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Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of brain lesions was performed using two techniques: (1) histological methods, such as TTC (Triphenyltetrazolium chloride), a time-consuming and inaccurate process; or (2) MRI imaging, a faster, 3D imaging method, that comes at a high cost. In the last decade, high-resolution micro-CT for 3D sample analysis turned into a simple, fast, and cheaper solution. Here, we successfully describe the application of brain contrasting agents (Osmium tetroxide and inorganic iodine) for high-resolution micro-CT imaging for fine location and quantification of ischemic lesion and edema in mouse preclinical stroke models. We used the intraluminal transient MCAO (Middle Cerebral Artery Occlusion) mouse stroke model to identify and quantify ischemic lesion and edema, and segment core and penumbra regions at different time points after ischemia, by manual and automatic methods. In the transient-ischemic-attack (TIA) mouse model, we can quantify striatal myelinated fibers degeneration. Of note, whole brain 3D reconstructions allow brain atlas co-registration, to identify the affected brain areas, and correlate them with functional impairment. This methodology proves to be a breakthrough in the field, by providing a precise and detailed assessment of stroke outcomes in preclinical animal studies.

Epoxy-embedded semithin sections are useful for the analysis of cell and tissue organization, as well as for the processing of samples for transmission electron microscopy. Because only a very limited number of staining protocols have been developed for epoxy-embedded sections; semithin sections are used infrequently compared to conventional paraffin sections. Here, we describe a simple and reproducible polychromatic protocol for the routine staining of epoxy-embedded semithin sections by adapting Twort's staining method (mixture of neutral red and fast green FCF). The method can be used for the visualization of cellular organization as well as for the detection of elastic and collagen fibers. The proposed protocol demonstrated the best results for samples fixed for transmission electron microscopy, which suggests, as we demonstrated here, that this staining protocol can also be used for correlative light and electron microscopy.

Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO4) treatment for improved SR-CLEM.

Knowledge about the three-dimensional fine structure of human heart, as a crucial vital organ of the body, is not only fascinating from the scientific or educational points of view, but has a very important clinical impact. Therefore, we decided to create a three-dimensional atlas of fine structure of the human heart. Tissue samples from ten human hearts were rinsed in phosphate-buffer solution, fixed by glutaraldehyde buffered solution, and post-fixed in osmium tetroxide solution. A gentle dehydration with ethanol in different concentration and drying at the critical point of CO2 were applied as next procedures. Non-conductive specimens were galvanized with thin gold layer and observed in scanning electron microscope. In this study, we present the three-dimensional ultrastructural architecture of the human heart from patients after myocardial infarction, end-stage failing heart as well as without apparent cardiac abnormalities at the time of autopsy. The results are presented as a histological atlas. Its images illustratively describe the fine structure of endocardium including the morphology of Purkyně (Purkinje) fibres, spatial arrangements of cardiac muscle cells inside myocardium or the arrangements of adipose tissue of epicardium. We present also figures of the ultrastructure of papillary muscles, intercalated discs as well as the connective tissue scars after myocardial infarction. The scanning electron microscopy could be a reliable technique to perform a more complete morphological data and to improve our knowledge of some pathological changes of tissues and cells, not always detected by conventional morphological examinations.

Lesion verification and quantification is traditionally done via histological examination of sectioned brains, a time-consuming process that relies heavily on manual estimation. Such methods are particularly problematic in posterior cortical regions (e.g. visual cortex), where sectioning leads to significant damage and distortion of tissue. Even more challenging is the post hoc localization of micro-electrodes, which relies on the same techniques, suffers from similar drawbacks and requires even higher precision. Here, we propose a new, simple method for quantitative lesion characterization and electrode localization that is less labor-intensive and yields more detailed results than conventional methods. We leverage staining techniques standard in electron microscopy with the use of commodity micro-CT imaging. We stain whole rat and zebra finch brains in osmium tetroxide, embed these in resin and scan entire brains in a micro-CT machine. The scans result in 3D reconstructions of the brains with section thickness dependent on sample size (12–15 and 5–6 microns for rat and zebra finch respectively) that can be segmented manually or automatically. Because the method captures the entire intact brain volume, comparisons within and across studies are more tractable, and the extent of lesions and electrodes may be studied with higher accuracy than with current methods.

Biological material itself appears with poor contrast in electron microscopy (EM), due to its composition mostly of light elements. Classical staining agents such as osmium tetroxide, uranyl acetate, and lead citrate preserve and/or stain cellular structures such as membranes, cytoplasm, and organelles well for EM. However, extracellular polymeric substances (EPS) show no or only poor contrast with these staining agents. The endothelial glycocalyx in blood vessels consists mainly of proteoglycans. It can be visualized by EM only by additional staining with heavy metal ions such as copper (Alcian blue, cupromeronic blue), ruthenium (ruthenium red), or lanthanum. Best results are achieved by combined perfusion of fixative and stain. Cationic hydrous thorium dioxide colloids (named here cThO2) trace acidic groups in EPS. We describe here the use of cThO2 to visualize the glomerular endothelial glycocalyx in the mouse kidney. cThO2 shows high electron density and binds to a continuous layer of up to a few hundred nanometers thickness on the glomerular endothelium, as well as on epithelia in other blood vessels in perfused animals. The observed staining pattern gives rise to periodic densities, with a spacing varying between 50 and 200 nm, depending on the overall layer thickness, which varies between below 50 up to 300 nm. Due to high electron density of the used cThO2 particles, the introduced method allows distinct imaging and precise fine structural analysis of the endothelial glycocalyx.

