Synthesis of a more stable osmium ammine electron-dense DNA stain

Specific DNA staining for electron microscopic observation is simplified by a shorter synthesis of the staining reagent. The new, more reliable reagent, osmium ammine-B, is stable for more than a year, dissolves completely in water, and does not require reoptimization of staining conditions for every batch, yet reproducibly gives strong contrast to DNA-containing structures.     

Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.     

Modeling the 3-D RNA distribution in the Balbiani Ring granule

Mature Balbiani Ring (BR) granules in situ were stained with the nucleic acid specific stain, osmium ammine-B, recorded by electron spectroscopic imaging and reconstructed by electron microscope tomography to examine the three-dimensional (3-D) distribution of BR heterogeneous nuclear RNA (hnRNA). The BR2 granules contain ca. 37 kb of mRNA. Reconstructed BR granules were selected to emphasize one of the prevalent conformations seen in the sectioned salivary glands, the en face or pin-wheel conformation. A variety of image processing and volume-rendering operations were applied to the set of reconstructed BR granules. Some of the conclusions of this study are the following: (1) RNA distribution is not uniform throughout the granule; (2) RNA is condensed into about ten particles per granule, which all appear to possess approximately the same RNA stain density; (3) heterogeneity exists in the positions and sizes of particles within the various BR granules. These data argue for the folding of a beaded ribbon, consisting of connected particulate condensations of BR mRNA, possessing considerable 3-D flexibility, even in the packaged state. A comparison of this beadedribbon model and a prior folded hnRNP fiber model is also presented.     

Electron spectroscopic imaging and X-ray microanalysis of phosphorus in mouse sperm chromatin.

The influence of HCl hydrolysis on DNA detection in a Feulgen-type reaction using osmium ammine has been analyzed at the electron microscopic level by means of electron spectroscopic imaging, electron energy loss spectroscopy and X-ray microanalysis in energy dispersive spectroscopy. Both the stained DNA and the phosphorus mapping for a given hydrolysis condition were studied in parallel on the same nucleus. We have found that the pattern of osmium ammine-stained DNA and phosphorus imaging can be superimposed for a short hydrolysis time. After long HCl treatment, DNA is barely detectable by osmium ammine while phosphorus is still present in the thin sections. Taking into account the fact that the cells are embedded in a plastic resin, it is reasonable to think that in this case DNA depolymerization does not completely correspond to DNA loss. An incomplete loss of this highly denatured and depolymerized DNA from the plastic sections will explain both the presence of phosphorus and the poor stainability with a Schiff-type reagent.     

Enhanced cellular membrane contrast in a marine alga by Osmium-azole complexes

Summary Addition of certain heterocyclic nitrogen-carbon compounds to standard osmium tetroxide solutions used as secondary fixative resulted in an enhanced general membrane contrast in cells of the marine algaEmiliania huxleyi (Lohmann) Kamptner.Ultrastructural cell morphology and the contrast distribution were compared between cells treated according to a standard secondary fixation procedure and cells post-fixed when above mentioned heterocyclic compounds were introduced; in both cases some of the ultrathin sections were post-stained.Different compounds were tested: 1,2,4-triazole (TRA), 3-amino-1,2,4-triazole (A-TRA), 5-amino-tetrazole (A-TEA) and 2,4,6-tri-amino-1,3,5-triazine (melamine).The results were interpreted to indicate the possible bonding types arising from interaction of the heterocyclic compounds with osmium tetroxide and with membrane constituents.Interpretations were partly inspired by considerations from coordination chemistry.All above tests which did not include post-staining of thin sections could be performed at alkaline pH, and consequently calcified structures were preserved.The enhanced osmium accumulation at membranes was verified with X-ray microanalysis, which also showed that in the cases where membranes were visibly contrasted, localization of probable sites of intracellular non-crystalline calcium was facilitated.     

Localization of the synaptonemal complex under the light microscope

The use of osmium tetroxide fixation followed by postreatment with p-phenylenediamine gives an opportunity of locating the synaptonemal complex (SC) under the light microscope in mouse testes and Allium cepa anthers. When semi-thin sections from these materials were observed under phase contrast optics or dark field microscopy, fine threads in the pachytene nuclei were clearly visible. Post-staining of semi-thin sections with ammoniacal silver increased the contrast of the SC and allowed for observations using a bright field illumination. Ultrathin sections of osmium tetroxide/ p-phenylenediamine treated material showed that, under the electron microscope, this technique stains preferentially elements of the synaptonemal complex, while the surrounding chromatin remains unstained.     

