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PubMed2000

Gold toning preserves integrity of silver enhanced immunogold particles during osmium tetroxide treatment for demonstration of a biogenic amine.

We describe a protocol that enhances immunolabelling of nervous tissue for ultrastructural study. Insect tissue is fixed, sectioned, and labelled with a polyclonal antiserum against serotonin and a secondary antibody conjugated with 1 nm colloidal gold. The gold particles are silver-enhanced to ease detection and then protected by gold toning. Finally, the tissue is post fixed in glutaraldehyde fixative followed by osmium tetroxide and further processed for electron microscopy. We demonstrated on insect nervous tissue that gold toning protects marker particles from the influence of osmium tetroxide. Use of buffered solutions throughout the protocol led to well preserved ultrastructural details, and marker particle size was not reduced with a short gold toning time. We also suggest use of this protocol for vertebrate or other invertebrate tissue.

Animals↗
PubMed1984

A comparison of standard lipid staining techniques used in electron microscopic studies of mammalian tissues.

Comparisons of several standard techniques for staining lipids in ultrastructural studies have been undertaken using the rat uterine epithelium as the experimental tissue. The best technique for clarity, retention of stain, and acceptability of cellular ultrastructure utilized p-phenylenediamine after primary fixation in glutaraldehyde and postfixation in osmium tetroxide. While osmium by itself stained only unsaturated lipids and p-phenylene-diamine stained no lipids in spot tests, when acting together, the staining of unsaturated lipids was enhanced and some staining of saturated lipids was seen. Further, the marked extraction of stained lipids normally found during dehydration did not then occur.

Animals↗
PubMed2002

Molecular "wiring" enzymes in organized nanostructures.

We report on the "molecular wiring" efficiency of glucose oxidase in organized self-assembled nanostructures comprised of enzyme layers alternating with layers of an osmium-derivatized poly(allylamine) cationic polyelectrolyte, acting as redox relays. Varying the relative position of the active enzyme layer in nanostructures alternating active enzyme and inactive apoenzyme we have demonstrated that the specific rate of bimolecular FADH(2) oxidation ("wiring efficiency") is limited by the diffusion-like electron hopping mechanism in the multilayers.

Electrochemistry↗
PubMed1986

The development of an 191Os----191mIr generator using an osmium chelate parent complex--I. Trans-dioxobismalonatoosmate(VI).

A 191Os----191mIr generator has been developed that has higher 191mIr yield and lower 191Os breakthrough than previous designs. These improvements have been realized through the use of the osmium chelate complex trans-dioxobismalonatoosmate(VI) as the parent species on the generator. The new generator provides an initial 191mIr yield of 40%/mL and 191Os breakthrough of 2-3 X 10(-3)% when eluted with a solution of 0.05 M malonic acid/0.10 M sodium chloride at pH 4. Other advantages of the new design include faster clearance of the 191Os breakthrough products and simpler assembly.

Angiography↗
PubMed1983

Parathyroid surgery: the role of chief cell intracellular fat staining with osmium carmine in the intraoperative management of patients with primary hyperparathyroidism.

The surgeon's gross evaluation, the hematoxylin and eosin stain, which assesses extracellular fat, and a new intracellular fat stain, osmium carmine, were comparatively evaluated in 30 consecutive patients who underwent surgery for primary hyperparathyroidism. The surgeon's gross judgment of enlarged and normal parathyroid tissue resulted in the correct functional assessment in 98% and 97% of the glands, respectively. The surgeon's functional assessment was correct in seven of 10 parathyroid glands that were considered by gross examination to be slightly enlarged (50 to 80 mg). The osmium carmine intracellular fat stain provided a correct functional assessment in all 10 of these glands. The osmium carmine stain also appears to have a role in providing scientific quantitative data to limit operations in patients who have a parathyroid adenoma to removal of the adenoma and biopsy of a grossly normal gland that demonstrates normal intracellular fat. Only one side of the neck was explored in 11 patients who fulfilled these criteria. There has been no persistent or recurrent hypercalcemia in these patients.

Adenoma↗
PubMed1983

Extraction of osmium-containing lipids by section staining for TEM.