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

The retina is a part of the central nervous system in terms of histological and embryological aspect. It is a thin layer of neural tissue lining the inner eye. This study assessed the histomorphometric changes in the thicknesses of the retina and its layers using light microscope (LM) and retinal photoreceptor cells using transmission electron microscope (TEM) as a consequence of diabetes and effect of aloe vera gel extract in male rats. Thirty Sprague-Dawley matured male rats (175 ± 25 g) in two age groups (12 and 16 weeks) were divided into three main groups as control, diabetic, and diabetic receiving 400 mg/kg aloe vera extract. There were two subgroups in each group. For inducing diabetes, a single dose of streptozotocin (STZ) (50 mg/kg) was injected through IP route. Their blood sugar was measured by a glucometer before STZ administration and 24 h thereafter. At the end, animals were anesthetized with sodium thiopental (40 mg/kg) via IP injection. The retina was collected for tissue processing following dissection of eyes, fixed in 4 % glutaraldehyde, postfixed in osmium tetroxide 1 %, dehydrated, and then embedded in TAAB resin. Semithin sections (1 μm) were stained with toluidine blue stain and viewed under light microscope. Ten slides were prepared from each animal. Ultrathin sections were prepared, stained, and viewed under TEM. The blood glucose levels and body weight in treated rats were reduced in comparison to diabetic groups. The neural retina thickness and its layers were also different. In the aloe vera-treated group, the thickness of the retina and its layers retained their normal histological structures. In diabetic groups, the vesicular mitochondria and degenerated cristae and dense nucleus were observed in photoreceptor cells. After treatment with aloe vera extract, cristae gained partially their arrangement and chromatin spread. It is concluded that the degenerated retina and the thickness of different layers of diabetic retina could be affected with long-term treatment of aloe vera gel extract.

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: 12,000 μm), 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: 3,500 μm), 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: 450 μm), high-resolution μCT is suitable for observation of the transversus muscle fiber fascicles.

Osmium tetroxide (OsO4) is one of the most toxic air contaminants but its environmental effects are poorly understood. Here, for the first time, we present evidence of osmium uptake in a common her ...

The electroactive complex osmium tetroxide bipyridine holds great promise as a covalent label for biosensor applications regarding nucleic acids and protein detection. Labeling can easily be performed in the laboratory. Until now, almost only DNA species have been investigated using this label. Thymine (which occurs exclusively in DNA) is known to react much faster than cytosine and uracil. In order to explore the possibilities to modify and detect also RNA species in a timely fashion, we have investigated the kinetics of reactions of osmium tetroxide bipyridine with the pyrimidine bases in the micromolar concentration range at different temperatures by means of spectrophotometry. Results were confirmed using voltammetric detection for the determination of labeled oligonucleotides. The modification reaction can be easily completed at room temperature within 7 h, even in case of cytosine and uracil. At 60 °C, 3 h are sufficient for complete modification of all pyrimidine bases that are found in natural nucleic acids. These findings will be important for future biosensor applications with RNA species as target molecules.

We developed a rapid and effective procedure for scanning electron microscopy of three delicate dinoflagellates, Karlodinium micrum, Akashiwo sanguinea, and Heterocapsa niei. Good results were obtained when the specimens were fixed with a modified Parducz’s fixative (2% osmium tetroxide:saturated mercuric chloride = 5:1 v/v) for 10 min, washed in 0.05 M sodium cacodylate trihydrate buffer for 2 min, dehydrated in tert-butanol for 10 min and dried with hexamethyldisilazane in air for 3 min in a fume hood because reagents are very toxic. This method could be completed in 25 min. Compared with other preparative techniques, the present protocol has significant advantages for SEM observation by limiting distortion of delicate specimens and reducing the preparation time.

Osmium tetroxide (OsO4) is a commonly used stain for unsaturated lipids in electron and optical microscopy of cells and tissues. In this work, the localization of osmium oxide and specific lipids was independently monitored in mouse adipose tissue by using time-of-flight secondary ion mass spectrometry with Bi cluster primary ions. Substance-specific ion images recorded after OsO4 staining showed that unsaturated C18 fatty acids were colocalized with osmium oxide, corroborating the view that osmium tetroxide binds to unsaturated lipids. In contrast, saturated fatty acids (C14, C16 and C18) and also unsaturated C16 fatty acids show largely complementary localizations to osmium oxide. Furthermore, the distributions of saturated and unsaturated diglycerides are consistent with the specific binding of osmium oxide to unsaturated C18 fatty acids. The abundance of ions, characteristic of phospholipids and proteins, is strongly decreased as a result of the osmium staining, suggesting that a large fraction of these compounds are removed from the tissue during this step, while ions related to fatty acids, di- and triglycerides remain strong after osmium staining. Ethanol dehydration after osmium staining results in more homogeneous distributions of osmium oxide and unsaturated lipids. This work provides detailed insight into the specific binding of osmium oxide to different lipids.

