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PubMed1983

Tannic acid- and thiocarbohydrazide-mediated osmium tetroxide binding in the preparation of human leucocytes for SEM observation.

Normal human leucocytes, successively treated with glutaraldehyde-tannic acid-osmium tetroxide-thiocarbohydrazide-osmium tetroxide-thiocarbohydrazide-osmium tetroxide, were prepared for scanning electron microscopy observation. These cells produced well-contrasted, non-charging scanning images compatible with metal-evaporated material. Further, the mononuclear and polymorphonuclear cells resisted shrinkage during dehydration and critical point drying, thus allowing much improved images at high magnification than those covered with evaporated metal. In all cases at least a second thiocarbohydrazide-osmium tetroxide treatment could not be avoided.

Fixatives↗
PubMed1982

Electron microscopic immunoperoxidase staining of insulin using 4-chloro-1-naphthol after osmium fixation.

Ultrastructural localization of insulin in B cells of guinea pig pancreas was accomplished after osmium fixation with an immunoperoxidase procedure that utilized 4-chloro-1-naphthol (CN) in the substrate solution. The principal features of this protocol were: a) osmium tetroxide postfixation; b) omission of hydrogen peroxide "etching"; c) use of CN instead of diaminobenzidine in the substrate solution; d) elimination of osmium tetroxide after the substrate reaction; e) uranyl acetate and lead citrate counterstaining. This procedure produces intense specific staining with low background using highly dilute antiserum, and appears to be useful for postembedding immunoperoxidase staining of a variety of peptide antigens in osmium-fixed tissue.

Animals↗
PubMed1984

Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings.

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.

Animals↗
PubMed1989

Antibodies to DNAs chemically modified with osmium structural probes.

It has previously been shown that osmium tetroxide, pyridine (Os,py) and osmium tetroxide, 2,2'-bipyridine (Os,bipy) are powerful probes of the DNA structure. To increase the possibilities of the detection of osmium-modified DNAs polyclonal antibodies against DNA modified with Os,py and Os,bipy were elicited in rabbits. Specificity of these sera or purified IgG was tested by ELISA and retardation of the DNA electrophoretic mobility in agarose gels. Antibodies against DNA-Os,py (anti-DNA-Os,py) reacted with single-stranded and double-stranded DNA-Os,py but they did not react with unmodified DNA; with DNA-Os,bipy only a weak reaction was observed. The specificity of the anti-DNA-Os,bipy was similar. Competition experiments with anti-DNA-Os,py showed a weak reaction with RNA-Os,py but no reaction with osmium-modified proteins and unmodified proteins and RNA. The results suggest that anti-DNA-Os,py may become an important tool in studies of DNA structure in situ.

Animals↗
PubMed1995

Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNA.

We present herein an improved method for the use of osmium ammine in a Feulgen-type reaction for specific staining of DNA at EM level and an analysis of its Schiff-type reagent behaviour. The activation of osmium ammine to a Schiff-type reagent (so far routinely performed by bubbling with gaseous SO2) can be accomplished by adding S0(2)-generating chemicals as for light microscopy. When used after HCI hydrolysis on epon or acrylate sections, activated osmium ammine behaves like a Schiff-type reagent, and the DNA staining can be selectively and completely abolished by aldehyde blocking agents. This preparation has the advantage of eliminating the use of gaseous SO2 thus rendering the technique more widely available to laboratories which cannot handle gas cylinders containing SO2. We recommend the use of osmium ammine in 8N acetic acid and 40mM sodium metabisulfite for 1 h at 37 degrees C for epon sections, and in 0.2N HCl and 0.2M metabisulfite for 30 min at room temperature for acrylate sections.

Adrenal Glands↗
PubMed1958

The contribution of lower oxides of osmium to the density of biological specimens in electron microscopy.

