The use of a simple method to avoid cell shrinkage during SEM preparation

In the course of preparing specimens for scanning electron microscopy, both glutaraldehyde and OsO₄-fixed cells exhibit a considerable shrinkage with a reduction of the mean cellular diameter of about 45% after critical point drying. However, if cells are successively treated with glutaraldehyde, OsO₄, tannic acid and uranyl acetate solutions, cellular shrinkage of only 5% is observed.     

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.     

Effects of glutaraldehyde fixative osmolarities on smooth muscle cell volume,and osmotic reactivity of the cells after fixation

The contribution of glutaraldehyde (GA) to the effective osmolarity of GA fixatives, the osmotic reactivity of the cells after fixation in GA, and also the duration of fixation in GA on cell volume, were investigated using cultured smooth muscle cells (SMC) and spiral aortic strips. Four fixation procedures were studied. We found that GA contributes to the total effective osmolarity of the fixatives, and that the type of buffers used for the fixatives can also affect the cell volume differently during GA fixation. After GA fixation, the cells were still osmotically reactive, regardless of the buffer types for making up the GA fixatives, so that the osmolarity of the wash buffer after GA fixation is important. However, OsO₄ eliminates osmotic responses, thus the osmolarity of OsO₄ fixative and wash buffer have negligible influence on the cell volume. Longer fixation time up to 4 h had no effect on the cell volume.     

A method for preparing quick-frozen,freeze-substituted cells for transmission electron microscopy and immunocytochemistry

A quick-freeze, freeze-substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO₄) in a ‘double-fixation’ protocol comparable to that used for conventional transmission electron microscopy. Cultured cells are quick-frozen in Freon 22 and freeze-substituted in an ethanolic solution of glutaraldehyde. Specimens destined for TEM are postfixed in OsO₄ in acetone, embedded in Epon-Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO₄ alone as a freeze-substitution fixative. However, if glutaraldehyde is used alone as a freeze-substitution fixative, specimens can be processed for immunocytochemistry without additional treatment with permeabilizing agents.     

Effects of fixation on electrophysiology and structure of human jejunal villi

The villi of human jejunum vary in size and shape during different functional conditions. In the base the lamina propria is isotonic with blood, in the tip hyperosmotic. Here we study electrophysiological and morphological effects of incubation in hypotonic, isotonic, or hypertonic solutions, and to test various isotonic fixatives for microscopy. Samples of jejunal mucosae, obtained during surgery in obese patients, were studied in Ussing chambers where electrical parameters were registered during incubation in Krebs solution at various osmolarities, and during fixation in formaldehyde, glutaraldehyde, or osmium tetroxide (OsO₄). The same fixatives were used for other jejunal specimens that were fixed directly for light microscopy. Morphometry was carried out to determine size and height of villi, proportion of lamina propria, and surface enlargement due to villi. Ussing chamber incubation in fluids with low osmolarity resulted in increased electrical resistance and epithelial swelling. Opposite results were obtained at high osmolality. Fixation was faster in formaldehyde than in glutaraldehyde or OsO₄. In biopsies processed directly for light microscopy the proportions of lamina propria of the mucosa, and of lamina propria of villi, were significantly larger in biopsies fixed in formaldehyde than after fixation in glutaraldehyde or OsO₄. The villus tips sometimes ended with a bleb with prominent spaces between the epithelial cells. In summary, jejunal villi swell in vitro when exposed to hypotonic solutions, and shrink in hypertonic solutions. Much of the morphological changes occurring during fixation can be related to the physiological hyperosmolar milieu in villus tips.     

Characterization of Erosion of Metallic Materials Under Cavitation Attack in a Mineral Oil

Cavitation erosion and erosion rates of eight metallic materials representing three crystal structures were studied. The erosion experiments were conducted with a 20-kHz ultrasonic electrostrictive oscillator in a viscous mineral oil. The erosion rates of the metals and alloys varied over three orders of magnitude. The erosion rates of brittle metals, iron, and molybdenum were higher than that of the titanium alloy but lower than the rest of the soft ductile metals and alloys. Studies with scanning electron microscopy indicated that the cavitation pits were initially formed at the grain boundaries and precipitates and that the pits formed at the junction of grain boundaries grew faster than the others. Transcrystalline craters formed by cavitation attack over the surface of grains and roughened the surfaces by multiple slip and twinning. Surface roughness measurements showed that the pits formed over the grain boundaries deepened faster than other pits. Computer analysis revealed that a geometric expression describes the normalized erosion curves during the time period 0.5 t₀ < t < 2.5 t₀, where t₀ is the incubation period. The fcc metals had very short incubation periods; the titanium alloy had the longest incubation period.     