Postfixation with osmium-ferrocyanide or OSO4 renders lipid droplets in rat liver and kidney homogeneously electron dense without additional section staining. In sections of the same block that have been single stained by uranyl acetate or lead citrate, lipid droplets show a more electron translucent center surrounded by a dense rim. In sections double stained with uranyl acetate and lead citrate, lipid droplets frequently appear as empty vacuoles, from which the electron dense content has been completely extracted.

Animals↗
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↗
PubMed2000

Ultrastructure of sea urchin calcified tissues after high-pressure freezing and freeze substitution.

The improvements brought by high-pressure freezing/freeze substitution fixation methods to the ultrastructural preservation of echinoderm mineralized tissues are investigated in developing pedicellariae and teeth of the echinoid Paracentrotus lividus. Three freeze substitution (FS) protocols were tested: one in the presence of osmium tetroxide, one in the presence of uranyl acetate, and the last in the presence of gallic acid. FS in the presence of osmium tetroxide significantly improved cell ultrastructure preservation and should especially be used for ultrastructural studies involving vesicles and the Golgi apparatus. With all protocols, multivesicular bodies, suggested to contain Ca(2+), were evident for the first time in skeleton-forming cells. FS in the presence of gallic acid allowed us to confirm the structured and insoluble character of a part of the organic matrix of mineralization in the calcification sites of the tooth, an observation which modifies the current understanding of biomineralization control in echinoderms.

Animals↗
PubMed1994

Preservation and contrast without osmication or section staining.

Conventional treatment of tissues for sectioning and transmission electron microscopy uses aldehyde fixation and osmium tetroxide postfixation. Although the result is aesthetically pleasing, osmication destroys some cell components and reduces the chemical activity of others, such as reaction with antibodies and lectins. We have found that aldehyde fixation followed by uranyl acetate preserves and contrasts most structures and visualizes some that are not easily seen after osmication. Aldehyde/UA treated tissues have enough contrast to be observed without section staining while retaining some of the chemical activity that is lost through osmication. Sections of tissues with good preservation and contrast can be used for immunogold and lectin-gold labelling of at least some molecules.

Aldehydes↗
PubMed1983

The use of radioimmune assay in investigating reagents to be used in the immunocytochemical localization of hepatitis B surface antigen in immune complexes in the kidney of patients with membranous nephropathy and Australia antigenaemia.

Radioimmune assay (RIA) was used to investigate the effect of fixatives on antigenicity of the hepatitis B surface antigen (HBsAg) and the effect of pronase on the elution of antibody (Ab) from the HBsAG-Ab complex. The effect of pronase on Ab elution was also tested on sections of kidney from a patient with the immune complex disease systemic lupus erythematosus (SLE). Immunoglobulin G (IgG) was located in pronase treated and untreated sections using the indirect immunoperoxidase technique. Glutaraldehyde was shown to be the fixative of choice for studies involving HbsAG. All fixatives were shown to have less effect on antigenicity at 4 degrees C than at room temperature. Osmium tetroxide reduced antigenicity to one-third, even at 4 degrees C. RIA and SLE kidney section studies showed that Ab was eluted from immune complexes by pronase. Pre-fixation of the antigen (Ag) by glutaraldehyde appears to have no effect on the final elution, although fixation after pronase treatment seemed to enhance the elution effects. The availability of an RIA kit with HBsAg- and AB-coated beads was of great assistance in evaluating reagents to be used in immunoperoxidase studies of HBsAg in immune complexes of patients with membranous nephropathy and Australia antigenaemia.

Antigen-Antibody Complex↗
PubMed1990

Quantifying synaptic number and structure: effects of stain and post-mortem delay.

