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PubMed1984

A sulphide-osmium procedure for detection of metal-containing synaptic boutons in the lizard cerebral cortex.

Two procedures of the Timm sulphide-silver method for the ultrastructural location of heavy metals and a sulphide-osmium procedure proposed here were applied to studying the Timm-reacting axonal boutons of the lizard cerebral cortex. It was shown that metal-containing boutons which would be labelled when using the Timm method, could be labelled by the sulphide-osmium procedure as well, i.e. perfusion and immersion in a glutaraldehyde fixative saturated with H2S vapour and then osmium postfixation. When the sulphide-osmium procedure was used, Timm-reacting boutons displayed an electron-dense loading in some of their synaptic vesicles. The histochemical significance of this labelling is suggested; metal contents (possibly zinc) in the synaptic vesicles are first transformed into metal sulphides that afterwards interact with OSO4 and render an electron-dense labelling. Moreover, chain-like electron-dense aggregates, which could not be detected in the Timm method, have been shown in endoplasmic reticulum cisternae using the sulphide-osmium procedure. The importance of both the Timm method and the sulphide-osmium procedure in ultrastructural studies in the lizard cerebral cortex could be paramount, as they define specifically one of the projection systems in the reptilian cortex. The sulphide-osmium procedure could be more useful than the Timm method since it is a simple procedure that avoids physical development, allows a better ultrastructural quality and a more precise location of the labelling.

Animals↗

PubMed2004

FTIR study of the ethanol electrooxidation on Pt(100) modified by osmium nanodeposits.

In the present work, ethanol electrooxidation on a Pt(100) electrode modified by different coverage degrees of osmium nanoislands obtained by spontaneous depositions, was extensively studied employing in situ FTIR spectroscopy. A collection of spectra of the ethanol adsorption and oxidation processes was acquired during the first series of a positive potential step, to determine the intermediate species, as well as the main products formed. The spectroscopic results obtained were correlated with conventional electrochemical results obtained by cyclic voltammetry. It was shown that the catalytic activity of Pt(100) for ethanol oxidation increases significantly after osmium deposition and that the mechanistic pathway for this reaction depends directly on the osmium coverage degree. Thus, for low osmium coverage (theta;( Os) up to 0.15) the formation of CO as an intermediate was favored and hence the full oxidation of adsorbed ethanol to CO(2) was increased. For higher osmium coverages (theta;(Os) up to 0.33), the higher the coverage is, the more the direct ethanol oxidation to acetaldehyde and acetic acid is favored. For osmium coverage degree of 0.40, the catalytic activity of the electrode for ethanol oxidation decreased. On an almost complete osmium layer (theta;(Os) = 0.92) obtained by electrodeposition at 50 mV vs reversible hydrogen electrode, the catalytic activity for ethanol oxidation shows a much lower value.

Electrochemistry↗

PubMed1983

Osmium-impregnation patterns of the Golgi complex in the epididymal epithelial cells of castrated and testosterone-injected mice.

After prolonged exposure of mouse epididymal epithelial cells to a solution of osmium tetroxide, reduced osmium compounds were detected in the Golgi cisternae and in the cytoplasm adjacent to the Golgi complex. Their appearance changed in time under certain conditions. Eight days after castration each cisterna of the regularly arranged Golgi lamellae fragmented into small vesicles, in which deposits of reduced osmium compound were in reduced amounts or completely absent, but no notable decrease of the fine reduced osmium particles in the cytoplasm adjacent to the Golgi complex occurred. The amount of deposit in the Golgi cisternae in castrated mice recovered to the normal level after the subcutaneous injection of testosterone for two weeks. On the other hand, the osmium particles observed in the cytoplasm adjacent to the Golgi complex during the recovery process of the lamellar structure increased in amount. This study showed that the reduced osmium compounds were of two kinds, and that the deposits contained in the Golgi cisternae were related to secretory products, and might be controlled by hormonal factors, but the fine reduced osmium particles that appeared in the cytoplasm adjacent to the Golgi complex might be regulated by other factors.

Animals↗

PubMed1989

Factors affecting preparation of chromosomes for scanning electron microscopy using osmium impregnation.

