Tannic Acid and Thiocarbohydrazide as Structural Reinforcement Agents in the Preparation of Rabbit Knee Articular Cartilage for the Scanning Electron Microscope

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.     

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.     

High resolution scanning electron microscopy of isolated and in situ cytoskeletal elements

Evidence is presented that cytoskeletal structures (actin filaments, intermediate filaments, and microtubules) can be resolved by scanning electron microscopy after osmium impregnation of biological material, using thiocarbohydrazide as a ligand, followed by critical-point drying. These different classes of filaments or tubules can be identified both as purified protein polymers and as structured organelles within cryofractured or detergent-extracted cells.     

Modified Thiocarbohydrazide Procedure for Scanning Electron Microscopy: Routine use for Normal, Pathological, or Experimental Tissues

A modified thiocarbohydrazide (TCH) technique to cross-link osmium layers has resulted in a reliable method for preparing a wide variety of soft biological tissues for scanning electron microscopy without the use of evaporated metal. The technique works equally well on tissues with smooth surfaces and those with abrupt changes in contour or cut surfaces, as in biopsy or autopsied material or pathologically altered tissues, and thus has wide applicability. Small surface structures and functional areas between cells are distinct. In addition, thin cells such as alveolar endothelium in the lung exhibit a transparent property, allowing the visualization of cells within the capillary lumen while retaining adequate contrast for study of the capillary wall itself. Absence of an evaporated metal coat makes the removal of tissue from the scanning electron microscope specimen easy for embedding and examination of the same material with the transmission electron microscope.     

Improved Fixation of Cellulose-Acetate Reverse-Osmosis Membrane for Scanning Electron Microscopy

Fixation of cellulose-acetate membranes with either glutaraldehyde-osmium tetroxide or glutaraldehyde-ruthenium tetroxide resulted in extensive electron beam damage. Beam damage was eliminated and the bacterial surface structure was preserved, however, when cellulose-acetate membranes were fixed with glutaraldehyderuthenium tetroxide and treated successively with thiocarbohydrazide and osmium tetroxide.     

The Use of Osmium-Thiocarbohydrazide for Structural Stabilization and Enhancement of Secondary Electron Images in Scanning Electron Microscopy of Pollen

Pollen grains stained in a sequence of osmium (O) and thiocarbohydrazide (T) solutions (collectively known as OTOTO) appear structurally stable and undistorted in the scanning electron microscope (SEM), and usually do not require special drying. In fact, OTOTO can be regarded as another special drying method in palynology. This sequential incubation also strikingly increases the electrical conductivity of pollen grains in the SEM. Compared to standard sputter-coating or vacuum evaporative procedures, OTOTO reduces charging and yields secondary electron images with significantly higher resolution.     

Chemical analysis of osmium tetroxide staining in adipose tissue using imaging ToF-SIMS

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.     

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.     

Localization of carbohydrates in thick and ultrathin LR white-embedded tissue sections oxidized by acetic anhydride in dimethyl sulfoxide

The applicability of acetic anhydride (AA) in dimethyl sulfoxide (DMSO) for the oxidation of polysaccharide and their subsequent visualization with thiocarbohydrazide (TCH) and silver proteinate (SP) was evaluated on LR White-embedded thick and ultrathin liver sections. The results of these studies indicated that AA-DMSO-TCH-SP reaction is chemically specific on LR White-embedded tissues and that it offers distinct advantages for the localization of minute glycogen aggregates.     

Studies of Osmium Deposits in the Carotid Body of the Cat

Double aldehyde fixed carotid bodies and small pieces of vagus nerve of cats were incubated in 3 mM copper sulfate and 0.5 mM potassium ferricyanide in 0.05 M acetate buffer (pH 5.6) for 30 minutes at room temperature. Several modifications of this procedure were also attempted. Tissues were then postosmicated with 2% unbuffered osmium tetroxide and heated to 50-55 C for ten minutes. Under the electron microscope carotid body cells exhibited fine osmium deposits within cisternae of endoplasmic reticulum, saccule and vesicles of Golgi complex, and cristae of mitochondria. Intense osmium precipitation was also noted in the mitochondria of nerve endings. In addition, much more intense, more conspicuous and more localized reaction wan noted in the intraperiod lines of the myelin sheath of nerves. Deposits here were rod-shaped, displaying considerable variation in length. These results are discussed in the light of previous findings on osmium deposits in various tissues. It was concluded that the osmium reaction is unspecific, and that histochemical methods employing hot osmium tetroxide to amplify enzymatic activities may therefore not be reliable.     