This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.

Complexation of osmium tetroxide (OsO4) with pyridine and its substituted derivatives (i.e. 4-picoline, 3 -picoline, 4-tertbutylpyridine, methyl nicotinate, 3,4-dimethylpyridine, 3-chloropyridine, and 3-phenylpyridine) has been studied at different temperatures in benzene. Spectrophotometric measurements have been carried out to obtain the equilibrium constants and thermodynamic parameters. The equilibrium constants of the resulting 1:1 complexes are found to vary in the order 3,4-Me2Py > 4-tBuPy > 4-MePy > 3-MePy > Py > 3- PhPy > 3-C(O)OMePy ≈ 3-ClPy. All complexes are enthalpy stabilized whereas the entropy changes counteract the complexation. The results are discussed in terms of different basicities of the substituted pyridines.

The lamellar body is a membranous structure periodically laminating in vesicles that is known as the most distinctive feature of type II pneumocytes by conventional preparation methods for transmission electron microscopy. The quick-freezing and freeze-drying method, followed by osmium tetroxide vapor-fixation (QF-FD-OsV), was performed to examine the in situ morphology of the lamellar body in type II pneumocytes of living mouse lungs. Typical lamellar structures were rarely seen in vesicles of the type II pneumocytes, but amorphous components and dispersed stripes were often detected in the vesicles, as revealed by the QF-FD-OsV method. To clarify how the lamellar body was formed during the conventional preparation steps, lung tissues of mice were treated with different fixation procedures, such as immersion-fixation with osmium tetroxide or perfusion-fixation with glutaraldehyde followed by osmium tetroxide, in combination with alcohol dehydration or QF-FD-OsV. In addition to lamellar bodies of type II pneumocytes in the specimens with alcohol dehydration, some lamellar structures were also formed even with the QF-FD-OsV method. These findings suggest that the labile lamellar body is easily modified and formed during both chemical fixation and alcohol dehydration steps.

In the present paper, we report the results we obtained using several cytochemical techniques to analyze the infective larva of Wuchereria bancrofti. An imidazole osmium tetroxide solution was used to visualize unsaturated fatty acids. A highly contrasted material forming a continuous structure was observed on the larval surface and over the epicuticle. A strong reaction was observed on the esophagus and also on the inner secreted material. Carbohydrates containing vic-glycol groups were not observed on the cuticle of the third-stage larva of W. bancrofti submitted to the Thiéry technique. Using a panel of eight gold-labeled lectins, we found that the cuticle exhibited slight labeling with all lectins used, indicating residues of N-acetyl-D: -glucosamine, N-acetyl-galactosamine, D: -galactose, D: -manose, and L: -fucose. Surface anionic sites were visualized by using cationized ferritin particles. Treatment with trypsin partially inhibited the reaction, whereas the treatment with chondroitinase ABC, a specific enzyme for glycosaminoglycans, completely abolished the labeling with cationic particles.

Chlorosomes are the light harvesting structures of green photosynthetic bacteria. Each chlorosome from green sulfur bacteria houses hundreds of thousands of bacteriochlorophyll molecules in addition to smaller amounts of chlorobiumquinone and carotenoids. In electron microscopy studies, chlorosomes exhibit different appearances depending on the fixation method used. Fixation with osmium tetroxide results in electron-transparent chlorosomes. Fixation with potassium permanganate results in clearly delineated electron-dense chlorosomes. This fixation method features an electron-transparent area in the interior of the chlorosome. In addition to electron density patterns that can be considered compositions of rod-shaped elements, chlorosomes exhibit a striation pattern that is oriented parallel to the longitudinal axis. Treatment with osmium tetroxide followed by potassium permanganate treatment results in a more diffused density distribution that outlines connecting elements between the chlorosome and the cytoplasmic membrane, and connecting elements between the cytoplasmic membrane and the outer membrane, which act as a diffusion barrier for electron density.

Two series of chlorin p6 13,15-cycloimides that differ in their substituents at the nitrogen atom of the additional six-membered ring were synthesized. The compounds of the first series have a hydroxyl, alkoxyl, or acyloxy group at the 13,15-cycloimide nitrogen and those of the second series, residues of aliphatic alcohols. The cycloimides synthesized are satisfactorily stable and display an intensive light absorption maximum at 710-718 nm. Treatment of the cycloimides with sodium periodate in the presence of osmium tetroxide and with the Vilsmeier reagent resulted in the formation of 3-formyl- and 3-(2-formylvinyl)derivatives, respectively. The conversion of vinyl into formyl group or 2-formylvinyl group leads to an additional bathochromic shift of the long-wave maximum by 30 nm on an average. An extra hydroxy group was introduced at position 18 of the macrocycle to increase the cycloimide hydrophilicity. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

Dynamic gas extraction of osmium tetroxide from aqueous solutions was studied. The possibility of shortening substantially the duration of the stage of gas-extraction recovery of OsO4 by reasonable choice of temperature, flow rate of extracting gas, and sample volume was also considered. The coefficients of OsO4 distribution between the aqueous and gas phases were determined in the temperature range 20-90°C.