Localization of Ca2+ -stores and tissue compartments with a Ca2+ -binding capacity in the organ of Corti of the guinea-pig by electron energy-loss spectroscopy

The addition of 10 mM CaCl₂ to glutaraldehyde fixative leads to the formation of small electron-dense deposits in the organ of Corti of the guinea-pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy-loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca²⁺ L_2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after conventional fixation (glutaraldehyde followed by post-fixation in OsO₄) revealed a small but significant calcium signal.     

A new fixation procedure for preserving the ultrastructure of marine invertebrate tissues

Addition of 0·05% OsO₄ to a conventional glutaraldehyde fixative for the first 10 min of fixation was found to improve greatly the preservation of ultrastructure in the eggs of Urechis caupo. Several workers have since confirmed this result in other marine invertebrate tissues. Specific protocols and techniques are given. We believe that the OsO₄ rapidly renders the plasma membranes of cells freely permeable to glutaraldehyde, allowing faster penetration of this fixative. This method should be applicable to a wide variety of tissues that are difficult to fix.     

Backscattered electron image of osmium‐impregnated/macerated tissues as a novel technique for identifying the cis‐face of the Golgi apparatus by high‐resolution scanning electron microscopy

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three‐dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis–trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis‐ or trans‐faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis‐Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis‐elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron‐mode SEM. Prolonged osmication by osmium impregnation (2% OsO₄ solution at 40°C for 40 h) and osmium maceration (0.1% OsO₄ solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.     

Chemical etching of natural fluorapatite crystals in acid solutions studied with the scanning electron microscope

Different aspects of the chemical etching process for natural fluorapatite (FAP) crystals in pure phosphoric acid solutions and for those with additives of Ca²⁺ and H₂SO₄ have been studied by means of the scanning electron microscope. The resulting experimental data obtained in the etching process are very sensitive to dissolving conditions, type of crystal face, and the presence or absence of any epitaxial coatings on the surface of dissolving crystals. It was found that the pyramidal crystal faces of the FAP have the highest dissolution rate value and, as a result, are not etched at all. The prism and pinacoid faces usually are covered by hexagonal etch pits under the same conditions. Additives of Ca²⁺ to the phosphoric acid solution result in transition from a chemical etching to a chemical polishing process, and additives of H₂SO₄ result in epitaxial coatings of CaSO₄· xH₂O (x = 0,0.5, 2) and shapeless pits formation.     

A procedure for rupture-free preparation of confluently grown monolayer cells for scanning electron microscopy

Monolayers of PtK-1 and HeLa cells grown on glass or plastic supports are extremely susceptible to lacerations, e.g., splits and cracks caused mainly by shrinkage when prepared for scanning electron microscopy (SEM). We find that a four-step fixation procedure including glutaraldehyde, OsO₄, tannic acid, and uranylacetate application, in combination with critical point drying, drastically reduces these structural damages. In addition, the conductivity of the specimens is enhanced, so that they can be investigated without gold coating. Transmission electron microscopy (TEM) investigation of perpendicular sections in the area of lacerations provides evidence that the subcortical cytoskeletal elements are of crucial importance in maintaining cell membrane stability during the preparations. Our relatively quick and simple procedure results in an improved structural appearance of the cells.     

The effects of osmium tetroxide post‐fixation and drying steps on leafy liverwort ultrastructure study by scanning electron microscopy

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO₄) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post‐fixed with 4% OsO₄, while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post‐fixation with 4% OsO₄ could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO₄ post‐fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.     

Morphology and lectin‐binding sites of pyloric caeca epithelium in normal and GnRH‐treated Atlantic bluefin tuna (Thunnus thynnus) Linnaeus 1758