上海市不同区县中小河道氮磷污染特征

以上海11个区县,共19条河道、65个点位进行1a的氮磷污染情况调查。结果表明:(1)上海河道为不完全感潮型河网水系。潮汐、降雨对氮磷污染物的分布影响具有时间差异性。(2)上海河道区县之间氮磷污染差异大(P0.05)。氮磷浓度从中心城区河道依次向外呈现"圆环状"稀释扩散趋势。黄浦江以南河道水质好于黄浦江以北河道;近江苏的河道总体氮磷浓度水平高于近浙江的河道(上海东南部)(P0.05)。水体中污染物浓度、扩散、降解与人为扰动和城镇化程度密切相关。(3)聚类分析(Cluster Analysis,CA)结果显示上海市河道污染水平在点位之间区别不大,而多维尺度分析(Multidimensional Scaling,MDS)显示上海市河道污染水平在点位之间具有一定差异,并与水质评价结果一致。(4)经生态修复后的河道水质优于修复前(P0.05),说明上海市人工水生态修复措施和生态型驳岸建设对改善河道水质有潜在价值。     

Ultrastructural Visualization of Lipids in Trypanosomatids1

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis, T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds

Summary Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study, I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32–34 s, final temperature between 40° C and 47° C.     

Immunocytochemical Evidence for the Presence of Barley Thionins in Rachis and Stalk of Wheat: Decrease of Antibody Binding at Fusarium culmorum Penetration Sites

In thin sections of wheat rachis and stem, embedded in Spurr's resin, prefixed with glutaraldehyde and postfixed with osmium, large amounts of barley thionins were localized in cell walls with anti-thionin-protein A/gold. Reduced binding to thionin was detected in cell walls penetrated by Fusarium culmorum, suggesting that cell wall thionins were degraded by the fungus.     

Periodic acid Schiff-p phenylenediamine staining of glycogen in chondrocytes

Summary This study reports a method whereby glycogen is identified in the chondrocytes of the secondary center of ossification prior to mineralization. The use of new fuchsin rather than basic fuchsin on one micron Spurr sections of femoral head cartilage fixed with potassium ferrocyanide-reduced osmium produced excellent identification of glycogen and when followed by p phenylenediamine, intensified cellular detail.     

The distribution of lipid in the cell structure: An improved method for the electron microscope

An improved partition method for visualizing lipid consists in fixing tissues in paraformaldehyde-glutaraldehyde followed by osmium tetroxide. Three progressive grades of lipid staining are then obtained: (i) by renewed osmium tetroxide alone, (ii) by partition in myrcene or farnesol solutions followed by renewed osmium, (iii) by saturated thymol in sucrose followed by partition and renewed osmium. No additional metallic stains are used. The thymol treatment in (iii) renders ‘masked’ lipid accessible to partition—the effect being regulated as required by time and temperature. Thymol used before the first osmium facilitates lipid extraction which provides a complementary test for lipid. The possibilities of the method have been demonstrated on sections of familiar tissues of insect (mainly Rhodnius, Hemiptera) and mammal (mouse). By and large the results support what is known already about the distribution of lipid in cells, but observations on lipid in muscle fibres, in the nucleolus and chromatin, in the cells of the adrenal cortex, in the lung and intestine suggest that the method might prove a source of new information.     

Crystallography and luminescence of divalent osmium complexes green osmium emitters and possible evidence for d-orbital backbonding

The preparation, photophysics, and solid-state structures of three osmium cored complexes are reported. The osmium complexes take the general form of [OsCl(N-N)(L-L)(CO)]⁺ hexafluorophosphate where N stands for a derivative of 1,10-phenanthroline and L stands for a phosphine type ligand. The emission of the complexes is shown to be blue shifted to the osmium emission of Os(bpy)₃ ²⁺. The emissions of the various complexes range from yellow (560 nm) to yellow-green (550 nm) to green (520 nm). The quantum yields vary between 60% and 75%. The complexes show lifetimes that are much longer than expected with ranges of 6.5-38 μs. Crystallographic results show that the carbonyl is trans to a phenanthroline nitrogen and the chloro ligand is trans to phosphorus. A discussion will be presented as to the nature of the bonding in these complexes based upon the data from the crystallography.     