Since the ability of alveolar epithelial cells to ingest inhaled fine particles has not been characterized in detail, the present study seeks to evaluate this physiological activity. We used a 0.2% suspension of intact or lecithin-coated polystyrene latex beads (240 nm in diameter). A 5-ml suspension of intact or lecithin-coated latex beads was intratracheally administered to rats using a compressor nebulizer. Thereafter, the lungs were perfused intratracheally with glutaraldehyde solution and cut into small pieces. The samples were postfixed with osmium tetroxide, embedded in epoxy resin and examined under an electron microscope. Both lecithin-coated and uncoated beads were incorporated into alveolar macrophages. Some of the ingested beads in the alveolar macrophages were sequestered within lysosomes. Types I and II alveolar epithelial cells selectively incorporated only lecithin-coated beads, which were also observed within the cytoplasm of monocytes in the capillary lumen. These findings suggest that alveolar epithelial cells can incorporate exogenous particles, which are then transferred from the alveoli to intravascular spaces by transcytosis.

Two basic specimen preparation protocols that allow field emission scanning electron microscope imaging of intracellular structures in a wide range of plants are described. Both protocols depend on freeze fracturing to reveal areas of interest and selective removal of cytosol. Removal of cytosol was achieved either by macerating fixed tissues in a dilute solution of osmium tetroxide after freeze fracturing or by permeabilizing the membranes in saponin before fixation and subsequent freeze fracturing. Images of a variety of intracellular structures including all the main organelles as well as cytoskeletal components are presented. The permeabilization protocol can be combined with immunogold labelling to identify specific components such as microtubules. High-resolution three-dimensional imaging was combined with immunogold labelling of microtubules and actin cables in cell-free systems. This approach should be especially valuable for the study of dynamic cellular processes (such as cytoplasmic streaming) in live cells when used in conjunction with modern fluorescence microscopical techniques.

Cytolytic granules are specific organelles of activated cytotoxic lymphocytes mediating storage and regulated excretion of lytic molecules for killing of target cells. A variety of the other granule components may also participate in granule-mediated cytotoxicity. In this study, the subcellular localization of lipids in the granules of human decidual CD56+ natural killer-like cells was determined by staining with malachite green aldehyde and imidazole-buffered osmium tetroxide. Lipids were shown, for the first time, to be a constitutive component of cytolytic granules. Lipids formed an additional structural microdomain, located between the granule-limiting membrane and the granule core. Images of the granules on serial sections suggested that intragranular lipids wrap the core. We speculate that granule lipids participate in packing of lytic molecules inside the granules, in autocrine signaling ending granule secretion, and in the killing process.

Onion (Allium cepa L. cv. Ebeneezer) roots from vermiculite culture were examined with transmission electron microscopy to detect the plasmodesmata in all tissues. In young root regions, plasmodesmata linked all living cells together in all directions. In old zones, the plasmodesmatal connections of the endodermis to its neighbor tissues were not interrupted by later suberin lamella and cellulosic wall deposition. Moreover, plasmodesmata in the fully mature endodermis usually exhibited a large central cavity. In the exodermis, however, upon deposition of suberin lamellae in long cells, all plasmodesmata that initially linked them to their adjacent cells were severed. Afterwards, the long cells lost the capability of forming wound pit callose and their protoplasts began to degenerate. The mature exodermal layer was symplastically bridged to its neighbors only by the short (passage) cells that lacked suberin lamellae. Compared to the long cells, the short cells not only had thicker cytoplasm surrounding their central vacuoles but also a higher density of mitochondria and rough endoplasmic reticulum, consistent with an active involvement in the transport processes of the root. The above results were obtained by an improved, extended transmission electron microscopy procedure devised to analyze plasmodesmata in cells with suberin lamellae. By prefixing root tissues in glutaraldehyde and acrolein, all cells were well preserved. Postfixation was carried out in osmium tetroxide at a low concentration (0.5%). Following dehydration in acetone and transfer to propylene oxide, infiltration with Spurr's resin was accomplished by incubating samples in the accelerator-free mixture for 4 days, then infiltrating samples in the accelerator-amended mixture for additional 4 days.