Centrifugally stratified eggs of the sand-dollar, Dendraster excentricus, have cytoplasmic structures segregated into distinct layers. Fat droplets, yolk granules, and mitochondria are separated by "hyaline" layers of protoplasm. Aggregations of particles of 150 to 200 A diameter ("heavy bodies") are found near the mitochondrial layer and a concentration gradient of free 150 to 200 A particles corresponds to a similar gradient of basophilia in thick sections. Eggs fixed in buffered osmium-tetroxide at pH 7.4 and embedded in methacrylate were sectioned and floated on a "bleaching" solution of acidified hydrogen peroxide. "Bleached" sections showed a considerable loss in general density and especially a loss in the sharp images of cellular membranes. It was shown that such loss is not due to sublimation of structure in the electron beam. Assuming that the "bleach" acts principally to reoxidize lower oxides of osmium, it was concluded that reduced and bound lower oxides of osmium play a major role in creation of the electron micrograph image, especially in the delineation of phospholipide components of cellular membranes. Particles of 150 to 200 A diameter showed little or no loss in density, but rather a high intrinsic electron density. Refractometric data were presented to substantiate the tentative conclusions.

Animals↗
PubMed2004

Road maps for nitrogen-transfer catalysis: the challenge of the osmium(VIII)-catalyzed diamination.

Although the Sharpless dihydroxylation has been used on laboratory and industrial scales for several decades, an analogous osmium-catalyzed diamination is unknown. To explore the reaction of osmium(VIII) oxo-imido complexes with C=C bonds, density functional calculations have been performed. The calculations predict a chemoselective and perispecific [3+2] addition of the NH=Os=NH moiety of diimidodioxoosmium(VIII) to ethylene, yielding dioxoosma-2,5-diazolidine. At first sight, this metallacycle seems extremely stable; it is more stable than diimidoosma-2,5-dioxolane by 40 kcal mol(-1). However, a comparison of the thermodynamic reaction profiles for catalytic model cycles of dihydroxylation, aminohydroxylation, and diamination reveals that, contrary to common belief, the instability of the metal=N bond in the osmium(VIII) imido complex rather than the stability of the metal-N bond in the osmium(VI) intermediate causes most of the energy difference between the metallacycles. Substituents on the substrate have a small effect on the thermodynamic reaction profiles, whereas substituents on the imido ligands allow steric and electronic control of the reaction free enthalpies in the range of up to 25 kcal mol(-1). The results of this study help identify potential challenges in the development of the as-yet hypothetical title reaction and provide a modular concept for exploring novel catalytic routes.

Journal Article↗
PubMed2003

Photosensitized generation of singlet oxygen from ruthenium(II) and osmium(II) bipyridyl complexes.

Photophysical properties for a number ruthenium(II) and osmium(II) bipyridyl complexes are reported in dilute acetonitrile solution. The lifetimes of the excited metal to ligand charge transfer states (MLCT) of the osmium complexes are shorter than for the ruthenium complexes. Rate constants, kq, for quenching of the lowest excited metal to ligand charge transfer states by molecular oxygen are found to be in the range (1.1-7.7) x 10(9) dm3 mol(-1) s(-1). Efficiencies of singlet oxygen production, fDeltaT, following oxygen quenching of the lowest excited states of these ruthenium and osmium complexes are in the range of 0.10-0.72, lower values being associated with those compounds having lower oxidation potentials. The rate constants for quenching of the excited MLCT states, kq, are found to be generally higher for osmium complexes than for ruthenium complexes. Overall quenching rate constants, kq were found to give an inverse correlation with the energy of the excited state being quenched, and also to correlate with the oxidation potentials of the complexes. However, when the contribution of quenching due exclusively to energy transfer to produce singlet oxygen, kq1, is considered, its dependence on the energy of the excited states is more complex. Rate constants for quenching due to energy dissipation of the excited MLCT states without energy transfer, kq3, were found to show a clear correlation with the oxidation potential of the complexes. Factors affecting both the mechanism of oxygen quenching of the excited states and the efficiency of singlet oxygen generation following this quenching are discussed. These factors include the oxidation potential, the energy of the lowest excited state of the complexes and spin-orbit coupling constant of the central metal.