Relationship between intracellular oxygenation and neuromuscular conduction during hypoxic hypoxia

Previous studies have shown that high-altitude hypoxic hypoxia is associated with reduced ventilatory capacity that may be related to skeletal muscle weakness. In the present investigation, ascent to high altitude (4, 000 m) was simulated experimentally by exposure of male rats (Sprague-Dawley, 250–350 g), anesthetized with thiopental sodium (25 mg/kg, i.p.), to a breathing gas mixture of 12% oxygen diluted in 88% nitrogen (F_iO₂ = 0.12). Determinations of oxygen saturation on micro- samples (250 ul) of arterial and central venous blood were made spectrophotometrically. Neuromuscular conduction latency was measured following electrostimulation of the sciatic nerve (1–5 V, 0.5 msec duration, 1–40 Hz) and recording of the electromyogram from the gastrocnemius muscle. Experimental hypoxia (F_iO₂ = 0.12) produced a highly significant increase in conduction latency from a control value (mean ± SEM) of 3.06 ± 0.16 msec to 4.02 ± 0.31 msec (n = 10, P < 0.001). Conduction latency increased with decreasing arterial oxygen saturation from a control value of 92.9% ± 0.18% to 83.2% ± 0.76% (P<0.001) in the absence of statistically significant changes in central venous oxygen saturation, central venous pressure, arterial and central venous pH, and heart rate. A significant decrement in the mean arterial blood pressure from a control value of 85 ± 1.5 mm Hg to 69 ± 1.5 mm Hg suggests that local ischemia may be a component of this model. These responses were accompanied by marked reduction in uptake of 3, 3′-diaminobenzidine (DAB) by gastrocnemius muscle mitochondria, suggesting decreased intracellular activity of cytochrome oxidase. It was concluded that exposure of rodents to hypoxic gas mixtures may provide a suitable model for studying the mechanism of skeletal muscle weakness associated with ascent to high altitude and of other conditions wherein the supply of oxygen to tissues is limited.     

Tissue fixation and staining with osmium tetroxide: the role of phenolic compounds.

It has been postulated that phenol-containing areas of plant and animal tissues were osmiophilic, but proof of direct interaction between osmium tetroxide and phenolic materials, or the nature of such reactions, has been lacking. We find that, under conditions similar to those of normal tissue fixation, osmium tetroxide reacts rapidly with those phenols containing o-dihydroxy groups (including such species found in plant tissues) to give very stable chelate complexes. We conclude that these complexes are responsible for the observed electron-density in phenol-containing areas of tissue treated with osmium tetroxide, so that such phenols are indeed osmiophilic.     

The chemical nature of osmium tetroxide fixation and staining of membranes by X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy was used to determine the oxidation states of osmium compounds present in erythrocyte ghost preparations and related systems treated with osmium tetroxide. Osmium tetroxide and cholesterol, codeposited at ?100 °C, began to react at ?70 °C, and Os(VI) was formed. Similarly, Os(VI) was detected for the known cholesterol-osmate ester prepared and purified chemically. However, osmium tetroxide applied in phosphate buffer (pH 7.2) gave rise to large proportions of Os(IV) and Os(III) species in addition to Os(VI) compounds. Egg phosphatidylcholine likewise produced a mixture of Os(VI), Os(IV), and Os(III), but dipalmitoyl phosphatidylcholine failed to give significant amounts of osmium containing products under identical conditions. Glutaraldehyde gave a mixture of compounds with the same osmium oxidation states when allowed to react with aqueous osmium tetroxide. Unfixed and glutaraldehyde-fixed erythrocyte ghosts also produced mixtures of Os(VI), Os(IV) and Os(III) under conditions identical to those of normal tissue processing. Additionally, the mixture of adducts initially formed by treatment with osmium tetroxide was further reduced by dehydration of the tissue with ethanol, resulting in a final mixture which was 50–60% Os(III).The results support a scheme for the reaction of osmium tetroxide with tissues in which the initial reaction site is the double bonds of unsaturated lipids to form Os(VI) derivatives. Subsequent hydrolysis and further reduction yield complexes of Os(IV) and Os(III). A mixture of these three states is present in membrane specimens during microscopic observation. Os(VI) and Os(IV) could be present as osmate esters and osmium dioxide, respectively; Os(III) could be present as an oxo- or amino complex(es). The photoelectron spectrum of intact erythrocyte ghosts can be synthesized from the spectra of phospholipid and cholesterol only, suggesting the predominance of the reaction with lipids in the fixation process.     