In urinary cytodiagnosis, pathological characterization of atypical cells is sometimes difficult because cells in urine samples tend to be degenerated. To overcome this problem, we adopted serial examination under light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Five patients with transitional cell carcinoma primarily developed in the bladder, three patients with dysplastic cells, and one patient with metastatic and infiltrative colorectal carcinoma were subjects of the present study. Sample cells were smeared on a film slide especially suitable for serial use (a film slide used in X-ray diagnosis), fixed in 1% glutaraldehyde (GA) for 30 min, stained with Papanicolaou (Pap) staining, and immediately processed for fixation in 2% paraformal-dehyde (PA) and 2.5% GA. Slides were postfixed in 0.5% osmium tetroxide. Then, cell samples were subjected to a series of observations under LM, SEM, and TEM. One of three patients in whom dysplastic cells were suggested under LM was finally diagnosed as transitional cell carcinoma by subsequent examination under SEM followed by TEM. Thus, serial use of LM, SEM, and TEM proved useful in discriminating transitional cell carcinoma (grade 1) from dysplastic cells and also in distinguishing metastatic tumor cells from primary tumor cells in urine samples.

Highly sensitive catalytic waves of OsO4-bromate linear sweep voltammetric and square wave voltammetric techniques were proposed for the determination of ultratrace amounts of osmium. Under optimized conditions, the catalytic linear sweep voltammetric procedure gives excellent reproducibility and limits of detection and a linear dynamic range and gives a low signal for background, whereas the square wave voltammetric technique gives higher sensitivity, but the background signal was high and the linear dynamic range was narrower. The methods are suitable for the determination of osmium as osmium tetroxide as low as 1.5×10-10 mol/l. The currents are linear for the concentration ranges of 0.10 - 210.0 ng/ml and 0.20 - 2.0 ng/ml for linear sweep voltammetry and square wave voltammetry, respectively.

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.

The formation of caoutchouc in teak sapwood and heartwood was examined in an increment borer core using light microscopy after staining with osmium tetroxide. The distribution of the polyisoprenoid in untreated and extracted heartwood, its influence on surface chemistry, contact angle with water and formation of radicals before and after irradiation with sunlight, and also on decay resistance were determined. Caoutchouc was not observed in the cambial zone and outer sapwood, but occurred from the middle of sapwood as black globule. In sapwood, if available, it was mainly found in the lumen of parenchyma. In heartwood the globule were also detected in fibers and vessel elements. They usually fused into one big or irregular formed structures. SEM of untreated heartwood cuttings showed that lumen of all cell types and also the cut-surfaces were covered with extraneous materials. Extraction with acetone which removed extractives except caoutchouc did not change the surface morphology markedly. Residual caoutchouc still covered cell lumen and cut-surfaces. Successive extraction with acetone and chloroform removed extraneous materials, and lumen surfaces and cut-surfaces became clear. It revealed that caoutchouc occurred also in cell walls. The XPS spectrum of untreated teak heartwood showed a rather high C1, a moderate C2 and a low C3 peak area. After acetone extraction the C1 peak area increased slightly but the C2 and C3 area decreased. Successive extraction with acetone and chloroform caused a significant decrease of C1, but an increase of both C2 and C3 values. Untreated teak heartwood cuttings showed a high contact angle with water, indicating hydrophobic properties. Extraction with acetone increased contact angle slightly. This is explained by removal of polar compounds and redistribution and enrichment of the apolar caoutchouc on the surface. Successive extraction with acetone and chloroform removed all extractives and contact angle was significantly reduced. The ratio of ESR relative signal intensities of free radicals in untreated specimens, acetone extracted and acetonechloroform extracted specimens which were irradiated and non-irradiated showed the highest values for acetone extracted ones, followed by untreated and acetone-chloroform treated specimens. Reconstituted panels made from ethanol extracted teak heartwood meal were decay susceptible. Specimens made from wood meal successively extracted with ethanol and chloroform showed slightly less resistance that specimens made from ethanol extracted ones. The high decay resistance of untreated teak heartwood is postulated due to a synergetic effect of decay active ethanol extracts and non-active caoutchouc.

A general mechanism for oxidizing reducing sugars (pentoses, hexoses and disaccharides) by OsO4 in alkaline medium is proposed. The reactions exhibit pseudounimolecular kinetics with respect to OsO4, are first order with respect to lower [sugar] and [OH−], but tend towards zeroth order with respect to both higher [sugar] and [OH−]. These results suggest the formation of an activated complex between the enediol andd osmium tetroxide, which slowly disproportionates to give osmium(VI) species and the intermediate products. The reaction products were identified. When an aldehydo sugar is treated with alkali, structural changes occur and an equilibrium is set up, mainly involving a keto sugar product and the starting aldehydo sugar consumed. This process is reversible. These changes are mainly due to the well-known Lobry-de-Bruyn-Alberda Van Ekenstein transformations.

