Prolonged ethanol replacement by CO2 increases splits on articular cartilage surface after critical point drying

Ethanol replacement by CO₂ of glutaraldehyde-fixed and ethanol-dehydrated rabbit articular cartilage specimens was monitored with both gas chromatograph and alcometer prior to critical point drying (CPD). The surface structure of the patellar specimens was also systematically registered with a semiquantitative scanning electron microscopic method. After a 2 h interval, when about 28 μl of ethanol/15 min CO₂ extract was removed, the articular surface was smooth, although small areas of striated surface and superficial splits were present. A long-term CO₂ treatment (16 h) removed ethanol completely, but increased superficial splitting of the articular surface after CPD. Air-drying of the specimens gave rise to inferior preservation of the cartilage: large areas with pitted and leafy surface qualities, but no superficial splits, were present on the surface. It was evident that prolonged ethanol replacement by CO₂, prior to CPD, degraded surface structure of the articular cartilage which should be taken into consideration in the planning and design of experiments. Ethanol removal by CO₂ could conveniently be monitored by an alcometer.     

Volume changes during preparation of mouse embryonic tissue for scanning electron microscopy

This paper describes the results of experiments in which the volume changes in mouse embryo limb samples were followed more or less continuously after fixation through dehydration and critical point drying, with in some instances data relating to post critical point drying shrinkage. 14 and 15 day p. c. mouse embryos were fixed in 3 % glutaraldehyde in cacodylate buffer and stored in this fixative until use. Single specimens were studied using a Quantimet image analysing computer to record the changes in projected area of the unmounted specimens as they were passed through the usual series of reagents according to various commonly used dehydration schedules. The area changes were converted to volume changes for the purposes of presentation in this paper. The Quantimet system could not be used to follow volume changes in the CPD bomb so that most experiments detail the volume in the intermediate fluid before CPD and the size of the specimen immediately after it was removed from the CPD bomb. A few experiments were conducted in which the specimens were measured whilst they were in the CPD bomb. The measurements relating to dehydration and CPD procedures were compared with measurements of air dried and freeze dried specimens. All three drying methods cause considerable shrinkage: freeze drying to 85 % of the glutaraldehyde fixed tissue volume; critical point drying to 41% (after 24 h); and air drying from a volatile solvent to about 18% of the fixed tissue volume. Air drying from water caused a shrinkage to about 12% of the original volume. There was no significant difference between the various commonly used CPD schedules or between GA only and GA + Os O₄ fixed tissue. CPD via cellosolve and CO₂ caused substantially more shrinkage than other methods. Dimensional changes during specimen preparation are probably associated with changes in shape and in relative relationships between organelles, cells and tissues having different compositions. This should be borne in mind by all those interpreting scanning electron micrographs of dried animal soft tissue specimens.     

Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy

Structure of bacterial biofilms may be investigated using several variants of scanning electron microscopy (SEM). We apply lyophilization to prepare nitrifying bacterial biofilm for conventional SEM imaging in high-vacuum mode (CSEM). We therefore replace standard biofilm fixation in glutaraldehyde cross-linking, ethanol dehydration, and critical-point drying (CPD) with less-invasive low-temperature drying by sublimation in vacuum. We compare this approach with: (1) standard preparation with glutaraldehyde fixation, ethanol dehydration, and CPD before CSEM, (2) cryo-sputter preparation of rapidly frozen biofilm in hydrated state (cryo-SEM), and (3) in situ observation without any sample pretreatment in environmental SEM. Combined imaging with these modalities revealed two distinct immobilization patterns on the polyurethane foam: (1) large irregular aggregates (flocs) of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) biofilm threads adherent to the surface of polyurethane foam. Our results indicate that lyophilization was suitable for preservation of bacterial cells and many forms of structure of extracellular matrix. The lyophilized material could be imaged with high resolution (using CSEM) to generate structural information complementary to that obtained with other SEM techniques.     

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells

Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.     

Use of peldri II as a sublimation dehydrant in place of critical-point drying in fracture-label cytochemistry and in backscattered electron imaging fracture-label

Peldri II, a fluorocarbon compound solid at room temperature, was used as a sublimation dehydrant in place of critical-point drying (CPD) in freeze-fractured cytochemistry. Its applicability to the fracture-label replica method was demonstrated using freeze-fractured rat pancreas labeled with Helix pomatia lectin-gold complex and phospholipase A₂-gold complex for the detection of glycoconjugates and phospholipids, respectively. No difference in morphology and labeling pattern was detected in CPD-treated and Peldri II-treated tissue samples. The use of Peldri II was further extended to freeze-fractured rat duodenum labeled with wheat germ agglutininovomucoid-gold complex. Again comparable results were obtained when duodenal samples were prepared according either to the conventional CPD method or to the Peldri II sublimation method. Freeze-fractured replicas of hamster ovaries labeled with Ricinus communis I-gold complex revealed that Peldri II could also be used as a sublimation dehydrant for preparing tissue samples for examination in the scanning electron microscope by the backscattered electron imaging mode.     