Fine Structure and Cytochemistry of the Hydrogenosome of Tritrichomonas foetus1

Fine structural studies of the hydrogenosomes of Tritrichomonas foetus using an improved fixative reveal that they are enclosed by two closely apposed 6 nm membranes, which separate at some regions forming a large intramembranous vacuole where Ca⁺⁺-binding sites are located. Fixation of the cells in a glutaraldehyde solution containing 5 mM CaCl₂ and postfixation in an osmium tetroxide-potassium ferrocyanide solution led to the appearance of a reaction product associated with certain regions of the membrane of the hydrogenosomes and in the cisternae of the endoplasmic reticulum, in the recurrent flagellum, and in the plasma membrane. Treatment of ultrathin sections with EGTA removed the reaction product. These results, in association with others previously described, indicate the existence of several similarities between the hydrogenosomes and the mitochondria.     

Facile Synthesis of (22R, 23R)-Homobrassinolide

A synthesis of (22R,23R)-homobrassinolide is described. The LC and the chemical correlation studies for the oxidation product of a stigmasterol-like side chain with osmium tetroxide are mentioned. A stereochemical view for the mechanism of osmium tetroxide oxidation of the side chain is proposed.     

Localization of l-fucose in the root cap and slime of Zea mays L. utilizing fluorescent lectin conjugates and colloidal gold-lectin techniques

Ulex europaeus lectin (UEA) labelled with fluorescein isothiocyanate (FITC), rhodamine or colloidal gold, localized l-fucose in maize root cap cells and secreted root cap slime. Free-hand sections of maize root apices stained with FITC-UEA or rhodamine-UEA and examined by fluorescence microscopy yielded satisfactory results as long as the stains were freed of unconjugated dye, the sections treated with osmium tetroxide vapour to quench autofluorescence, and the samples incubated at 37°C. This resulted in successful labelling with a lower concentration of fluorochrome-lectin conjugate than reported by previous workers. Rhodamine-UEA was superior to FITC due to the lower primary fluorescence of the root tip observed under green light.Thin sections from glutaraldehyde fixed and Spurr's resin embedded maize root tips were treated with UEA bound to colloidal gold. Gold particles were found within sloughed cells and root cap cells, particularly concentrated over the Golgi complex, Golgi-derived vesicles and within the secretory slime products.     

PHOSPHORUS STATUS AND CYTOPLASMIC STRUCTURES IN SCENEDESMUS (CHLOROPHYCEAE) UNDER DIFFERENT METABOLIC REGIMES1

Phosphorus deficiency affects the anatomy of the unicellular green alga Scenedesmus obtusiusculus Chad. and influences the distribution of other inorganic elements in the cell in addition to phosphorus. Scenedesmus was grown under standard conditions with or without phosphorus. Cells were then cultured with phosphorus under conditions favouring glycolysis, respiration, or photophosphorylation for 2 h or photosynthesis for up to 8 h. The dominating features of phosphorus starvation, were loss of phosphorus and coions from polyphosphate bodies, accumulation of starch, decrease in the volume density of ribosomes both in the chloroplast and cytoplasm, and an increase in wall thickness. Under conditions favoring photosynthesis the mass fraction for phosphorus is low after 1 h, exceptionally high by 2 h, and diminishes by 8 h. High amounts of phosphorus are also regained under conditions favoring glycolysis and photophosphorylation but not respiration. After 2 h under photosynthetic conditions the volume densities of the chloroplast, cytoplasmic ribosomes, the vacuole, and the mitochondrion increased over controls. By 8 h the relative volume of the single ramified mitochondrion had decreased slightly and recognizable segments of it were sequestered within the vacuome. The autophagic nature of the vacuole was further evidenced by the presence of ribosomes and whorls of lamellae within it. Serial sections showed that all polyphosphate granules and sequestered materials were located within a continuous vacuolar cisterna.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and balbiani rings in Chironomus salivary glands

Summary This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.