Ruthenium red as a stain for electron microscopy. Some new aspects of its application and mode of action.

Commercial ruthenium red has been tested for its purity by spectrophotometry. Impurities detected by this method could be abolished by nitric acid-precipitation of ruthenium brown. This substance has no effect on cell surface staining and converts almost completely to ruthenium red under the conditions used in electron microscopy. It was found, by photometric analysis, that in the ruthenium red-osmium tetroxide-cacodylate combination, generally used for cell surface staining, chemical reactions between ruthenium red and osmium tetroxide occur. As aerial oxidation of hexammineruthenium2+ leads to a product with some surface staining capability, it is suggested that an oxidized product of ruthenium red is responsible for binding to cellular components, and that a reduced product of osmium tetroxide gives an additional contrast enhancement. In ruthenium red-osmium dioxide combinations ruthenium red seems to bind to cell surfaces without any molecular alteration, and contrast is gained by the model proposed by Blanquet (1976b). The latter method could open a way for investigating the binding of ruthenium red to certain natural compounds involved in calcium transport, as postulated by a number of authors. Both ruthenium-osmium combinations differ in their cell surface staining ability. The ruthenium red-osmium dioxide combination tends to form distinct subunits, whereas the osmium tetroxide variety stains homogeneously. In combination with osmium dioxide, the surface staining is affected by EDTA, and, in contrast to osmium tetroxide, a successive application of ruthenium red and osmium dioxide as possible.     

Ruthenium red as a stain for electron microscopy. Some new aspects of its application and mode of action

Summary Commercial ruthenium red has been tested for its purity by spectrophotometry. Impurities detected by this method could be abolished by nitric acid-precipitation of ruthenium brown. This substance has no effect on cell surface staining and converts almost completely to ruthenium red under the conditions used in electron microscopy. It was found, by photometric analysis, that in the ruthenium red-osmium tetroxide-cacodylate combination, generally used for cell surface staining, chemical reactions between ruthenium red and osmium tetroxide occur. As aerial oxidation of hexammineruthenium²⁺ leads to a product with some surface staining capability, it is suggested that an oxidazed product of ruthenium red is responsible for binding to cellular components, and that a reduced product of osmium tetroxide gives an additional contrast enhancement.In ruthenium red-osmium dioxide combinations ruthenium red seems to bind to cell surfaces without any molecular alteration, and contrast is gained by the model proposed by Blanquet (1976b). The latter method could open a way for investigating the binding of ruthenium red to certain natural compounds involved in calcium transport, as postulated by a number of authors.Both ruthenium-osmium combinations differ in their cell surface staining ability. The ruthenium red-osmium dioxide combination tends to form distinct subunits, whereas the osmium tetroxide variety stains homogeneously. In combination with osmium dioxide, the surface staining is affected by EDTA, and, in contrast to osmium tetroxide, a successive application of ruthenium red and osmium dioxide as possible.     

Scanning electron microscopical comparisons of insect virus occlusion bodies prepared by several techniques

Samples of baculoviruses, a cytoplasmic polyhedrosis virus, and an entomopoxvirus were prepared by several techniques for study in the scanning electron microscope. The techniques which gave satisfactory definition of surface ultrastructure were: (1) double fixation in glutaraldehyde and osmium tetroxide with critical point drying; and (2) double fixation as in (1) with a further postfixation by ligand-mediated osmium binding using thiocarbohydrazide as the ligand (OTO method) followed by critical point drying. The latter technique was superior to the former technique. Air drying of samples gave acceptable but inferior definition when compared with critical point-dried samples prepared by these techniques.     

Ruthenium Red-Mediated Osmium Binding for Examining Uncoated Biological Material Under the Scanning Electron Microscope

A simple technique for examining uncoated soft biological material under the scanning electron microscope is described. Rat tissues were initially fixed in 2.5% glutaraldehyde either by intravascular perfusion or by immersion. The samples were placed in buffered 2.5% glutaraldehyde containing 0.05% ruthenium red and postfixed in buffered 1% osmium tetroxide containing 0.05% ruthenium red. The samples were alternately incubated 3 to 5 times in 1% O₃O₄ and 0.1% ruthenium red solutions with continuous shaking at room temperature. The specimens were dehydrated, critical point dried, mounted and examined under the scanning electron microscope. Contour details were clearly defined at both the external and cut surfaces of the tissues. The specimens could be observed for more than 30 minutes without excessive charging or glow effects and the material remained stable under the beam at 20-25 kV and at various magnifications.     