Osmium impregnation techniques have become useful for imparting conductivity to tissue specimens for SEM, thereby avoiding coating with gold or other metals. Such techniques have been developed to produce aesthetically pleasing images of mammalian (particularly human) chromosomes prepared by standard cytogenetical methods which use methanol-acetic acid fixation. The present study was designed: (1) to examine changes in the appearance of chromosomes as a result of preparation by osmium impregnation techniques; (2) to assess the function and importance of the various stages of chromosome preparation; and (3) to identify the chemical groups responsible for osmium binding. Methanol-acetic acid fixed chromosomes are known to have lost many proteins during fixation, and appear to be flattened down on the substrate. Osmium impregnation swells these flattened chromosomes to a variable extent, but the result is inevitably an artefact, albeit a useful one, and not a true representation of the chromosome in vivo. The size of chromatin fibres, for example, is the consequence of the degree of protein extraction during fixation, the loss of material during pre-treatments (e.g. trypsin), and the amount of osmium uptake during impregnation. Trypsin pre-treatment removes a surface coating of protein from the chromosomes as well as exposing chemical groups which can react with osmium. The principal reactive site appears to be amino groups, which bind glutaraldehyde, which in turn binds thiocarbohydrazide, to which the osmium becomes attached. Pre-treatments other than trypsin can be used to extract chromosomal material and to reveal different aspects of chromosome structure.

Animals↗

PubMed1984

Osmium-induced alteration in DNA structure.

In the presence of pyridine and other ligands osmium tetroxide binds covalently to pyrimidine bases in DNA. Properties of osmium-modified native and denatured calf thymus DNA, and plasmid Co1E1 DNA were investigated by means of differential pulse polarography, absorption spectrophotometry, circular dichroism, agarose gel electrophoresis, and nuclease S1 digestion. A great difference in the reaction kinetics of native and denatured DNAs with osmium, pyridine was observed. On the ground of the slow stepwise reaction kinetics of native DNA in the initial stage of its modification by osmium it has been suggested that the primary reaction sites do not include bases contained in the intact double helix. Osmium binding to sporadic primary reaction sites (represented e.g. by bases in the vicinity of a single-strand break) in native calf thymus DNA resulted in local changes in DNA conformation limited to a close neighbourhood of the binding site. At higher osmium/nucleotide ratios disordering of the DNA structure over a region extending beyond the immediate binding site was observed. With denatured DNA the same type of structure disordering was detected already in the initial stage of the reaction at osmium/nucleotide ratios as low as 0.01. Osmium binding to the supercoiled Co1E1 DNA resulted in its relaxation without nicking and it increased its sensitivity to linearization by cleavage with nuclease S1. The behaviour of Co1E1 DNA has been explained by the formation of a denatured region in the molecule (accompanied by a coupled loss of duplex and superhelical turns). It has been suggested that osmium can be used to label and to visualize distorted regions in the DNA double helix.

Animals↗

PubMed1998

Direct measurement of femtomoles of osmium and the 187Os/186Os ratio in seawater

Two depth profiles of the osmium concentration and the 187Os/186Os isotopic ratio in the Indian Ocean showed that the osmium concentration seems to be unaltered by chemical or biological processes occuring in seawater; accordingly, osmium is conservative. These data were obtained from an experimental method that eliminated the problems related to osmium preconcentration. This method led to a new evaluation of the concentration of osmium in seawater; the mean concentration of osmium and the 187Os/186Os ratio are equal to 10.86 +/- 0.07 picograms per kilogram and 8.80 +/- 0.07, respectively. The results suggest the existence of an organocomplex that dominates the speciation of osmium in seawater.

Journal Article↗

PubMed2006

New osmium-based reagent for the dihydroxylation of alkenes.

The cis dihydroxylation of alkenes is most efficiently accomplished by reaction with osmium tetroxide. Recently, the expense and toxicity of osmium tetroxide have led to a number of attempts to harness alternative osmium-based reagents, including microencapsulation and solid support techniques. We describe here the development of a new nonvolatile, stable, and recoverable osmium-based reagent devised for the stoichiometric cis dihydroxylation of alkenes. Although attempts to make this new dihydroxylation work with catalytic amounts of this reagent were unsuccessful, we did develop a sensitive test for free osmium tetroxide leached from the reagent in situ: this test may well have uses in probing future applications of derivatized osmium reagents.

Alkenes↗

PubMed1984

Cellular membrane contrast and contrast differentiation with osmium triazole and tetrazole complexes.