The chlorine water/ethanolamine-silver nitrate method introduced by Coppick and Fowler for the detection of lignins was evaluated for cyto- and histochemical work using different reagents and fixatives for specimens embedded in epoxy resin. Fixation schedules tested included ethanol, glutaraldehyde, and glutaraldehyde followed by OsO4 as a post-fixative. Chlorine water, sodium hypochlorite, and calcium hypochlorite were the oxidising agents evaluated for their efficacy as part of the Coppick and Fowler procedure. The Coppick and Fowler method was tested against stem woody tissue ofLophomyrtus obcordata, and haustorial xylem tissue of the sucker of its attached dwarf mistletoeKorthalsella lindsayi. The presence of lignins in walls of these cells was indicated in thin sections for transmission electron microscopy by fine electron-dense deposits. Post-staining thin sections did not affect the lignin reaction, but tended to mask its effect due to increased wall contrast. In histological preparations lignified walls stained orange/brown. Counter-staining in methylene blue/azur B caused lignified walls to appear dark green/brown and non-lignified walls blue. Fixation in either ethanol or glutaraldehyde produced identical staining for lignins. Penetration by chlorine water was sometimes irregular, more so with glutaraldehyde fixation, with parts of tissues consequently not responding to the lignin reaction. Post-fixation in osmium tetroxide following primary fixation in glutaraldehyde slightly improved penetration of chlorine water. However, osmium caused greater amounts of extraneous stain deposits compared with other fixative regimes. Chlorine water was confirmed as the most effective oxidising agent for reacting with groups in lignins to produce reducing residues in the Coppick and Fowler method. Sodium hypochlorite caused no reaction. Calcium hypochlorite exhibited limited oxidative capacity resulting in slight staining for lignins. The Coppick and Fowler procedure was concluded to be a suitable method for demonstrating lignins in cyto- and histochemical preparations using material fixed in either ethanol or glutaraldehyde, and with embedding in epoxy resin.

Confocal laser scanning microscopy and transmission electron microscopy were used to study the inner membrane complex of tachyzoites of Toxoplasma gondii. DiOC6, a lipophilic cationic fluorescent dye used to visualize the endoplasmic reticulum of eukaryotic cells, labeled cytoplasmic structures in a reticulated pattern and the periphery of the nucleus of the host cell. Intracellular and extracellular tachyzoites were stained. Observation of several focal planes showed labeling of the most peripheral region of the protozoan. Reaction product was observed in the outer nuclear membrane, in profiles of the endoplasmic reticulum, and in the inner membrane complex of tachyzoites subjected to the KI-OsO4 technique. Taken together, these observations suggest that the inner membrane complex may represent a specialized region of the endoplasmic reticulum of tachyzoites of T. gondii.

A five-step synthesis of both enantiomers of 1,2-di(2,4,6-trimethylbenzylamino)-1,2-diphenylethane,i.e., Corey (R,R)- and (S,S)-controllers for enantioselective dihydroxylation of olefins by osmium tetroxide, starting from α,α′-diphenylglyoxime, has been developed. The key operations in the synthesis are the optical resolution of intermediaterac-1,2-diamino-1,2-diphenylethane into two enantiomers using only (R,R)-tartaric acid and the subsequent enhancement of the enantiomeric purity to >98% by crystallizations of the corresponding Schiff's bis-bases. Analysis of the enantiomeric purity of the controllers can be easily performed using1H NMR spectra of their salts with (R)-α-methoxy-α-(trifluoromethyl)phenylacetic acid (MosherR-acid).

We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purple-red (lambda max = 520 nm) to violet (lambda max = 539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultratain sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated specimens. In conclusion, the ability to bind selectively and specifically to protein-aceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level.

A new version of Timm's sulphide silver method involving in vivo binding of zinc ions in zinc enriched terminals is presented. By injecting sodium sulphide into the vena cava of deeply anaesthetized animals, it is possible to bind chemically the vesicular zinc, i.e. chelatable zinc (zinc ions), in secretory and synaptic vesicles, in the form of zinc sulphide crystal lattices. Four minutes after the intravenous injection the animal is perfused transcardially with a phosphate-buffered solution of glutaraldehyde, glutaraldehyde and formaldehyde, or with a saline solution. The nanometer-sized catalytic crystals can then be silver-amplified in cryostat and vibratome sections by exposure to an autometallographic developer. It is demonstrated that contemporaneously with silver enhancement, the zinc sulphide crystals are transformed to the corresponding silver sulphide crystals. For ultrastructural studies, autometallographic development of vibratome sections is recommended. From these sections tissue blocks are cut from the areas of interest, blockstained with osmium tetroxide and embedded in Epon. This approach results in a zinc-specific autometallographic staining of the sections of a hitherto unseen, high technical quality.

Triacylglycerol is accumulated by Streptomyces spp. when grown in submerged culture. Ultrastructural studies using transmission electron microscopy (TEM), staining and freeze-fracture/freeze-etch procedures, and light microscopy confirmed the accumulation of neutral lipid by S. lividans and S. coelicolor during the stationary phase and its storage within membrane-bound globular structures within the cytoplasm. These structures were of various sizes and occupied up to approximately 80% of the total cell volume at that time. There was no evidence of such material within cells examined during the early exponential phase of growth. The globules visualised by TEM were electron-transparent since they comprised lipids containing saturated fatty acids that did not react with osmium tetroxide. The globules appeared to be bounded by a single membrane.