Journal Article↗
PubMed1996

Osmium Recycling in Subduction Zones

Peridotite xenoliths from the Cascade arc in the United States and in the Japan arc have neodymium and osmium isotopic compositions that are consistent with addition of 5 to 15 percent of subducted material to the present-day depleted mantle. These observations suggest that osmium can be partitioned into oxidized and chlorine-rich slab-derived fluids or melts. These results place new constraints on the behavior of osmium (and possibly other platinum group elements) during subduction of oceanic crust by showing that osmium can be transported into the mantle wedge.

Journal Article↗
PubMed1984

Electron staining of the cell surface coat by osmium-low ferrocyanide.

In aldehyde-fixed liver and renal cortex of rat and mouse several variations of postfixation with osmium tetroxide plus potassium ferrocyanide ( FeII ) were tried. Depending on the ferrocyanide concentration different staining patterns were observed in TEM. -Osmium-High Ferrocyanide [40 mM (approximately 1%) OsO4 + 36 mM (approximately 1.5%) FeII , pH 10.4], stains membranes and glycogen. Cytoplasmic ground substance, mitochondrial matrices and chromatin are partially extracted, cell surface coats remain unstained. Membrane contrast, but extraction too, are higher with solutions containing cacodylate- than phosphate-buffer. -Osmium-Low Ferrocyanide [40 mM (approximately 1%) OsO4 + 2 mM (approximately 0.08%) FeII , pH 7.4], stains cell surface coats and basal laminae, but not glycogen, except for special cases. The trilaminar structure of membranes is poorly delineated. Signs of cytoplasmic extraction are not visible. The surface coat staining is stronger and more widespread with solutions containing phosphate- instead of cacodylate-buffer; it is enhanced by section staining with lead citrate. The cell surface coat stain does not traverse tight junctions nor permeate membranes.

Animals↗
PubMed1979

The zinc iodide-osmium tetroxide staining-fixative of Maillet. Nature of the precipitate studied by x-ray microanalysis and detection of Ca2+-affinity subcellular sites in a tonic smooth muscle.

A mechanism of osmium reduction during zinc iodide-osmium tetroxide (ZIO) fixation is proposed. X-ray powder microanalyses of ZIO precipitates formed both in the presence or absence of tissues are identical with those of CuOsO4 and CuRuO4. Therefore, and based on indexation methods, ZnOsO4 was found to be the formula of the ZIO mixture reduction; this zinc osmate has an orthorhombic crystalline lattice. In smooth muscle preparations, ZIO electron dense deposits are localized in both cisternae of the sarcoplasmic reticulum and in mitochondria after a short fixation time. According to the microanalysis results, the zinc osmate has been associated to Ca2+ high affinity sites since Zn2+ is either replacing Ca2+ and/or displacing it by having a higher affinity for Ca2+ binding sites. Consequently, the ZIO mixture might be useful in revealing some Ca2+ storage sites in cells. This hypothesis was tested in ABRM preparations by selectively depleting sites which are known to bind Ca2+. In this case, the sarcoplasmic reticulum only retains the staining deposits after a short ZIO fixation. It is likely that OsO4 alone, used as fixative in cytology might be due to the formation of metallic osmates (e.g., divalent osmates like CaOsO4). In addition, of course, reduction of osmium during tissue fixation is accompanied by oxidation of double bonds of lipoproteic complexes or unsaturated lipids, and oxidation of sulfhydryl groups and amino groups.

Animals↗
PubMed1985

Osmium tetroxide postfixation in relation to the crosslinkage and spatial organization of stereocilia in the guinea-pig cochlea.

