the problem of low contrast in polymer thin sections, heavy metal stains such as osmium tetroxide and ruthenium tetroxide are used to selectively increase mass at specific locations. These stains, however, have limited application and carry the potential for creating artifacts. When staining is effective or unnecessary, there is the other concern of how the embedding resin has effected polymer morphology. Previous work with biological material has shown the effect of different epoxies on contrast and the correlation between the mechanical properties of various resins and sectioning dynamics, but the potential alteration of polymer ultrastructure has largely been ignored.

Ultrastrctural identification of viral infections in paraffin sections stained by immunohistochemistry and in situ hybridization

Confirmation of viral infections in surgical biopsies is sometimes limited to routine paraffin sections because a specimen may not have been set aside for culture or culture results are negative. Since viral infections are frequently focal and localized to only a few cells, definitive histopathological diagnosis can be difficult to achieve even with immunohistoGhemistry (IHC), in situ hybridization (ISH) or electron microscopy. In this paper, we would like to demonstrate a siirple and direct approach to confirming viral infections in paraffin sections , using two immuno-labelling techniques coupled with electron microscopy.Paraffin sections of surgical specimens are deparaffinized in xylene and rehydrated through graded ethyl alcohol. Herpes simplex virus (Figure 1) and Toxoplasma gondii are localized immunohistochemically by the peroxidase antiperoxidase technique and Pneumocystis carinii (Figure 2) by 3 step ipdirect method, using 3,3'-diaminobenzidine tetrahydrochloride as the chrmogen, CMV ENA probe (ENZO Biochemicals) is used for in situ hybridization, following kit instructions (Figure 3 & 4). After the final chromogen reaction, wet sections are examined with a light microscope. Slides in vrfiich focal positive staining is difficult to interpret as specific are fixed in osmium tetroxide, dehydrated with graded acetone, and infiltrated with Poly/Bed 812. After polymerization, the epoxy blocks containing area of interest can be easily separated from the glass slides by heating on a 100 C hotplate for approximately 20 seconds . The area of interest should be positioned in the center of the block face for ultrathin sectioning. With careful adjustments of knife and microtcme, the area of interest can be precisely sectioned without loss of tissue. Ultrathin sections are collected on uncoated mesh grids for higher contrast and then directly examined by TEM. Peroxidase-labelled microorganisms or viral DHA can be easily localized and identified without heavy metal staining. Cellular details can be further increased by staining with uranyl acetate and lead citrate. Tissue remaining in block face after sectioning is available for IM comparison with the EM micrographs.

Same-section correlative light and electron microscopy

Epoxy embedded biological material, sectioned for conventional, intermediate or high-voltage electron microscopy (EM), can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy (LM). The contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation (glutaraldehyde with and without osmium postfixation), by heavy metal (uranyl and lead) staining, and by the section thickness. The ability to examine the specimen with the LM, within a section cut for EM, allows each section to be rapidly screened for content prior to examination in the EM. We have found this approach to be particularly useful for studies requiring the ultrastructural examination of a selected area or structure which is large enough to be visualized with the LM but which comprises only a small volume of the embedded material (e.g., centrosomes and nuclei within oocytes; specific regions of large protists.)Same section LM-EM can also be extended to sections cut from cells stained prior to embedding for the immunofluorescent localization of antigens using fluorescein, rhodamine or Texas red-conjugated antibodies. Under these conditions the sections show high resolution patterns of antigen-specific fluorescence against a background void of autoflouorescence. The fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section can be extended to structures as small as a single microtubule or just a few actin filaments . This approach of same section correlative fluorescence microscopy (FLM) and EM can eliminate the need, in many instances, to employ more complex procedures such as immunoferritin or immunogold for labeling antigens for ultrastructural detection.

The net distribution of elements in multiphase polymer materials via element‐specific electron microscopy

AbstractApplications of the Electron Spectroscopic Imaging technique (ESI) within the field of multiphase polymer materials are discussed. Imaging with elastically scattered electrons as well as imaging with electrons with defined energy loss are presented. From the latter the net distribution of elements can be derived.Several examples are given: Glassy diblock copolymers polystyrene‐block‐poly(2‐vinyl pyridine), polyethylenes, interpenetrating networks, interdiffusion couples, blends of polystyrene and polystyrene‐graft‐poly(oxyethylene), carbon black reinforced natural rubber, and epitactic growth of poly(1‐pentenylene) on isotactic poly (propylene) surface.Visualization of the rigid amorphous phase in polyethylene is presented, as well as electron spectroscopic diffraction (ESD) of oriented PE. One of the features of ESI is due to the fact that discrimination between true morphology and artefacts is possible in an unbiased manner, even for minor details: Heavy metal staining is abandonable, and structural analysis by one element may be proved by analysis with other elements, which may be present in the sample or which may be added by chemical reaction or interaction with groups within the molecules.

Electron spectroscopic imaging of DNA

Electron spectroscopic imaging (ESI) was carried out using a fixed beam electron microscope equipped with a parallel electron energy filter to form micrographs of purified plasmid DNA without the use of heavy metal stains and shadows. Inelastically scattered electrons that have ionized the phosphorus L II,III shell electrons were used to form phosphorus distribution maps of DNA and deoxyribonucleoprotein complexes. Signal-to-noise values of the net phosphorus content over single DNA molecules compared to two and four interwound DNA strands directly reflect the known stoichiometric levels of phosphorus content, illustrating that ESI can be used to determine the relative levels of nucleic acid in nucleoprotein complexes. An initial attempt to characterize nucleosomal and transcriptionally active chromatin from Saccharomyces cerevisiae with this technique reveals three distinct ultrastructural classes of the basic chromatin fiber.

Demonstration by staining and radioautography of cyclical distributions of protein at the enamel surface in rat incisors

Staining patterns in the enamel during the maturation stage of amelogenesis reflect the banded distribution of ruffle-ended and smooth-ended ameloblasts. This study investigated the possibility that proteins at the enamel surface may be distributed cyclically according to cyclical changes in ameloblast morphology. Dissected lower rat incisors were wiped free of their enamel organs and immediately immersed in fixative containing one of the following heavy metal and histological stains: uranyl acetate, lead citrate, Coomassie blue, alcian blue and ruthenium red. Other animals were injected with [ 35S]-methionine to label newly-formed enamel proteins. Their incisors were dissected, the enamel organs were wiped from the enamel surface, and the teeth were processed as whole mounts for radioautography. Teeth stained by heavy metals were also viewed by back-scattered electron imaging. The in-situ staining revealed that proteins were distributed in bands and stripes across maturing enamel. Radioautography revealed that the proteins in the stripes were newly-synthesized and secreted into the enamel by certain maturation ameloblasts. We conclude that the enamel organ expresses cyclical activity in part through secretion of proteins.

Ultrastructure and Analytical Microscopy of Silicon in the Leaf Cuticle of Ficus lyrata Warb.

Leaves of Ficus lyrata contain large deposits of silicon (Si) in the adaxial and abaxial cuticles. Si was also found in the external and internal periclinal and anticlinal walls of epidermal and subepidermal cells. The Si in the cuticle filled large interstices that were sometimes confluent with each other and with the underlying cell wall material. Si deposits were more abundant in the adaxial than in the abaxial surface. Samples prepared without heavy metal stains showed that Si deposits have high intrinsic electron density. Sections incubted in 5% HFI had Substantially less Si than adjacent serial, nontreated sections. The Accumulation of the Si in the cuticle of F. lyrata is consistent with the proposed mechanism of apoplastic movement of monosilicic acid in the transpiration stream.