Rapid primary microwave-osmium fixation. I. Preservation of structure for electron microscopy in seconds

Microwave irradiation (MWIr) of tissues immersed in aldehydes has been used to preserve fine structure in seconds. The purpose of this study was to extend these findings to include rapid primary osmium fixation in a microwave (MW) device with a high volume exhaust. Blocks of rat heart and liver were trimmed to approximately 4 mm3 and exposed to 0.2 M symcollidine-buffered 2% osmium tetroxide for a period of 6-7 sec during MWIr (final solution temperature approximately 45 degrees C). We also evaluated rapid fixation of tissues exposed to MWIr simultaneously with immersion in dilute Karnovsky's fixative (6-7 sec to approximately 50 degrees C) followed by MWIr of specimens immersed in osmium (7 sec to approximately 45 degrees C). Tissues were stored in 0.1 M sodium cacodylate buffer (pH 7.3, 4 degrees C) up to 2 weeks and were stained en bloc in uranyl acetate, dehydrated in a graded series of alcohols, and embedded in propylene oxide-Epon sequence. Thin sections were stained with lead citrate and examined by transmission electron microscopy. We demonstrate that fine structural preservation of tissue blocks can be achieved by MWIr in aldehyde and/or osmium in seconds.     

Pressurized gas transport in two Japanese alder species in relation to their natural habitats

Oxygen uptake measurements have shown that pressurized gas transport, resulting from the physical effect of thermo-osmosis of gases, improves oxygen supply to the roots of the seedlings in two alder speciesAlnus japonica (Thunb.) Steud. andAlnus hirsuta (Spach) Rupr., which are both native in Japan. When gas transport conditions were established by irradiation of the tree stems the internal aeration was increased to a level nearly equal to the oxygen demand of the root system in leafless seedlings ofA. hirsuta, but was higher inA. japonica so that excess oxygen was excreted into the environment. An increase of superoxide dismutase (SOD) activity, which protects plants from toxic oxygen radicals and post-anoxic injury, has been observed in root tissues ofA. japonica when the seedlings were flooded for 3 days. The increase of SOD activity, in concert with high gas transport rates, may enable this tree species to grow in wet sites characterized by low oxygen partial pressure in the soil and by varying water tables. A less effective gas transport, flood-induced reduction of SOD activity in root tissues, and reduced height growth in waterlogged soil may be responsible for the fact thatA. hirsuta is unable to inhabit wettland sites.     

Myofibrillogenesis in vitro as seen with the scanning electron microscope

The present study describes an experimental approach whereby myofibrillogenesis and the three-dimensional arrangement of myofibrils present within cultured skeletal muscle cells can be examined using the scanning electron microscope. This procedure uses cells that have been cultured on gold-coated coverslips, and treated with Triton X-100 to extract the cell membrane and the soluble cytoplasm. Subsequent electroconductive staining by treatment with thiocarbohydrazide and osmium allows the myofibrils to be visualized. The images of myofibrils in various states of development observed by this method generally accords to those previously reported by transmission electron microscopy. Cell elongation and adhesion to the substrate causes mechanical stress from different directions which meet at branchings of the cultured myotubes. Many myofibrils are observed to run in the direction of the inferred stress lines.     