Guinea-pig cochleae were fixed in glutaraldehyde, followed by short (30 s), intermediate (3 min) or long (24 h) periods of postfixation in 1% osmium tetroxide. The hair cells of the organ of Corti were examined by scanning electron microscopy. Following short periods of postfixation in osmium tetroxide, the preservation of crosslinks between the stereocilia could be enhanced, compared to the picture seen without postfixation. Moreover, in many cases the stereocilia parted slightly, revealing many lateral links between the stereocilia, which were otherwise hidden. The tip of each shorter stereocilium on a hair cell gave rise to a single, vertically pointing link, which ran upwards to the taller stereocilium of the adjacent row on the hair cell. It is suggested that distortion of such links is associated with sensory transduction. An extensive array of lateral links, connecting the stereocilia of the same and different rows on the hair cell, was also seen. In addition, the surface membranes of the stereocilia had a granular appearance. Following intermediate and long periods of postfixation in osmium tetroxide, crosslinks were rarely seen, and the stereocilia had smooth or wavy rather than granular surface membranes. Moreover, the spatial organization of the stereocilia, particularly on inner hair cells, was disrupted. In tissue postfixed for short periods, it was possible to map the directions of the vertically pointing links, and see the three-dimensional arrangement of the stereocilia. It was shown that the V-shape of the rows of stereocilia on outer hair cells, and the straight-line arrangement of the stereocilia on inner hair cells, were both appropriate for a maximal sensitivity of the hair cells to deflection of their stereocilia in a radial direction. It is suggested that the differences in the shape of the rows on inner and outer hair cells are derived from the different packing of the stereocilia on the two types of hair cell.

Animals↗
PubMed1983

Determination of nanogram quantities of osmium-labeled nucleic acids by stripping (inverse) voltammetry.

Modification of nucleic acids with OSO4 in the presence of pyridine results in a formation of a covalently bound electroactive center in a polynucleotide chain detectable by polarographic (voltammetric) methods. It has been shown that DNA modified with osmium (DNA-Os) accumulates at the hanging mercury-drop electrode during a waiting time in a wide range of potentials between 0 and -1.0 V (against the saturated calomel electrode) and produce at neutral pH a well-developed reduction peak at about -1.2 V due to scanning in the cathodic direction. Using the differential-pulse stripping (inverse) voltammetry, nanogram quantities of single-stranded DNA-Os can be determined at relatively short waiting times (1-3 min). Double-stranded DNA is modified with osmium to a much lesser extent as compared to single-stranded polynucleotides. The degree of modification of double-helical DNA is influenced by the presence of single-stranded and distorted double-stranded regions in the DNA molecules and by the environmental conditions which influence the DNA conformation. Osmium can thus be used as a probe of the DNA structure, and a few micrograms of double-helical DNA sample suffice for the voltammetric analysis.

Animals↗
PubMed1982

Zinc iodide-osmium staining of membrane-coating granules in keratinized and non-keratinized mammalian oral epithelium.

Specimens of keratinized and non-keratinized oral epithelium were examined in the electron microscope after being stained with zinc iodide-osmium. In both types of tissue, reaction was seen in unmyelinated nerves, in the specific granules of epithelial Langerhans cells and within lysosome-like organelles and small vesicles associated with Golgi systems. In keratinized epithelia, the reaction was also present in the membrane-coating granules and between the deepest cells of the keratinized layer. In contrast, the membrane-coating granules of non-keratinized epithelia lacked Zn iodide-osmium staining despite the presence of reaction in adjacent Golgi systems. It is suggested that Zn iodide-osmium stains glycolipid or glycoprotein material in the cell. This material is elaborated in the Golgi systems from which lysosomes and the membrane-coating granules of keratinized tissues are probably derived.

Animals↗
PubMed1989

Echinomycin-induced hypersensitivity to osmium tetroxide of DNA fragments incapable of forming Hoogsteen base pairs.

To show conclusively that the critical structural deformation of double-helical DNA that is induced by the binding of quinoxaline antibiotics does not involve the formation of Hoogsteen base pairs, we have prepared a DNA fragment containing the nucleoside analog 7-deaza-2'-deoxyadenosine in one of the two strands. This DNA fragment was subjected to treatment with the thymidine-specific reagent osmium tetroxide and to DNase I "footprinting" in the presence or absence of micromolar concentrations of echinomycin. We report that this anti-tumor antibiotic binds to DNA containing the nucleoside analog as well as to natural DNA and that the previously reported hypersensitivity to osmium tetroxide of certain thymidine residues adjacent to echinomycin binding sites is maintained in analog-containing DNA. Since these thymidines are rendered incapable of participating in Hoogsteen base pairs by the incorporation of 7-deaza-2'-deoxyadenosine, we conclude that this unusual base-pairing scheme is not the cause of the observed hypersensitivity to osmium tetroxide and that it therefore results from a large local unwinding of the DNA in the presence of the antibiotic. Moreover, preventing the possibility of Hoogsteen base pairing does not preclude echinomycin binding.