Current research indicates the importance of synaptic number and structure in plastic processes such as development, learning and memory, and aging. As such, the examination of these neural features has become an important factor in research on human conditions such as mental retardation, aging and Alzheimer's disease. Synaptic research in human tissue typically involves delayed post-mortem fixation, therefore the current research was designed to examine the effect of post-mortem delay on synaptic number and structure in tissue stained with either routine osmium lead citrate/uranyl acetate (osmium) or ethanol phosphotungstic acid (EPTA). Results indicate that synaptic density shows either a gradual decline (EPTA) or an initial marked drop followed by a plateau (osmium) up to 10-15 h post-mortem depending on the stain used. The number of synaptic vesicles per synapse also undergoes a gradual decline. Measures of synaptic structure were more stable, with the primary change being an initial increase in the cross-sectional length of the synapse. Maximal height of the pre- and postsynaptic dense elements were not affected by post-mortem delay. The EPTA stain gave the best estimates of synaptic parameters with short post-mortem delays. These results indicate that different synaptic measures (and stains) show different responses to post-mortem fixation delay, and that experimental or statistical methods must be used to control for post-mortem effects.

Animals↗
PubMed2005

DNA mismatch detection by resonance energy transfer between ruthenium(II) and osmium(II) tris(2,2'-bipyridyl) chromophores.

Octahedral tris-chelate complexes [M(II)(bpy)(3)](2+) (M = Ru or Os, bpy = 2,2'-bipyridyl), covalently attached to the 3'- and 5'-phosphates of two oligonucleotides, are juxtaposed when hybridized contiguously to a fully complementary DNA target. Visible metal-to-ligand charge-transfer (MLCT) excitation of the [Ru(II)(bpy)(3)](2+) unit leads to resonance energy transfer to the MLCT state of the [Os(II)(bpy)(3)](2+) moiety, with the energy transfer efficiency depending on the degree of hybridization. The extent of attenuation of the intense red luminescence from the Ru(II) chromophore hence allows highly sensitive structural probing of the assembly and constitutes a novel approach to DNA sensing which is capable of detecting mutations.

Base Sequence↗
PubMed1981

Backscattered electron imaging of human leukocytes.

Methods of backscattered electron imaging (BEI) have been applied to the study of normal and malignant human leukocytes. Four different elements, principally of high atomic number, have been used: iron, silver, lead and osmium. Iron, in the form of iron carbonyl, has been found to be an excellent marker for the phagocytic activity of granulocytes and monocytes. Silver staining permitted a clear observation of the shape of the nuclei and of the nucleo-cytoplasmic ratio and made the identification of polymorphonuclear cells and of certain normoblasts possible. Lead phosphate precipitates, deposited as a result of a classic acid phosphatase reaction, were detected in several cases of myelocytic and monocytic leukemias. Precipitates of osmium/diaminobenzidine complex on peroxidase-containing granules permitted the unambiguous identification of several classes of myeloid precursors. In all these techniques, the surface morphology of the cells was adequately preserved and could be correlated with the presence and/or the distribution of the various markers.

Acid Phosphatase↗
PubMed1992

Evaluation of osmium(II)-nitrosyl complexes as a method to increase the yield of the 191Os-191mIr generator.

The nitrosyl complexes pentachloronitrosylosmate(II), [OsCl5(NO)]2-, and hydroxytetranitronitrosylosmate(II), [Os(OH)(NO2)4(NO)]2-, were evaluated as parent species for use on the 191Os-191mIr generator in an attempt to increase the 191mIr yield of the generator by providing a direct route to a chemically stable 191mIr daughter. The uptake of the 191Os-labeled complexes by the inorganic ion-exchangers ZrO2, SnO2, PbS, MnO2 and Al2O3 and the organic resin AG MP-1 was measured and prototype generators were prepared using those exchangers that demonstrated greater than 90% uptake of the 191Os-labeled complexes. The 191mIr(III)-nitrosyl complexes produced subsequent to beta- decay of the 191Os-nitrosyl parent complexes were found to undergo secondary chemical reactions to form nitro (NO2-) complexes that were tightly retained on the ion exchanger limiting 191mIr yield to less than 5%.

Iridium Radioisotopes↗
PubMed1982

Morphometric study of cardiac muscle: the problem of tissue shrinkage.