Ruthenium red-mediated osmium binding for examining uncoated biological material under the scanning electron microscope

A simple technique for examining uncoated soft biological material under the scanning electron microscope is described. Rat tissues were initially fixed in 2.5% glutaraldehyde either by intravascular perfusion or by immersion. The samples were placed in buffered 2.5% glutaraldehyde containing 0.05% ruthenium red and postfixed in buffered 1% osmium tetroxide containing 0.05% ruthenium red. The samples were alternately incubated 3 to 5 times in 1% OsO4 and 0.1% ruthenium red solutions with continuous shaking at room temperature. The specimens were dehydrated, critical point dried, mounted and examined under the scanning electron microscope. Contour details were clearly defined at both the external and cut surfaces of the tissues. The specimens could be observed for more than 30 minutes without excessive charging or glow effects and the material remained stable under the beam at 20-25 kV and at various magnifications.     

Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride.

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris-(bipyridyl)ruthenium (II) chloride (TRC), tris (bipyridyl) iron (II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post-fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e., RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post-fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post-fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.     

Comparative effect of osmium tetroxide and ruthenium tetroxide on Penicillium sp. hyphae and Saccharomyces cerevisiae fungal cell wall ultrastructure

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.     

The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate

Synopsis A densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.     

Preservation of Tracheal Mucus by Nonaqueous Fixative

Two nonaqueous fixatives, composed of fluorocarbon solvents with dissolved osmium tetroxide, were used to determine the feasibility of preserving the mucous coat in bovine and rat trachea for light and electron microscopy. Aqueous fixatives, while providing excellent cytological preservation, wash away the mucous lining, precluding ultrastructural analysis. Inclusion of ruthenium red or alcian blue within aqueous fixative improved retention of mucus, but provided incomplete, patchy results. Fixation with nonaqueous fluorocarbon solvent and dissolved osmium tetroxide preserved a continuous mucous epiphase layer above a clear hypophase layer. Subcomponents of the mucus included an electron dense surface layer, interrupted patches of mucus above the surface layer and electron dense membrane-like material within the mucus. This method of fixation will preserve mucus for light, scanning and transmission electron microscopy, using either intratracheal or immersion methods of fixation. The latter would enable use of materials from large animal models, autopsy or an abattoir.     

A method for the chemical treatment of cells for the purpose of the subsequent study of the same cell culture preparation by light, scanning and transmission electron microscopy]

Cells cultured on transparent conductive substrates (glass coated with indium oxide) were fixed with aldehyde and osmium tetroxide and then treated with tannic acid, uranyl acetate and lead citrate. The same cell culture preparation could be sequentially studied by light microscopy (in water immersed condition), SEM (after dehydration and critical point drying) and TEM (after embedding in an epoxy resin). This method ensures the preservation of intact cell morphology, cell surface topography and intracellular structures. The treatments used render the cells conductivity and permit to carry out successfully SEM of uncoated cells cultured on conductive substrates. This method also provides a higher contrast of TEM images.     

The fine structure of the glycocalyx of equine spermatozoa: a high-resolution cytochemical study.

Equine spermatozoa were obtained from ejaculates of young stallions. The seminal plasma was removed and the sperm pellets washed three times with 0-15 M-NaCl solution before final centrifugation at 4500 g for 15 min. The pellets were fixed in a mixture of 2-5% glutaraldehyde in 0-1 M-cacodylate buffer, pH 7-4, with 0-5% Alcian blue and post-fixed in 1% osmium tetroxide with 1% lanthanum nitrate; other samples were treated with ruthenium red. All samples were dehydrated in ascending concentrations of ethanol, embedded in araldite and thin sections examined in an electron microscope. Electron dense deposits of lanthanum were present on the surface plasmalemma of the head, mid-piece and tail of 70% of mature spermatozoa, and similar deposits were seen in ruthenium red-treated samples. No glycocalyx was observed in untreated spermatozoa.     

On processing field and culture samples of desmids (Desmidiales,chlorophyta) for scanning electron microscopy

A procedure is outlined for preparing both field collected and laboratory grown desmid populations for scanning electron microscopy which avoids the use of the potentially hazardous chemicals glutaraldehyde and osmium tetroxide and reduces preparation time substantially. FAA is utilized both in fixation and mucilage removal, and living material can be prepared for critical point drying in as little as 90 min. Limitations of this procedure are outlined briefly and the use of SEM material in systematic studies is commented upon.     

Potassium permanganate and tetraethylammonium chloride are a safe and effective substitute for osmium tetroxide in solid-phase fluorescent chemical cleavage of mismatch.

Whilst chemical cleavage of mismatch (CCM) detects all point mutations in DNA, its widespread use has been hampered by the complex multistage methodology and the need for toxic chemicals, in particular osmium tetroxide. Here we show that osmium tetroxide can be replaced by potassium permanganate, giving the same spectrum of mutation detection, but with greater sensitivity. The use of potassium permanganate is compatible with solid phase capture and fluorescent detection, giving a safer method of mutation detection. We present here a comparison of CCM with osmium tetroxide and with potassium permanganate, tested on a complete set of single base pair mismatches and a number of small insertion/deletions.     