Aminoglycosides↗
PubMed1984

Histiocytic and dendritic reticulum cells shown by a zinc iodide-osmium technique.

Structures corresponding to histiocytic and dendritic reticulum cells have been shown in human tonsillar tissue, "reactive" lymph nodes and spleens by means of a zinc iodide-osmium technique. These cell types have been shown in various locations in these tissues using paraffin and resin embedded sections produced after fixation/staining of the tissue in zinc iodide-osmium. The quality of morphology attained by this procedure is much improved compared with the demonstration of the two cell types by means of alpha-naphthyl acetate esterase reactions performed on frozen sections. The zonal architecture of the lymphoid follicle is emphasised by this technique. In lymph nodes, sinus lining cells are also shown. Lymphoid cells, polymorphs, fibroblasts, and endothelial cells are negative with the zinc iodide-osmium method. In addition, interdigitating cells are not stained. The results of this procedure are compared with those with those of other methods for the demonstration of histiocytic and dendritic reticulum cells.

Cytological Techniques↗
PubMed1988

Osmium tetroxide probing of local DNA structure in linear and supercoiled plasmids containing curvature-inducing sequences.

Recombinant plasmids pK1A108, pK3A108, pK4A108 and pK5/6T217 containing 80 +/- 1 base pair inserts with different curvature-inducing sequences were studied using the DNA structure probe osmium tetroxide in the presence of pyridine (Os, py). The insertion sequences of the plasmids pK1A108, pK3A108, and pK4A108 are strongly related while the degree of curvature increases from pK1A108 (no curvature) less than pK3A108 less than pK4A108 less than pK5/6T217. The Os, py probe reacts selectively with single-stranded and distorted double-stranded regions in the DNA double helix. Nuclease S1 was used to recognize and cleave regions made permanently single-stranded due to osmium recognize and cleave regions made permanently single-stranded due to osmium modification. In linearized plasmids treatment with Os, py produced no S1-detectable site-specific modification. This result is in agreement with models suggested for DNA curvature; in general, continuous base pairing and base stacking is considered through different sequence blocks as well as through structural junctions. Os, py-probing of the plasmids in the supercoiled state also resulted in no S1-detectable site-specific modification within the inserts of pK1A108, pK3A108, and pK4A108 plasmids (while the regions containing inverted repeat nucleotide sequences in these plasmids were site-specifically modified). In contrast, supercoiled pK5/6T217 DNA was site-specifically modified within the curvature-inducing insert sequence. The nucleotide sequence of the insert of this plasmid strongly differs from the insertion sequences of the other three plasmids; it is extremely AT-rich and contains regularly arranged dAGAGA and dATATA sequences. The structural distortion observed in supercoiled pK5/6T217 is most probably due to the presence of these sequences in a particular arrangement in the insertion sequence.

Base Sequence↗
PubMed2004

The transillumination possibility of imidazole-osmium postfixed muscle tissue and its consequences for the handling of muscle tissue samples.

The osmium postfixation of tissue leads to good results for transmission electron microscopy, but also produces completely blackened tissue samples that do not allow the recognition of internal structures. With imidazole-osmium postfixation, one achieves comparable results in high electron microscopic resolution as with routine osmium postfixation. But the tissue samples are not blackened and can, therefore, be transilluminated with point light sources. The new postfixation technique makes it possible to recognize histological details such as vessels, nerve fibers, and the cross-banding pattern in an untrimmed block. This makes it possible to screen-embedded tissue samples for appropriate ultrastructural processing.

Animals↗