Comparison of data from morphometric studies dealing with the heart is complicated by the fact that little information dealing with cell size changes during tissue processing is available. To investigate these changes, isolated cardiac myocytes were adhered to glass cover slips of Sykes Moore chambers and photographed after each step of processing for transmission electron microscopy. Six different experiments with a minimum of 10 cells each were followed through the entire procedure after fixation with isoosmolar glutaraldehyde. Cellular dimension changes were determined by tracing individual isolated myocytes after each step of the procedure with a sonic digitizer. Significant cell volume changes occurred after osmium (16 per cent swelling), postosmium wash (10 per cent swelling), and uranyl acetate (25 per cent shrinkage). Hypertonic aldehyde solutions resulted in cellular shrinkage during fixation not found with isotonic solutions. Changes in cell cross-sectional area rather than length were largely responsible for altered cell volumes during any given phase of processing. The results indicate that, although cell volume changes occur during processing, final cell dimensions of embedded cells were not different from unfixed cells. In whole tissue blocks, inclusion of propylene oxide in the procedure resulted in tissue shrinkage which was not observed in isolated myocytes, suggesting that different tissue components react in a variable manner to propylene oxide. After each of the other steps in processing, tissue blocks reacted in a similar manner to the isolated myocytes.

Animals↗
PubMed2006

Structure and thickness dependence of "molecular wiring" in nanostructured enzyme multilayers.

Supramolecular organized multilayers composed of glucose oxidase (GOx) and osmium-derivatized poly(allylamine) redox polymer have been self-assembled electrostatically from Os-polyelectrolyte solutions of variable pH (5.5-8.8) leading to a decrease of the linear charge density in the PAH-Os with increasing pH. The layer-by-layer enzyme multilayers were studied by ellipsometry, quartz crystal microbalance, AFM, cyclic voltammetry, and electrocatalytic oxidation of beta-D-glucose. At higher adsorption solution pH, an increase in the film thickness, enzyme loading, and redox charge was observed. While the electrocatalytic response increases with the increase of the adsorption solution pH (decrease of the polyelectrolyte linear charge), the FADH2 oxidation bimolecular rate constant has a maximum in the pH range 7.0-7.5 where a change in the film growth mechanism is observed.

Electrochemistry↗
PubMed1999

Synthesis and spectral characterization of a long-lifetime osmium (II) metal-ligand complex: a conjugatable red dye for applications in biophysics.

There is a need for luminescent probes, which display both long excitation and emission wavelengths and long decay times. We synthesized and characterized an osmium metal-ligand complex which displays a mean decay time of over 100 ns when bound to proteins. [Os(1,10-phenanthroline)2(5-amino-1,10-phenanthroline)[(PF6)2 can be excited at wavelengths up to 650 nm, and displays an emission maximum near 700 nm. The probe displays a modest but useful maximum fundamental anisotropy near 0.1 for 488-nm excitation, and thus convenient when using an argon ion laser. [Os(phen)2(aphen)](PF6)2 is readily activated to the isothiocyanate for coupling to proteins. When covalently linked to bovine serum albumin the intensity decay is moderately heterogeneous with a mean decay time of 145 ns. The anisotropy decay of the labeled protein displays a correlation time near 40 ns. This relatively long lifetime luminophores can be useful as a biophysical probe or in clinical applications such as fluorescence polarization immunoassays.

Biophysics↗
PubMed1987

An osmium-191/iridium-191m radionuclide generator using an oxalato osmate parent complex.

A new osmium-191/iridium-191m (191Os/191mIr) radionuclide generator has been developed that offers high 191mIr yield (greater than 20%/ml) and low 191Os breakthrough (less than 5 X 10(-4)%/ml) when eluted with a solution of 0.001 M oxalic acid and 0.9% (normal) saline. This is the first 191Os/191mIr generator that combines the advantages of high 191mIr yield, extremely low 191Os breakthrough, and an eluate that does not require buffering prior to injection. These improvements in performance were accomplished through use of the chelate transdioxobisoxalatoosmate(VI) as the parent complex on the generator. The clinical result of the combination of higher yield and lower breakthrough is a 100-fold decrease in the estimated patient radiation dose compared with the same study performed with technetium-99m (99mTc), and the injectable eluate makes the generator easier to use. Acute and subacute toxicity studies performed on this generator eluate have shown no adverse effects attributable to the eluate.

Iridium↗