The alveolar-lining layer in the lung of the axolotl,Ambystoma mexicanum

Summary Lungs of neotenic larvae of Ambystoma mexicanum were prepared for maintaining the air-tissue boundary during aldehyde fixation. Four methods of postfixation were applied: 1) osmium tetroxide followed by en-bloc staining with uranyl acetate and phosphotungstic acid, 2) ruthenium redosmium tetroxide, 3) osmium tetroxide-ferrocyanide, and 4) tannic acidosmium tetroxide.Three types of cells line the inner surface of the axolotl lung: 1) pneumocytes, covering the capillaries with flat cellular extensions and containing two types of granules: the osmiophilic lamellar bodies, precursors of extracellular membranous material, and apical granules of unknown significance; 2) ciliated cells, also containing osmiophilic lamellar bodies; and 3) goblet cells filled with secretory granules as well as osmiophilic bodies.The extracellular material forms membranous whorls as well as tubular myelin figures, consisting of membranous backbones combined with an intensely stained substance. This material strikingly resembles the surfactant of amphibian lungs.     

Ultrahistochemical study on the ruthenium red surface staining. II. Nature and affinity of the electron dense marker.

The electron-dense marker which is thought to produce the ruthenium red surface staining is studied. This stain is prepared under conditions which should give its rise in cell surface membrane, and its nature and charge are tested electrophoretically and by measuring the turbidity, respectively. It is a positive colloid resulting from the recharging of colloidal osmium dioxide by RR polycations. Controls on the affinity are carried out by applying positive sol to gelled agarose sections containing hyaluronic acid, polyvinyl sulfate or polylysine. Controls are also carried out on ascites Ehrlich carcinoma and Zajdela ascites hepatoma cells subjected to prior enzymatic and chemical treatments. It is found that the osmium-RR system visualizes all acidic groups in the outer hydrophilic leaflet, that is the greater part of compounds in this external cell layer. A model is presented for the mechanism underlying its rise in cell surface membrane.     

Ultrastructure and permeability characteristics of the membranes of mucous granules of the hagfish

Mucous granules secreted by the slime glands of the hagfish. Eptatretus stouti, were studied after ultrarapid cryofixation and freeze substitution in diverse media (osmium tetroxide in acetone; several aqueous glutaraldehyde-based media with or without osmium). Only freeze substitution with osmium tetroxide-acetone preserved the granules intact, allowing visualization of its single unit membrane. Tests of the rupture or stability of freshly secreted mucous granules in sea water and other aqueous media showed the membranes of the granules are permeable to all inorganic cations tested, ranging in relative mass from ammonium to barium. They are permeable to the univalent anions chloride, nitrate, and bicarbonate, but not to the di- or trivalent anions sulfate, phosphate, and citrate. Moreover, in solutions where nonpenetrant anions were present, rupture occurred if the osmotic pressure was below a critical level (about 800 mOsmol/l). The structural and permeability characteristics of the granules account for the explosive speed with which they rupture, releasing their mucous contents, on contact with sea water.     

Digestibility of elastic fibers by elastase in semifine and fine sections as a function of fixation mode and embedding media

Summary As a preparatory study for investigations upon disorders of elastic fibers, the paper compares in a systematic manner the influences which different fixation agents and embedding media have on the digestibility of elastic fibers. The results establish that the fixation agents including osmium tetroxide have less influence than have the embedding media. Previous oxydation with periodic acid does not enhance the digestibility. Epoxyresin-embedded material proved to be affectable by elastase, whereas glycolmethacrylate-embedded samples resisted digestion.     

Cosmos pollen ontogeny: A scanning electron microscope study

Summary Ontogenetic data concerning pollen not only clarifies the mode of deposition of the elaborate walls but has considerable functional and taxonomic relevance. Hitherto such studies have used optical or transmission electron microscopy but here a recently devised preparative technique has enabled pollen development inCosmos bipinnatus to be studied using the scanning electron microscope. The technique involves freeze-fracturing of osmium fixed, cryoprotected anthers, maceration in dilute osmium tetroxide, critical point drying, sputter coating and examination. The processes of pollen wall development can then be observed in three dimensions, an important aid to understanding the spatial relationships involved in the determination of ornamentation and apertures. Details of the pollen and tapetum are described at various stages between meiosis and anthesis. A close conformity is demonstrated between the results obtained and those of earlier transmission electron microscopic studies of the same and related species although very different interpretations are made.     

Oxidation of pyrimidine nucleosides and nucleotides by osmium tetroxide

1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0° by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO₃NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient.