Use Of Osmium Tetroxide Research Articles

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components.

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.

Methods of preparing flower stem samples for scanning electron microscopy

Brazil has great capacity for expansion in the floriculture sector. Studies on postharvest cut flowers contribute to development of the sector, helping to maintain the quality of domestic production. Scanning electron microscopy (SEM) is a powerful tool that allows viewing of flower structures and also microorganisms. The aim of this study was to evaluate methods of preparing flower stem samples for viewing in SEM as a support for studies on postharvest cut flowers. Ways of cutting, fixing, and drying samples were tested. Cutting with a stainless steel blade and through freeze-fracture were tested; fixation was carried out without the use of osmium tetroxide (OsO4); and drying of the samples was performed through freeze-drying and through critical point dryingwithCO2. Cutting with a stainless steel blade proved to be a satisfactory method for stem samples, with low cost and simple application compared to freeze-fracturing. Good fixation and high image contrast were obtained without the use of osmium tetroxide, thus avoiding the use of this toxic compound. Freeze-drying allowed the structure and morphological composition to be viewed, while critical point drying withCO2 preserved the microorganisms present in the samples.

Three-dimensional observation of mouse tongue muscles using micro-computed tomography

The aim of this study is to obtain information about the mouse tongue muscle rendered using micro-computed tomography (μCT) at low, middle, and high magnifications. Three-dimensional (3D) μCT is used in various fields. Most μCT observations are restricted to hard tissue in biomaterial samples. Recently, with the use of osmium tetroxide, μCT has been effectively employed to observe soft tissue; it is now believed that μCT observation of soft tissue is feasible. On the other hand, the structure of the tongue muscle has been well studied, but cross-sectional imaging enhanced by 3D rendering is lacking. We chose the mouse tongue as a soft tissue case study for μCT and generated cross-sectional images of the tongue enhanced by 3-D image rendering with histological resolution. During this observation, we developed new methods of low-magnification observation to show the relation between the tongue muscles and surrounding tissues. We also applied high-resolution μCT in high-magnification observation of muscle fiber fascicles. Our methodological techniques give the following results: (1) For low-magnification observation (field of view: 12,000μm), pretreatment with decalcification and freeze drying is suitable for observing the area between the muscle of the tongue and the bone around the tongue using μCT. (2) For middle-magnification observation (Field of view: 3,500μm), the use of osmium tetroxide to observe the muscle arrangement of the tongue by μCT is suitable. (3) For high-magnification observation (Field of view: 450μm), high-resolution μCT is suitable for observation of the transversus muscle fiber fascicles.

ChemInform Abstract: Steroids. Part 354. A Route to 24-epiBrassinolide (VII) from Ergosterol Avoiding the Use of Osmium Tetroxide.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Fixation and Drying Protocols for the Preparation of Cell Samples for Time-of-Flight Secondary Ion Mass Spectrometry Analysis

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a promising tool for subcellular chemical analysis of biological cells. However, to obtain relevant information, the method used for sample preparation is critical. In this work, we have used TOF-SIMS, scanning electron microscopy (SEM), and interference reflection microscopy (IRM) to study the effects of different fixation and drying methods on the morphology and chemical structure of human fibroblast cells (hTERT) adhered to a silicon surface. Specifically, two fixation techniques (chemical fixation with glutaraldehyde and cryofixation by plunge freezing) and two drying techniques (freeze drying and alcohol substitution drying) were investigated. Cryofixation followed by freeze drying was determined to produce dried cells with preserved cell morphology, intact cell membranes, and retained sodium/potassium ion concentration gradients across the plasma membrane. By washing samples in an aqueous solution of ammonium formate (AF) before cryofixation, the accumulation of salts on the sample surface during drying could be suppressed. IRM measurements showed that the cell morphology was preserved during washing with ammonium formate, although some swelling occurred. Compared with cryofixation, cells fixed with glutaraldehyde showed finer structures on the cell surface in SEM and similar lipid distributions in TOF-SIMS, but the sodium/potassium ion gradients were not retained. Alcohol drying was determined to remove cell membrane phospholipids significantly, although the use of osmium tetroxide as a post-fixative was shown to decrease this effect.

Efecto comparativo del tetróxido de osmio y del tetróxido de rutenio como fijadores sobre la ultraestructura de la pared celular de hongos

The fungal cell wall viewed through the electron microscope appears transparent when fixed by the conventional osmium tetroxide method. However, ruthenium tetroxide post-fixing has revealed new details in the ultrastructure of Penicillium sp. hyphae and Saccharomyces cerevisiae yeast. Most significant was the demonstration of two or three opaque diverse electron dense layers on the cell wall of each species. Two additional features were detected. Penicillium septa presented a three-layered appearance and budding S. cerevisiae yeast cell walls showed inner filiform cell wall protrusions into the cytoplasm. The combined use of osmium tetroxide and ruthenium tetroxide is recommended for post-fixing in electron microscopy studies of fungi.