The Critical Oxygen Pressures for Respiration in Intact Plants

Two methods for determining critical respiratory oxygen pressure in whole plants are described. By a polarographic method involving the use of cylindrical platinum electrodes the following critical oxygen pressures for root respiration were found: Rice (cv. Norin 36). 0.024 atm: Rice (cv. Norm 37). 0.026 atm: Eriophorum angustifolium. 0.02 atm. These values contrast markedly with those obtained in vitro, and support earlier criticisms of in vitro measurements: they call into question the use of such data in the modelling of root aeration. When the results were assessed by an electrical analogue system, it was concluded that the respiratory activity in the intact root does not follow the normally accepted hyperbolic relationship with oxygen partial pressure. The experimental data were simulated most closely by assuming the critical oxygen pressure to be a function of respiratory responses in the low porosity (high diffusional impedance) tissues of the root meristem and stele, and respiratory activity in the moderately porous root cortex to be unaffected at values greater than 0.001 atm. A critical oxygen pressure of 0.025–0.04 atm for E. angustifolium was found from analyses of the gas phase oxygen in the leaves of whole plants after submergence in the dark. It was concluded that the higher value found by this method was most likely a function of respiratory responses in root tissue remote from the leaf and should not be regarded as the critical oxygen pressure for leaf respiration. The form of the oxygen concentration vs. time plot again suggested a very much lower critical oxygen pressure for certain of the plant tissues.     

An Approach to a Function of Photorespiration in C3 Plants

Intact attached leaves of wheat were illuminated at 2000 μmol m-2·s-1 in CO2-free gas for 3 hours, inhibition percentage of photosynthesis in these leaves by illumination was related lo oxygen concentration in the gas. (1) The damage to the leaves became less gradually when oxygen concentration rose from 0 to 10%. (2) Almost no damage occurred between 10%–50% O2. (3) The damage appeared again when oxygen concentration exceeded 50%. The duration of CO2 outburst of wheat leaves in CO2-free gas containing 8%–11% O2 was 0nly about 15–30 min. However, no photoinhibition could be observed under this condition. Oxygen also could prevent isolated chloroplasts from the damage by strong light. No matter what concentration of oxygen in CO2-free gas was during photoinhibition treatment, the photodamaged site was always in PSⅡ. It is concluded from the results that the way in which photoinhibition was alleviated by oxygen seems not only to be photorespiration, but also the other unknown mechanisms waich may play more important part in it.     

Cellular membrane contrast and contrast differentiation with osmium triazole and tetrazole complexes

Summary Addition of heterocyclic nitrogen compounds to the classical osmium tetroxide postfixation medium, applied after glutaraldehyde fixation, results in enhanced membrane contrast in ultrathin sections of liver tissue. The addition of similar compounds to potassium osmate solutions, results in contrast differences in some cellular membranes. The membranes of the rough endoplasmic reticulum, the nuclear envelope and the plasma membrane acquire contrast, while the mitochondrial membranes do not. The apolar regions of membranes are contrasted when osmium tetroxide is combined with heterocyclic nitrogen compounds, whereas the polar regions are contrasted by combinations of potassium osmate with these compounds. This polar membrane contrast is probably due to the presence of an amino-group in the heterocyclic nitrogen compounds. Compounds without the amino-group do not contrast membranes, although the glycogen is contrasted.X-ray microanalysis served to establish the relative osmium content in contrasted glycogen, and showed that such nitrogen compounds play a role in complexation of cations in aldehyde-fixed tissues. Electron spectroscopy for chemical analysis (ESCA) measurements of isolated muscle glycogen show that after treatment with various osmium tetroxide or potassium osmate solutions, hexavalent and quadrivalent osmium species are present in the glycogen. The presence of (heterocyclic) nitrogen compounds in such solutions stabilizes certain osmium valency species, and this may account for the contrast observed.     

Ultrastructural Details in Germinating Sporangiospores of Rhizopus stolonifer and Rhizopus arrhizus

Electron microscope examination of sporangiospore sections from Rhizopus stolonifer (Ehrenb. ex Fr.) Lind. and R. arrhizus Fischer revealed details on intracellular organization not previously reported. Aldehyde fixation followed by chromeosmium postfixation permitted clear depiction of ribosomes hitherto unrevealed in these cells. Mitochondria were diversiform. Spore wall structures in the two species were generally similar, but outer contours differed sufficiently to permit easy species identification in examination of sections. The spores of both species abounded in cytosomes, corresponding in size, shape, and heavy-metal "stain" affinities to spherosomes in cells of higher plants. The osmiophilic response of these spherosome-like inclusions was intensified by treatment of sections with thiocarbohydrazide solution and subsequent application of aqueous osmium tetroxide, which strengthens an assumption that they are lipid-rich. The margins of the spherosome-like inclusions in lead citrate-stained sections included dense particles, about 60 A across, whose crystalline-like arrangements suggested that protein as well as lipid was present. Frequent and close associations between the spherosome-like inclusions and various cell membranes suggested that such bodies participate in membrane elaboration during germination.