Addition of heterocyclic nitrogen compounds to the classical osmium tetroxide postfixation medium, applied after glutaraldehyde fixation, results in enhanced membrane contrast in ultrathin sections of liver tissue. The addition of similar compounds to potassium osmate solutions, results in contrast differences in some cellular membranes. The membranes of the rough endoplasmic reticulum, the nuclear envelope and the plasma membrane acquire contrast, while the mitochondrial membranes do not. The apolar regions of membranes are contrasted when osmium tetroxide is combined with heterocyclic nitrogen compounds, whereas the polar regions are contrasted by combinations of potassium osmate with these compounds. This polar membrane contrast is probably due to the presence of an amino-group in the heterocyclic nitrogen compounds. Compounds without the amino-group do not contrast membranes, although the glycogen is contrasted. X-ray microanalysis served to establish the relative osmium content in contrasted glycogen, and showed that such nitrogen compounds play a role in complexation of cations in aldehyde-fixed tissues. Electron spectroscopy for chemical analysis (ESCA) measurements of isolated muscle glycogen show that after treatment with various osmium tetroxide or potassium osmate solutions, hexavalent and quadrivalent osmium species are present in the glycogen. The presence of (heterocyclic) nitrogen compounds in such solutions stabilizes certain osmium valency species, and this may account for the contrast observed.

Amitrole↗

PubMed1976

Studies of osmium deposits in the carotid body of the cat.

Double aldehyde fixed carotid bodies and small pieces of vagus nerve of cats were incubated in 3 mM copper sulfate and 0.5 mM potassium ferricyanide in 0.05 M acetate buffer (pH 5.6) for 30 minutes at room temperature. Several modifications of this procedure were also attempted. Tissues were then postosmicated with 2% unbuffered osmium tetroxide and heated to 50-55 C for ten minutes. Under the electron microscope carotid body cells exhibited fine osmium deposits within cisternae of endoplasmic reticulum, saccula and vesicles of Golgi complex, and cristae of mitochondria. Intense osmium precipitation was also noted in the mitochondria of nerve endings. In addition, much more intense, more conspicuous and more localized reaction was noted in the intraperiod lines of the myelin sheath of nerves. Deposits here were rod-shaped, displaying considerable variation in length. These results are discussed in the light of previous findings on osmium deposits in various tissues. It was concluded that the osmium reaction is unspecific, and that histochemical methods employing hot osmium tetroxide to amplify enzymatic activities may therefore not be reliable.

Acetates↗

PubMed1991

Influence of calcium and amino acids on the osmium impregnation of the endoplasmic reticulum.

The aim of this study was to define further the interaction between osmium and organelle content in cells prefixed with glutaraldehyde. We have studied the reaction of osmium with divalent or trivalent cations (calcium, barium, zinc, aluminum, and iron) and various amino acids in the same conditions prevalent in histological techniques, in particular with Thiéry's technique of metal impregnation. Experiments were carried out in vitro in test tubes, on cellulose acetate discs, or with an immunodiffusion apparatus. Some experiments were also carried out with tissue extracts (kidney and intestine). Our studies suggest that calcium is in general essential for the formation of osmium black, but also that lysine is reactive even in the absence of calcium and that a few amino acids--such as tryptophan, ornithine, cysteine, and aspartic acid--are only slightly reactive in the absence of calcium. Other amino acids do not seem to participate in the endoplasmic reticulum osmium impregnation even in the presence of calcium ions. Our studies also suggest that osmium reactivity reflects calcium binding sites and not only calcium content.

Amino Acids↗

PubMed1981

Osmium dependent argentaffin staining of lysosomes.

Lysosomes stain with the argentaffin reaction after fixation with glutaraldehyde followed by osmium tetroxide. The reaction works well both at the level of the light and electron microscope. Control experiments show that this argentaffinity is caused by reduced osmium tetroxide. No staining could be observed in freeze-dried material, in tissues fixed only with glutaraldehyde, or after bleaching of the sections with hydrogen peroxide solutions. In the electron microscopy, the population of lysosomes appears heterogeneous as related to the density of silver deposits over the organelles. No correlation is found between size and argentaffinity of lysosomes. X-ray microanalysis of sections from glutaraldehyde/osmium tetroxide fixed material reveals significantly higher amounts of osmium in lysosomes, as compared to other cell organelles (e.g. peroxisomes or mitochondria). A significant peak for silver is observed in lysosomes after treatment of the sections with ammoniacal silver solution, whereas the signal for osmium is reduced. Amounts of sulphur are too low to be detected in lysosomes. It is concluded that argentaffin staining of lysosomes is an osmium dependent reaction.