Preservation of EDTA-expanded grid-mounted chromosomes and nuclei for electron microscopy using a specially designed freeze-dryer

We describe methods for freezing and drying EDTA-expanded, fixed metaphase chromosomes and nuclei, attached to grids as whole-mounts, for transmission electron microscopy. These methods use a special apparatus that is simple to construct. While separate freezers and dryers are commercially available, one for freezing blocks of tissue by slamming them against a cold metal surface, and the other for vacuum drying the frozen tissue, our apparatus is designed for gentler, cryogenic liquid plunge freezing and drying, sequentially, in the same apparatus, thus avoiding any compression or damage to the sepcimen. Use of a cryoprotectant is not essential; however, good results are obtained more often when 20% ethanol is used. Freezing is accomplished by rapid propulsion of the grid, with specimens attached, into slushy N₂ (-210°C) within the drying chamber; drying is automatic, by either sublimation under vacuum or by solvent substitution using absolute ethanol followed by acetone, which, in turn, is removed with a critical-point dryer. The apparatus offers a means of drying chromosomes and nuclei in an expanded state, and avoids the shrinkage of these structures that occurs during stepwise passage through increasing concentrations of ethanol or acetone.     

A practical device for processing Polaroid 55 P-N film used with scanning electron microscopes

Nematodes and mildew-infected barley leaves when examined in the scanning electron microscope after critical point drying (CPD) from sulphur dioxide (critical temperature 157–7°C) showed no obvious physical damage, but the specimens had a surface deposit which was probably heat damaged natural waxes. The nematode Caenorhabditis elegans and clover roots (Trifolium subterraneum) showed no physical or heat damage after CPD from monochlorodifluoromethane (Freon 22, critical temperature 96°C). The hyphæ and conidia of unfixed mildew on barley were damaged after CPD from Freon 22, probably due to the Freon extracting lipids from the cell walls. Freon 22 was preferred for most specimens as it is cheap, easy to get and not very toxic.     

Phase separation in alcohol/liquid carbon dioxide solvent systems facilitates critical point drying

Carbon dioxide and methanol or ethanol, although miscible, form alcohol/CO₂ solutions that do not easily mix with additional pure liquid CO₂. If the CO₂ inlet is situated at the top of a critical point drying apparatus chamber, pure CO₂ will entirely displace the alcohol/CO₂ phase (which is more dense) while keeping the chamber filled with liquid. This unexpected phenomenon is invaluable in critical point drying delicate biological tissues which remain continuously immersed, avoiding surface or convection currents. By providing an objective criterion for intermediate solvent displacement, the protocol also eliminates ambiguous ‘flushing’ steps.     

Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.     

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.     

Tribological Behavior and Tribochemistry of Self-mated Ti3SiC2 in Ethanol

The tribological behavior of self-mated Ti₃SiC₂ in low viscosity fluids, that is, water and ethanol, is investigated. The results show that both water and ethanol effectively reduce the friction and wear of self-mated Ti₃SiC₂. The friction coefficient and total wear rates of Ti₃SiC₂ tribo-pair are 0.82 and 2.17 × 10^?2 mm³/N m under dry condition, 0.41 and 6.50 × 10^?4 mm³/N m in water and 0.16 and 2.22 × 10^?4 mm³/N m in ethanol. As sliding distance is prolonged from 0.5 to 22.9 km, the total wear rates of Ti₃SiC₂ in ethanol can be further reduced. Mechanical wear of Ti₃SiC₂ is partly inhibited in water and greatly inhibited in ethanol. Surface polishing of Ti₃SiC₂ pin and tribochemistry are the two distinct characteristics of self-mated Ti₃SiC₂ in ethanol. The surface film in ethanol is composed of TiO₂ and SiO. Tribo-products in ethanol, that is, silica gel and titanium dioxide, are found. Polycondensation of ethanol by the catalysis of TiO₂ and SiO_2?x gives birth to paraffins and olefins.     