Mucosal epithelium of pyloric caeca was studied in normal and in GnRH‐treated Atlantic bluefin tuna Thunnus thynnus L., using morphological analysis, conventional and lectin glycohistochemistry. The lining epithelium consisted of columnar (absorptive) cells, goblet cells and intraepithelial leucocytes. The epithelium from normal animals was significantly taller than GnRH‐treated samples. Conventional histochemistry displayed the same staining pattern in normal and hormone‐treated specimens which showed a mixture of neutral and sulphated acidic glycoconjugates in the luminal surface and goblet cells, and neutral glycans in apical granules of enterocytes. Lectin histochemistry revealed a different glycoconjugate pattern in normal and GnRH‐treated tunas. In normal specimens the luminal surface expressed sialoglycoconjugates which bound MAL II, SNA, KOH‐sialidase‐PNA, KOH‐sialidase‐SBA as well as asialoglycans stained with HPA, SBA, GSA I‐B₄, LTA. N‐linked glycans were highlighted by Con A and KOH‐sialidase‐WGA. In GnRH‐treated tunas the luminal surface did not react with SNA, SBA and LTA. The columnar cells of normal tunas bound KOH‐sialisase‐PNA in the apical region, KOH‐sialidase‐PNA, KOH‐sialidase‐DBA, HPA, SBA, KOH‐sialidase‐SBA and KOH‐sialidase‐WGA in apical granules, GSA I‐B₄ and LTA in the supranuclear region. GnRH‐treated specimens showed some columnar cells that stained with KOH‐sialidase‐WGA in the apical granules and with GSA I‐B₄ in the supranuclear region. The goblet cells of normal animals produced mucins positive to PNA, HPA, KOH‐sialidase‐DBA, SBA, GSA II. The latter three binding sites lacked in GnRH‐treated tunas. The results suggest that the mucosal epithelium of Thunnus thynnus L. pyloric caeca expresses a complex glycan pattern that is affected by GnRH‐treatment. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

An investigation on component and formation of tribochemical film in Si3N4-white iron sliding pair lubricated with distilled water

A tribofilm was formed during wear tests of a Si₃N₄-white iron pair lubricated with distilled water. In order to clarify the formation of the film, the wear tests for Si₃N₄-white iron pair with different sliding distances were carried out on a ring-block tester, using distilled water as lubricant. The worn surfaces of white iron specimens were observed under SEM. Furthermore, the component and structure of the film were analyzed by using AES, XPS, FTIR and XRD. From the investigation, the following results are presented. During the wear tests of Si₃N₄-white iron pair lubricated with distilled water, the oxidation and hydrolysis of Si₃N₄ occur on the wearing surfaces, and a tribochemical film, which mainly consists of silica gel, is formed on the wearing surface. The reason for the film formation is that the carbides in cast iron spall off during the wearing tests and the spalling pits are left on the wearing surface of the white iron. Then, the debris of Si₃N₄ or its oxidized product are embedded into the pits, and are further oxidized and hydrolyzed. The products of reactions are concentrated in the pits and polycondensed into silica gel, and a silica gel film is formed on the wearing surface. The film protects both Si₃N₄ and white iron, and makes the paired surfaces smooth. Therefore, the friction coefficient of the pair is down to 0·02, and the wear rates of Si₃N₄ and iron are near zero. However, because the resultants of oxidation and hydrolysis of Si₃N₄ can not be enriched on the wearing surface of carbon steel to form an effective tribofilm, both friction coefficient and wear rate of Si₃N₄-carbon steel pair lubricated with distilled water are still high in value.     

Boric Acid Self-Lubrication of B2O3-Filled Polymer Composites

Utility of boric oxide particles in PTFE and epoxy composite materials, in sliding contact with stainless steel, is explored. Boric oxide filler can provide PTFE with a two-decade reduction in wear rate, to 10^?5 mm₃/N-m. With adequate ambient humidity reduced wear rate can be achieved without inducing counterface abrasion, and the friction of PTFE is further reduced slightly. In such environments, boric oxide fillers can also reduce friction coefficient of epoxy from μ>0.7 to as low as μ=0.07. This lubrication mechanism results from replenishment of lubricous boric acid lamellar solid provided to the sliding interface by reaction of boric oxide with ambient water. Maintenance of the lubricating effect depends upon a sufficient rate of boric acid formation, relative to subsequent removal by wear. It is demonstrated that this formation/removal balance is affected by relative humidity and volume fraction of boric oxide filler, as well as normal load and sliding speed.     

A new freeze-drying device for platinum replica studies of cell surface and cytoskeleton: An example using immunogold-labeled human erythrocytes

We designed and built a freeze-drying device that ensures the protection of the specimens against contaminants during mounting on the cold stage of the freeze-fracture machine, transferring into the vacuum chamber and deep etching. The device consists of a copper cap that covers the specimen and a thermal connection that ensures thermal transfer between the microtome arm and the copper cap. This device was used to study the ultrastructural features of the erythrocyte membrane skeleton and the immunocytchemical localization of spectrin in an “in situ” approach, by freeze drying and platinum rotary shadowing. Human erythrocytes adhered to polylysine-coated coverslips and were broken by a stream of buffer that mimics the intracellular ionic environment (“inside buffer”). The samples were prefixed in periodate-lysine-paraformaaldehyde fixative, labeled with antispectrin 5-nm gold particles, fixed in glutaraldehyde, mordanted in tannic acid, postfixed in OsO₄, repeatedly washed in water, rinsed quickly in 30% ethanol, freeze-dried, and rotary-shadowed. Electron microscopic examination of the replicas revealed the skeletal network on the inner surface of the erythrocyte membrane. Immunocytochemical labeling proved that spectrin represents a fibrillar component of the network. Our data confirm the speculative model of the molecular organization of the erythrocyte skeleton, based on studies on in vitro association of proteic constituents. Both the technique and the device developed by us may lead to a deeper understanding of the spatial organization of the cytoskeletal network of more complex cell types.     