In situ Generation of Ruthenium Tetroxide and Osmium Tetroxide for the Physical Sciences and Their Reaction Indicators

Abstract Recently, there was a suggestion on the MSA listserver about the use of osmium tetroxide (OsO4 and how to handle it. One suggestion was that ampoules be scored, placed in a glass jar, and the ampoule smashed to release the contents. This seemed like a very unsafe way to use osmium tetroxide or ruthenium tetroxide. The purpose of this article is to suggest a way to generate smaller amounts of these compounds in a safer manner than smashing ampoules and wondering about what to do with the unused portion after staining or storing. Another purpose is to discuss a new reaction indicator for mainly osmium tetroxide. The use of a reaction specific indicator was mandatory for judging the level or degree to which staining had proceeded in thin sections for the transmission electron microscope (TEM).

Process Development of a Dual MMP/TNF Inhibitor (SDZ 242-484)

The compound (2R,3S)-N-4-((S)-2,2-Dimethyl-1-methylcarbamoyl-propyl)-N-1-hydroxy-2-hydroxymethyl-3-(4-methoxy-phenyl)succinamide (1; SDZ 242-484) shows antiinflammatory effects due to inhibition of matrix metalloproteases (MMP) and tumor necrosis factor-α activity (TNF). We describe the development of a chromatography-free process for a multikilogram-scale pilot-plant production. Two of the three chiral centers are introduced in a diastereoselective Claisen−Ireland rearrangement. It was found that the selectivity of this step was significantly enhanced by the addition of catalytic amounts of Lewis acids. The resulting enantiomeric carboxylic acids were resolved with (S)-(−)-phenylethylamine. The use of osmiumtetroxide was considered to be problematic for pilot-plant use. Therefore, it was necessary to find an alternative method for the oxidative cleavage of the terminal olefin functionality. For the last chemical transformation, a hydrogenolytic cleavage of a benzyl group in the presence of a hydroxamate,...

A novel immunocytochemical staining method that preserves cell membranes: Application for demonstrating Ca2+ pump ATPase in the liver

We have devised a method for immunogold staining of unosmicated, plastic-embedded cells that gives high levels of specific staining without sacrificing cell ultrastucture. Important conditions include PLP (periodate-lysine-paraformaldehyde) fixation, postfixation with uranyl acetate to preserve membrane phospholipids, dehydration with acetone, low-temperature embedding in LR gold resin, and use of osmium tetroxide to stain thin sections after immunogold labeling. We developed this method to localize plasma membrane calcium pump ATPase in rat hepatocytes and hepatic sinusoidal endothelial cells. Most gold particles of Ca2+ pump ATPase were easily assigned to bile canalicular membranes in rat hepatocytes, and the gold particles of Ca2− pump ATPase were located on the labyrinthlike structures of the endothelial sinusoidal fenestrae in rat hepatic sinusoidal endothelial cells. In these studies, it was useful to preserve the cell membrane for postembedding methods.

Freeze-substitution of rye grass and ragweed pollen grains

Rapid freezing and freeze-substitution have been employed as alternatives to chemical fixation because of the improved structural preservation obtained in various cell types. This has been attributed to biomolecular immobilization derived from the extremely rapid arrest of cell function. These methods allow the elimination of conventionally used fixatives, which may have denaturing or “masking” effects on proteins. Thus, this makes them ideal techniques for immunocytochemistry, in which preservation of both ultrastructure and antigenicity are important. These procedures are also compatible with cold embedding acrylic resins which are known to increase sensitivity in immunolabelling.This study reveals how rapid freezing and freeze-substitution may prove to be useful in the study of the mobile allergenic proteins of rye grass and ragweed. Most studies have relied on the use of osmium tetroxide to achieve the necessary ultrastructural detail in pollen whereas those that omitted it have had to contend with poor overall preservation.

Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution

Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure during freeze-substitution of select Escherichia coli and Bacillus subtilis strains. Radioisotopes were specifically incorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and into the peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed by liquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator of cell structural integrity and retention of cellular chemical composition. Subsequent visual examination by electron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol) osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate; 2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate. All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituents depended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2% gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In both gram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids in which 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alone were used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence of different chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone for routine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalytical processing necessitates the omission of osmium.

An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.

A route to 24-epibrassinolide from ergosterol avoiding the use of osmium tetroxide

A synthesis of (22R,23R)-2α,3α,22,23-tetrahydroxy-5α-ergostan-6-one (V) from the dienone IV via the diepoxide XXII is described. The tetrol V is a key intermediate in the synthesis of 24-epibrassinolide. The reactivity of the side chain was studied on 5α-ergost-22-en-6-one (XIV) as a model compound. 1H and 13C NMR spectra of 24-epibrassinolide derivatives are discussed.

ChemInform Abstract: Steroids. Part 344. An Alternative Route to 2α,3α‐Diols from 2,3‐Unsaturated 5α‐Steroids Avoiding the Use of Osmium Tetroxide.