Spermatozoa from several members of the closely related Mycetophilidae and Keroplatidae were examined by electron microscopy using a fixative that contains glutaraldehyde and tannic acid, followed by a post-fixative that consists of uranyl acetate rather than osmium tetroxide. With this fixative, the detailed architecture of the flagellar axoneme and its various microtubules could be resolved. The so-called accessory tubules, which surround the central 9+2 unit of the sperm axoneme, were found to have 16 protofilaments in several examined Mycetophiloidea, but in no other Diptera. As 16 is the common number in holometabolic insects, it is presumably the plesiomorphic condition in Diptera. Other fungus gnats have accessory tubules with 15 or 14 protofilaments. The intertubular material situated between the accessory tubules is smaller in the examined members of the Mycetophilidae than in the Keroplatidae. The acrosome consists of an apical vesicle, which in one species, Macrorhyncha ancae, has three microtubular doublets in its anterior part and two large and three small extensions which extend posteriorly along the sperm axoneme.

The effect of deafferentation on glycogen metabolism was studied in the sensory cells of mormyrid Knollenorgan electroreceptors. Glycogen was visualized in the sensory cells after fixation in a solution containing potassium ferricyanide and osmium tetroxide. The density variations of glycogen were evaluated by a morphometric method. Sectioning of the afferent nerve results in a cessation of the spontaneous receptor cells activity after 48 h and the glycogen content of these cells increases three fold in the first 5 days after nerve transection. From day 5 on, the glycogen concentration diminishes progressively until day 13. After the sensory cells had become completely deformed, the quantification of glycogen particles was no longer possible and the degeneration of the sensory cells was complete within 20 days after nerve section. These results show that (1) the afferent nerve fibre is indispensable for the anatomo-functional maintenance of the sensory cells and (2) the nerve has only an indirect influence on glycogen variations within the sensory cells.

We describe a DNA-specific staining procedure, using a blue platinum organic dye, which allows DNA imaging of chromosomes by detection of back-scattered electrons in the scanning electron microscope. DNA distribution is visualized within chromosomal details of the centromeric and satellite regions, or in interphase chromatin, with high-resolution (10-15 nm) for comparison with the corresponding secondary electron image representing DNA plus protein.

A method of quantitatively determining the volume of fat emboli in a tissue using an image analysis system (I.B.A.S.) was developed. This procedure is an interactive, semi-automated tool allowing the quick and accurate gathering of large quantities of data from sections of different tissue samples stained by osmium tetroxide. The development of this procedure was aimed at producing a system which is reliable, reproducible and semi-automated thereby enabling epidemiological and serial studies to be made of a large number of histological sections from different tissues. The system was tested in a study of tissue sections from a series of fatalities from an aircraft crash in an attempt at correlating the incidence of fat emboli with the presence of multiple fractures and soft tissue injuries, the correlation to be made being between the quantitative presence of fat emboli and the extent and severity of injuries suffered.

Osmium tetroxide (OsO4) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO4 hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO4 will also identify other torsional perturbations of DNA. In this study, OsO4 was used to footprint an AT-rich region (between -780 and -500) of the maize Adh1 promoter. Hyperreactive sites were identified both in vitro and in vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO4 hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei.

This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosomal organelle that stains metachromatically with Toluidine Blue and which is present in Kurloff cells (a blood cell unique to the guinea pig). Splenic tissues were fixed with 1% cetylpyridinium chloride (CPC) added to 4% paraformaldehyde and examined either after Spicer's high-iron diamine staining for sulphated anionic sites followed by post-fixation with ferrocyanide-osmium tetroxide or after a simple post-fixation with ferrocyanide-osmium tetroxide. CPC-precipitated sulphated sites were preferentially located at the periphery of the Kurloff body but, unexpectedly, were absent in the central matrix. Although their electron opacity was lower, these anionic sites were readily observable in the absence of HID-staining after sole post-fixation by ferrocyanide-reduced osmium. CPC-precipitated sulphated anionic sites were either associated with the myelin figures or constituted unexpected structures. They contained (i) tightly-stacked lamellae, with a very regular 4 nm periodicity, and (ii) groups of 2, 3, 4 short dense lines with a 3–5 nm periodicity. By taking into account the susceptibility of these HID-reactive structures towards chondroitinase ABC, these different sulphated components were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell. Their colocalization with phospholipidic structures was suggested following, observation of sections treated by a chloroform-methanol mixture.

Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding "vesicles" from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO4, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO4 (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO4 for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).

An ultrastructural study was performed on Arthrinium aureum. The fungi were treated with glutaraldehyde and osmium tetroxide fixation. The hypha and conidia has a concentric membrane system which consisted of multiple membranes of a myelinoid appearance, and continued to the conidia and hypha plasma membrane. The fungi were also treated with periodic acid-alkaline bismuth (PABi) staining after glutaraldehyde and osmium tetroxide fixation. PABi positive materials were found on the marginal glycogen granules, the concentric membrane system and the conidia plasma membrane.