Animals↗

PubMed1985

Ultrastructural demonstration of calcitonin in osmium-fixed human medullary carcinoma of thyroid by the protein A-colloidal gold technique.

In two medullary carcinomas of the thyroid gland two types of secretory granules were found electron microscopically in the cytoplasm of the tumour cells. The sizes of the granules in one case ranged 103-345 nm in diameter; they were round in shape, and they co-existed in the same tumour cell. They could not, therefore, be distinctively subdivided into two types. In another case, secretory granules in the cytoplasm closely resemble EC granule in morphology. Using the protein A-colloidal gold (PAG) technique the content of secretory granules could be identified as calcitonin irrespective of their sizes or morphology. Immunoreactivity at the ultrastructural level was fairly well preserved even in the osmium-fixed tumour cells. The labelling index, expressed as a mean number of gold particles per unit square area of the secretory granule, was higher in the non-osmium-fixed tumour cells than in the osmium-fixed. Non-osmium-fixed tumour cells embedded either in epoxy or methacryl resin were almost equally labelled with gold particles. The result indicates that the PAG method is practicable to demonstrate the ultrastructural localization of calcitonin even in the osmium-fixed, epoxy resin embedded material.

Adult↗

PubMed2005

Electrochemical DNA sensing using osmium complexes as hybridization indicators.

A surface-based method for the study of the interactions of DNA with redox-active osmium complexes is described. The study was carried out using gold electrodes modified with DNA by adsorption and [Os(bpy)3]3+/2+ (bpy=2,2'-bipyridyl) or [Os(phen)3]3+/2+ (phen=1,10-phenantroline) as electrochemical indicators. The method, which is simple and reagent saving, allows the accumulation of osmium complexes on the DNA layer. The amount of osmium complex bound by the layer of double-stranded (dsDNA) or single-stranded DNA (ssDNA) adsorbed at gold electrodes was estimated from the cyclic voltammetric (CV) peak charge of osmium complex reduction. The dissociation constants (K) for the oxidized and reduced forms of a bound species are also estimated. [Os(phen)3]3+/2+ was applied to a probe for electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 70 pmol/cm2 on a gold electrode. Following hybridization with the complementary DNA, the osmium complex was electrochemically accumulated on the double-stranded DNA layer and the differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of [Os(phen)3]3+/2+ that was bound to the double-stranded DNA on the electrode.

Adsorption↗

PubMed2005

DNA biosensor for detection of Helicobacter pylori using phen-dione as the electrochemically active ligand in osmium complexes.

A surface-based method for the study of the interactions of DNA with redox-active 1,10-phenantroline-5,6-dione (phen-dione) osmium complexes is described. The study was carried out using gold electrodes modified with DNA via adsorption and [Os(bpy)(2)(phe-dione)](3+/2+) (bpy = 2,2'-bipyridyl) or [Os(phen)(2)(phen-dione)](3+/2+) (phen = 1,10-phenantroline) as electrochemical reported molecules. The method, which is simple and reagent-saving, allows the accumulation of osmium complexes within the DNA layer. The amount of osmium complex bound by the adsorbed layer of DNA was determined from the voltammetric charge associated with the osmium redox process of the immobilized metal complex. The quinone moiety of the phen-dione ligand was useful as an indicator for electrochemical DNA sensing because of its redox response at low potentials. A thiol-linked single-stranded Helicobacter pylori DNA probe was immobilized, through S-Au bonds on to a gold electrode (density of modification 86 pmol/cm(2)). Following hybridization with the complementary DNA sequence, the osmium complex was electrochemically accumulated within the double-stranded DNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range where the quinone moiety was redox active (i.e., at very low potentials, -0.020 V vs SSCE); with this approach, a sequence of the H. pylori could be quantified over the range from 5 to 20 pmol with a linear correlation of r = 0.9888 and a detection limit of approximately 6 pmol.

Biosensing Techniques↗

PubMed1985

Recognition of the structural distortions at the junctions between B and Z segments in negatively supercoiled DNA by osmium tetroxide.