ChemInformVolume 20, Issue 50 Natural Products ChemInform Abstract: Steroids. Part 344. An Alternative Route to 2α,3α-Diols from 2,3-Unsaturated 5α-Steroids Avoiding the Use of Osmium Tetroxide. V. CERNY, V. CERNY Inst. Org. Chem. Biochem., Czech. Acad. Sci., 166 10 Prague 6Search for more papers by this author V. CERNY, V. CERNY Inst. Org. Chem. Biochem., Czech. Acad. Sci., 166 10 Prague 6Search for more papers by this author First published: December 12, 1989 https://doi.org/10.1002/chin.198950293Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume20, Issue50December 12, 1989 RelatedInformation

ChemInform Abstract: A Convenient Method for Dihydroxylation of Olefins by the Combined Use of Osmium Tetroxide and Dihydroxyphenylborane.

AbstractThe dihydroxylation of indene (I) with osmium tetroxide/N‐methylmorpholine 1‐oxide is performed in the presence of the dihydroxyboranes (II), yielding the dioxaborolanes (III), whereas in the presence of other additives, only the cis‐diol (IV), is obtained.

An alternative route to 2α,3α-diols from 2,3-unsaturated 5α-steroids avoiding the use of osmium tetroxide

A method for preparation of 2α,3α-dihydroxy-5α-steroids from 2,3-unsaturated 5α-steroids without the use of OsO4 is described. It involves the sequence of epoxidation, cleavage of the oxirane ring by HI, acetylation, oxidative replacement of iodine by hydroxyl and alkaline hydrolysis (I → VI) without purification of the intermediates. The presence of a 6-keto group does not decrease the yields. The yields are comparable with those obtained by osmylation and the method can find general use, particularly in the synthesis of brassinolide analogs.

A Convenient Method for Dihydroxylation of Olefins by the Combined Use of Osmium Tetroxide and Dihydroxyphenyiborane

Abstract cis-Dihydroxylation of olefins with a catalytic amount of osmium tetroxide and N-methylmorpholine N-oxide proceeds smoothly in an organic solvent by the use of dihydroxyphenyiborane, giving the corresponding 2-phenyl-1,3,2-dioxaborolane derivatives (phenylboronic esters) in good yields.

A simple method for combining HRP-TMB histochemistry with tritiated thymidine autoradiography on the same tissue section

In order to determine the sequence of development of various types of spinal neurons defined by their projection, we have developed a method for combining tritiated thymidine autoradiography for birthdate determination, with the demonstration of retrogradely transported horseradish peroxidase (HRP) using tetramethylbenzidine (TMB) as the chromogen. Because of its greater sensitivity, TMB is the chromogen of choice for the demonstration of HRP. However, the HRP-TMB reaction product is unstable and completely destroyed when the tissue is processed for autoradiography. The present study describes the use of osmium tetroxide as a postreaction stabilizing agent which preserves the HRP-TMB reaction product in the form of a dark brown precipitate which is not destroyed when the tissue is subsequently processed for autoradiography. Background levels of autoradiographs stabilized with the osmium procedure are extremely low.

Synthesis of “K-region” quinones and arene oxides of polycyclic aromatic hydrocarbons

A new general synthesis of arene oxides recently reported’ from this laboratory has allowed the synthesis of the previously inaccessible “K-region” oxides (@, ,b) of the potent carcinogens ‘7,12-dimethylbenzraY anthracene (‘7.1 P-DMBA) and benzola] pyrene2. This approach, however, still suffers from the limitation of requiring the use of osmium tetroxide in the initial step. This reagent is both costly and hazardous, affords only moderate yields, and necessitates time-consuming operations in handling the air sensitive products6. As a consequence, the quantities of the oxides available for biological studies remain severely limited.

Coumarins XII: Synthesis of (±)-cis- and trans-3ʹ,4ʹ-Dihydroxy-3ʹ,4ʹ-dihydroxanthyletin and Their Diesters

(±)-as-3ʹ,4ʹ-Dihydroxy-3ʹ,4ʹ-dihydroxanthyletin was synthesized from xanthyletin by the use of osmium tetroxide. It was also obtained, unexpectedly, by acid or base hydrolysis of (±)- fran.s-3ʹ-acetoxy-4ʹ-(m-chlorobenzoyloxy)-3ʹ,4ʹ - dihydroxanthyletin and, spontaneously, by recrystallization of the product from epoxi- dation of xanthyletin with trifluoroperacetic acid. (±)-trans-3ʹ,4ʹ- Dihydroxy-3ʹ,4ʹ-dihydroxanthyletin was obtained together with the (±)-frarti-3ʹ-hydroxy-4ʹ-acetoxy derivative by peracetic acid treatment of xanthyletin. The diacetates, dipro'pionates, and di-n- butyrates of both the cis- and trans-diols were prepared together with the rra//.v-3ʹ-propionoxy-4ʹ-acetoxy- and 3ʹ-n-butyroxy-4ʹ- acetoxy-3ʹ,4ʹ-dihydroxanthyletins. NMR studies of the diols and the diesters were carried out to establish chemical shifts of specific groups, with special emphasis on the 2'-gem-dimethyls together with they values for the 3ʹ- and 4ʹ-protons as diagnostic features for the cis- or f raws-configuration.