Measurements of critical point shrinkage of glutaraldehyde fixed mouse liver

The dimensional changes of small cubes of glutaraldehyde fixed mouse liver tissue were measured using a light microscope image projected into the Quantimet 720 Image Analysing computer system. The dimensional changes occurring in the critical drying bomb could be followed at all stages when violent turbulence was not occurring. The results show that liver tissue blocks shrink in four stages whilst in the critical point drying bomb: (1) during substitution of the intermediate solvent with the transitional fluid; (2) when the transitional fluid is warmed above the critical temperature; (3) when the transitional fluid, now a gas, is allowed to escape from the CPD bomb – the rate of shrinkage increasing as atmospheric pressure is approached; (4) at atmospheric pressure when all the gas has been allowed to escape from the bomb. Taken together with the authors' previous findings, it would seem that substantial shrinkage of animal soft tissue specimens must occur whilst they are undergoing “critical point drying”. This fact should be taken into account when interpreting SEM images of CPD tissues.     

Cryofixation and ultra-low-temperature freeze-drying as a preparative technique for TEM

We have developed cryofixation and ultra-low-temperature molecular distillation drying as a method for preparing biological samples for electron microscopic analysis. To validate this approach, we have investigated the relationship between the drying characteristics and ice phases present within frozen samples. Two sample types were investigated. In the first, pure deuterium oxide (D₂O), or heavy water, was vapour condensed under vacuum conditions onto a gold-coated copper sample holder held at ?175 or ?110°C. Additionally, D₂O was slow-rate cooled from room temperature under an ultra-pure dry nitrogen gas atmosphere. The second sample type was rat liver biopsies from animals after 5 days of feeding with D₂O loaded water and ultra-rapid cooling by metal-mirror cryofixation. Ice forms present in the latter samples, determined by electron diffraction of frozen-hydrated cryosections, were amorphous, cubic, and hexagonal. Drying of samples was achieved using a molecular distillation configuration with continuous, microprocessor-controlled sample heating. The vacuum contents of the drying column were monitored by residual gas analysis (RGA) throughout the drying cycle. D₂O vapour in the vacuum chamber, as analysed by RGA, was found to increase in a phasic manner across a broad temperature range. These phases had characteristic onset temperatures and could be removed sequentially. For condensed D₂O samples, these onset temperatures were — 160, — 148, — 125 and — 90°C. Rat liver samples also demonstrated phasic drying patterns which were more complex than those detected with pure D₂O samples. Ultrastructural analysis of samples cryofixed and dried in this manner demonstrated a morphology consistent with the ice phases demonstrated in the frozen-hydrated cryosections. This, together with the RGA results, suggests the absence of devitrification or ice crystal growth during the drying procedure.     

The preparation and tribology of spherical nanometric particles of diantimony trioxide SbOx(OH)y

Spherical diantimony trioxide SbO_x(OH)_y with a particle size of 30–70 nm has been prepared using a technique known as ‘ethanol supercritical fluid drying’. The tribological properties of an SN500 oil containing these nanometric particles were measured using a four-ball and block-on-ring tribotester. The wear resistance and load-carrying capacity of the oil were higher, and its friction coefficient lower, than that of the base oil. An excessive nanometric particles content was found to be disadvantageous to the load-carrying capacity of the oil. The spherical nanometric diantimony trioxide appears to work by deposition on the rubbing surfaces and through the rolling action of the spherical particles.     

The fixation,dehydration, drying and coating of cultured cells for SEM

Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues. Studies on cultivated cells indicate that the glutaraldehyde component of a glutaraldehyde-based fixative does not contribute to the effective osmotic pressure of the fixative and thus the osmolarity of the buffer, and other components, must be equalized to that of the medium in which the cells grow. Even small deviations from this ideal effective osmotic pressure will result in osmotically induced artefacts. Disturbances of pH and temperature of the cultures prior to and during fixation will result in changes in the appearance of many cellular structures such as microspikes and ruffles. We find that osmium fixation is advisable in most instances for best possible membrane preservation and that even long periods of glutaraldehyde fixation do not compensate for osmium fixation. Dehydration always results in shrinkage. Freeze drying (FD) and critical point drying (CPD) also give rise to shrinkage, the former to a lesser degree than the latter. A gold-palladium alloy gives a less granular coating that does gold alone. When cultured cells are studied, a metal thickness of between 5 and 15 nm is usually sufficient to give rise to an adequate secondary electron production and to avoid charging even at accelerating voltages of 30–40 kV. Without treatment with OsO₄ a thicker metal coating is required.     

Effects of fullerene C60 on the friction and wear characteristics of ceramics in ethanol

For the combinations of a pin of Si₃N₄ and five kinds of disk, the friction and wear test was carried out in ethanol, and in ethanol containing C₆₀ particles (1 wt%). A topographical analysis was also performed on the micro-asperities of the wear surfaces to estimate the behavior of C₆₀ particles, and the degree of surface damage. As a results, the following facts were found. (1) The addition of C₆₀ particles in ethanol decreased the wear rates of such ceramic disks as Al₂O₃, SiC and TiC and of the mating Si₃N₄ pins. (2) The addition of C₆₀ particles decreased the mean coefficient of friction for SiC, Si₃N₄, and TiC disks. (3) The wear rates of pin and disk depended on the topographies of worn surfaces such as the mean depth of micro-grooves, the mean tip radius of micro-asperities and so on.     