It has been shown for the first time that conformational junction between contiguous right-handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)13 and (dC-dG)16 blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease S1 cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)13 segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.

Binding Sites↗

PubMed1988

Vasopressin modifies osmium impregnation of the endoplasmic reticulum in cultured kidney collecting duct cells.

The ultrastructure of collecting duct epithelia was studied with the osmium impregnation technique in renal cortical explants grown in culture in the form of globular bodies. When this technique was applied to 7-day-old globular bodies, the endoplasmic reticulum (ER) of the superficial layer cells was faintly impregnated in the presence or absence of arginine-vasopressin (AVP) in the incubation medium; the ER of the cells located in the core of the globular bodies was densely impregnated with osmium. When these globular bodies were sectioned in 2 fragments and one was incubated in AVP for 30 min while the other was used as a control, a marked increase in osmium impregnation occurred: osmium black deposits were then noted in the lumen of the endoplasmic reticulum of two-thirds of the cells in the superficial layer. Various patterns of impregnation were observed. Cryptlike formations gave rise to mature epithelial cells showing the same pattern of osmium impregnation. When cyclic adenosine monophosphate (cAMP) was substituted for AVP in the incubation medium, the treated globular bodies revealed the same ultrastructural characteristics. Our data suggest that this primary culture of collecting duct epithelia is made up of heterogeneous cells with characteristics of principal and intercalated cells and that the AVP has a stimulatory effect on ER maturation, which is mediated by the adenylate cyclase system.

Animals↗

PubMed1978

Endonuclease activities from a permanently established mouse cell line that act upon DNA damaged by ultraviolet light, acid and osmium tetroxide.

The activity of damage-dependent endonuclease in mouse plasmacytoma cells (line MPC-11) has been studied using damaged phi X 174 RFI DNA as substrate. The DNA was treated with ultraviolet light, acid, or osmium tetroxide to introduce different types of lesions. Ultraviolet light-damaged DNA was cleaved at approx. 1.1 sites per 35 thymine-containing dimers by the extract, which indicates no specificity towards this type of lesion. The acid-treated DNA, which contains apurinic sites, was enzymatically broken in every alkalilabile site and this strongly suggests the presence of an apurinic-specific endonuclease activity in the nuclear extract. The activity which acts on ultraviolet-irradiated DNA and that which acts on acid-treated DNA have different specificities as shown by their salt requirements and the extent to which they are stimulated by magnesium. While the ultraviolet-endonuclease activity was very little affected by reducing the KCl concentration, the apurinic-specific activity was almost completely abolished. Osmium tetroxide renders the DNA an excellent substrate for endonucleolytic activity in the mouse cell extract. The response to KCl and MgCl2 of the osmium tetroxide-specific endonuclease activity is qualitatively similar to that of the endonuclease activity, which acts on ultraviolet-irradiated DNA. Treatment of DNA with osmium tetroxide is known to produce 5,6-dihydroxydihydrothymine which is a minor photoproduct in DNA after irradiation, suggesting that the ultraviolet-specific endonuclease activity acts upon this lesion.

Acetates↗

PubMed1981

Comparative demonstration of pulmonary fat emboli by "en bloc" osmium tetroxide and oil red O methods.

The presence and localization of fat in human lung tissue was evaluated by the "en bloc" staining procedure with osmium tetroxide performed with formalin fixed tissue with subsequent paraffin sectioning and with the oil red O technique performed with frozen sections. Fifty-one autopsy cases were divided into three clinical groups: group A, consisting of 17 patients with severe skeletal trauma; group B, consisting of 14 patients with minor skeletal trauma; and group C, consisting of 20 control patients without trauma. Adjacent sections of lung were selected from all cases, stained with the "en bloc" osmium tetroxide and oil red O methods, coded, and examined under the light microscope without knowledge of the clinical grouping, Stainable fat was graded on a 1 to 4+ scale, and attention was given to histologic localization in tissue sections. The "en bloc" osmium tetroxide technique revealed greater amounts of stainable lipid in clinical groups A and B and was most effective in demonstrating lipid when present in small quantities (group B). Since the method permits the employment of paraffin sections, evaluation of fine histologic detail is an advantage that is not always obtained in frozen sections. It is concluded that the "en bloc" osmium tetroxide technique is superior to the oil red O technique in terms of visualization and histologic localization of minute amounts of lipid in lung tissue.

Adipose Tissue↗