Further evaluation of boric acid vis-à-vis other lubricants for cold forming applications

The effectiveness of boric acid as lubricant has been evaluated in ring forging and deep drawing operations. The elapsed lay-by time between boric acid coating preparation and forming operation has significant influence on the friction factor. Fresh surfaces provide least friction since longer lay-by time permits absorption of moisture from the environment and tend to increase friction. Forging speed has no effect on the performance of boric acid lubricant. The average of peak forces obtained with tools having different surface coatings indicated that the hardness of the coating also has no effect on the effectiveness of the boric acid lubricant.     

Effects of cold rolling process variables on final surface quality of stainless steel thin strip

In cold rolling some surface defects, known as pits, are due to lubricant that, entrapped in the deep valleys of the surface roughness, is nearly incompressible and acts like an inclusion avoiding microcavity elimination. During the rolling process, when specific favorable conditions can be set up, the lubricant may be expelled by the microplasto-hydrodynamic lubrication (MPHL) mechanism and pits may be recovered. In this paper the Λ_m parameter, index of the MPHL, is investigated together with the neutral point position to better understand the practical process recommendations for surface defect recovery. By means of finite element analysis of a Sendzimir’cold rolling process, the sensitivity of these objective functions are studied by means of a design of experiment analysis changing the major process variables like back tension, friction coefficient, reduction parameter, initial thickness, and roll diameter.     

Optimal preparatory procedures of cryofixation for immunocytochemistry

To examine the optimal preparatory procedures of cryofixation for immunocytochemistry, the labeling density over the antigenic sites in cells processed by various protocols of freeze-substitution and embedding was quantitatively evaluated. Fresh tissue blocks of gerbil parotid gland were quickly frozen by a metal contact method using liquid helium and freezesubstituted with one of the following media: 4% OsO₄ in acetone or 0.4% OsO₄ in acetone or 0.3% glutaraldehyde in acetone. They were then embedded in either an Epon-Araldite mixture or Araldite 6005, which were polymerized at 60°C and 50°C, respectively. Some frozen samples substituted with aldehyde-containing acetone were embedded in Lowicryl K4M (polymerized at —30°C). Immunocytochemical localization of amylase was examined by indirect immunostaining by using antigerbil parotid amylase antibody and protein A/gold complex. Thin sections of epoxyresin-embedded materials were treated with oxidizing agents before immunostaining. The central dense core of heterogeneous secretory granules in the acinar cells was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The labeling density on thin sections of all the cryofixed materials examined was about 1.5 times or more as high as in those processed by conventional chemical fixation. The highest value of the labeling density was obtained from material which was substituted with 0.3% glutaraldehyde in acetone and embedded in Araldite 6005. Substitution with osmium-containing acetone appeared not to seriously affect immunoreactivity of the antigenic sites and was advantageous because of the distinctive images of membranes. Advantages and disadvantages of the individual protocols employed are discussed.     

A comparative study on the performance of boric acid with several conventional lubricants in metal forming processes

During metal forming, lubricants are necessary to prevent direct contact, adhesion, transfer, and scuffing of workpiece materials and tools. The lubricating action of boric acid is due to its layered crystalline structure, and is similar to the structure of MoS₂ and graphite. However, boric acid lubrication could offer distinct advantages in terms of its application prior to forming and removal after a forming operation since it can be dispensed using water and alcohol solutions, raising the possibility of being environmentally friendly. Its effectiveness under actual conditions of metal forming operations such as rolling, forging, and sheet metal drawing and stretching has been evaluated in this study with both ferrous and non-ferrous work materials. It was found that boric acid provided lowest friction in sheet drawing and stretching operations, which is attributed to its lattice layer structure that facilitates easy sliding between molecular layers. Under predominantly compressive conditions of forming, liquid or semi-solid lubricants have performed better as they could squeeze out along with forwarding workpiece surfaces. The findings presented in this paper increase the prospect for developing boric acid as an effective lubricant in the cold forming of materials under certain conditions.