Substitution of surfactant in immature lungs has two functional targets: the reduction of the overall alveolar surface tension and the mechanical stabilization of the system of alveoli having different diameters. Indeed, the lowering of the surface tension facilitates the inflation of the lungs, but according to Laplace's law small and large alveoli are not in pressure equilibrium as long as the surface tension is equal in both small and large alveoli. In the present work, we tried to stabilize the lungs and to compare the effect of bolus surfactant substitution with the two-step substitution of fluorocarbons and surfactant. In all, 24 fetal immature lungs were used. For our experiments we used fluorocarbon 72 (FC-72) with a surface tension of 12 mN/m. In groups 1 and 2, a mixture of dipalmitoylphosphatidylcholine (DPPC): cholesterol 9∶1 (molar ratio) or DPPC: phosphatidylglycerol (PG) 9∶1 (molar ratio) was administered intratracheally as a bolus. In the case of groups 3 and 4, the immature lungs were rinsed first with FC-72. After removing the fluorocarbon, the lungs were artificially ventilated and the DPPC: cholesterol 9∶1 (group 3) or DPPC: PG 9∶1 mixture (group 4) was given in aerosol form. Static pressure-volume curves (p-v) of the mean values of the 6 lungs in each group were registered at the beginning (0 min) and after 20 and 40 min of artificial ventilation. Airway opening pressure, weight-specific end-inspiratory lung compliance, and phospholipid contents were investigated. Statistically significant differences in weight-specific end-inspiratory compliance were found between groups 1 and 3 and between groups 2 and 4; between the FC groups (3 and 4) no statistically significant improvement could be observed, although the compliances of group 4 were better than those of group 3 in three p-v diagrams registered at the beginning and after 20 and 40 min of artificial ventilation. The date presented suggest that the initial transient opening of the immature lungs following the application of fluorocarbon seems to be necessary for subsequent replacement of phospholipids precisely where they are needed; i.e., at the air-liquid interface of the alveoli. The additional substitution of phospholipids (in our present study DPPC: PG is better than DPPC: cholesterol) is necessary to stabilize the lungs after the fluorocarbon has been evaporated.

The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18–21 particles/1000 nm of LRE) with fairly regular interspacing (45–65 nm) as reported previously. This is the first report to identify the three-dimensional ultrastructure of anionic sites of GBM, and provides new information on the location and distribution of anionic sites in the glomerular capillary wall. In addition, these studies suggest that several chemical procedures used in conventional transmission electron microscopy to visualize PEI tracers, may produce structural changes and disarrangement of PEI particles that can be avoided with the quick-freezing and deep-etching method.

Reaction of furan with 3,4-di-O-acetyl-L-rhamnal (1) afforded a mixture of epimeric 3-C-(2-furyl) glycals3 and4 and 1-C-(2-furyl)-hex-2-enopyranoses5 and6. Glycals3 and4 were transformed to 2-deoxy-glycosides9–13 and 3′-deamino-3′-C-(2-furyl)daunorubicines15 and16. Hex-2-enopyranosyl5 wascis hydroxylated with osmium tetroxide/N-methylmorpholine N-oxide to C-glycoside14.

For the first time, immunoreactivity to 1,25(OH)2D3-like molecules and their receptors has been investigated in a calcium transporting epithelium of a crustacean, Orchestia, using vertebrate antisera on ultrathin cryosections of posterior caeca previously fixed in glutaraldehyde, then postfixed in osmium tetroxide. Both immunoreactivities were located mainly in the nuclei of epithelial cells. Quantitative differences in 1,25(OH)2D3-like immunoreactivity were noticed from one stage of the molt cycle to another. These results, together with other data, contribute to evidence that immunoreactive 1,25(OH)2D3-like molecules may be involved in the regulatory processes of calcium metabolism in this terrestrial crustacean and suggest an involvement of these substances in the regulation of calcium movements in the posterior caeca.

Samples from seven sectors of the rabbit knee articular cartilage were shaved and prepared for the scanning electron microscope using either tannic acid, thiocarbohydrazide or nothing (control). Surface morphology was found to be more typical to a given sector and less so to a specific preparation procedure. Rough areas were recorded from load-bearing sectors, while smooth areas appeared on load-free ones. However, fibrillations were discerned on control load-bearing sectors only, and pits and humps were never detected. Tannic acid and thiocarbohydrazide may have exerted their structural reinforcing effect on the tissue preservation by enhancing the binding of osmium tetroxide to it, possibly along with that of other soluble tissue constituents.

The brush cells (BC) of the mouse gallbladder were studied using light and electron microscopy (transmission and scanning) to determine their shape and distribution. Specimens were fixed in glutaraldehyde and postfixed in ferrocyanide-reduced osmium tetroxide. BC selectively stained with toluidine blue could be identified by means of light microscopy and subsequently studied in serial semithin and ultrathin sections. The results revealed that the shape of the BC is flask-like. A slender, occasionally branched cytoplasmic process emerges from the bulk cell body and extends through the basal region of neighboring epithelial elements to the basement membrane. Examination of the entire gallbladder epithelial surface by scanning electron microscopy revealed that the BC are numerous in the neck region of the organ but only scanty or even absent in wide areas of the corpus region. Their number increases again in the fundic region. These results demonstrate a preferential regional distribution of BC in the gallbladder, which is discussed in relation to a possible functional significance of the BC.