Indoor indirect solar dryer for ceramic craft industry

We report the development of a prototype indirect solar dryer for plaster molds for the ceramic craft industry. The prototype was designed considering the reduction of the drying time against the traditional technique, working indoors and using minimal conventional energy. The indoor drying chamber has a capacity for 32 plaster molds with a total weight of 425 kg. The drying rate through day and night, drying efficiency and drying constant were of 0.54 kg-H₂O/h, 0.29±0.02% and 0.0094 h^?1, respectively. The solar energy supplied to heat the air of drying was 58% and the remaining 42% was provided by the ambient air. To meet the requirements of the moisture content of potters 5.5 days (132 hours) was needed, resulting in a reduction of two-thirds of the traditional drying time of plaster molds (21 days). The continuous rate of drying reveals the solar dryer ability to dry reasonably rapidly under indoor conditions.     

Microwave drying and moisture diffusivity of white mulberry: experimental and mathematical modeling

The literature surveyed revealed that drying kinetics of white mulberry under microwave treatment has not been investigated. In present study, both experimental study and mathematical modeling on microwave drying of white mulberry was performed. The microwave drying process which reduced the moisture content of mulberry from 3.76 to 0.25 (g water/g dry matter) was carried out at 90, 180, 360, 600, and 800 W in a modified microwave drying set-up. The effects of microwave drying technique on the moisture ratio and drying rate of white mulberry were investigated experimentally. Both the effects of microwave power level (under the range of 90–800W) and initial sample weight (50–150g) were studied. No constant rate period was observed. Mathematical modeling of thin layer drying kinetics of white mulberry under microwave treatment was also investigated by fitting the experimental drying data to eight thin layer drying models. Among the models proposed, Midilli et al. model precisely represented the microwave drying behavior of white mulberry with the coefficient of determination higher than 0.999 and mean square of deviation (χ²) and root mean square error (RMSE) lower than 1.1×10⁻⁴ and 8.9×10⁻³, respectively for all the microwave drying conditions studied. The effective moisture diffusivity (D_eff) of white mulberry varied from 0.45×10⁻⁸ to 3.25×10⁻⁸ m²s⁻¹. Both the drying constant (k) and D_eff increased with the increase of microwave power level.     

Preparation and tribology of aerogel silica and alumina-silica as lubricant oil additives

Aerogel silica and an aerogel mixture of aluminium and silica were prepared using the ethanol supercritical fluid drying technique. Their physical properties were characterised using XRD, transmission electron microscopy, (TEM), and N₂ adsorption. The tribological properties of a 500 SN oil containing the aerogel and a dispersing agent were evaluated using a four-ball machine and a block-on-ring tribotester. Results indicated a greater wear resistance and load-carrying capacity and a lower friction coefficient than for the basic oil. In the oil, the aerogel worked by its deposition on the rubbing surface. Copper stearate was superior to dodecyl alcohol as a dispersing additive, and ultrasonic treatment helped promote the dispersion of the aerogel in oil. There existed an optimum ratio of aluminium isopropyl alcoholate to tetraethanol silicate. The corresponding aerogel mixture gave a maximum value of P_E.     

A gas sensor made from a graphite paper with a nanotube film

A technique for obtaining gas sensor samples from graphite paper with a nanotube film is described. Current-induced annealing of the graphite paper and additional evaporation of a nanotube graphite film in a hydrogen atmosphere are combined in the developed technique. The current?voltage characteristics of the samples have been measured at room temperature in air, in vacuum, and at low concentrations of NH₃, ethanol, and acetone. Experiments demonstrate that these samples containing carbon multiwall nanotubes can be used as a gas sensor to detect the presence of NH₃ and acetone. They are characterized by high sensitivity and selectivity, fast response, restoration, and stability of the characteristics. The estimated sensor sensitivities to NH₃, acetone, and ethanol at a current of 96.8 mA are ~15, ~12, and ~1 mV/Torr, respectively. Their sensitivity is determined by the difference in the behavior of their current?voltage characteristics under exposure to NH₃, ethanol, and acetone. The sensor features fast response (5–20 s) and restoration (within 5 min, restoration to the initial state before the exposure to NH₃ is 100.2%), as well as the stability of its characteristics (the pressure ranges from 1 × 10^–6 to 760 Torr).