On estimating freezing times during tissue rapid freezing

For the study of morphological changes that are associated with fast physiological processes, it is important to know the times at which the surface regions of specimens are frozen during rapid freezing. A simple physical model has been used to estimate the freezing times and the cooling rates at 10 μm depths in specimens. The calculations indicate that cooling rates in excess of 4 × 10⁴ K s^?1 are associated with freezing times of less than 0.5 ms. Using the same model, experimental measurements of freezing times at much larger depths have been extrapolated to a depth of 10 μm, the times obtained are 0.1-0.6 ms for freezing by rapid immersion in cryogenic liquids, and 0.1 ms or less for freezing on a metal block. It is concluded that the delay time between contact with a cryogenic source and specimen freezing is less than 0.5 ms. The uncertainty in the time of freezing may be larger than this, because of an uncertainty of about ± 0.5 ms in determining the exact time of contact and, for freeze fracture studies, because of an uncertainty of up to 0.5 ms due to imprecision in the depth of fracture. At the same time it is estimated that the time during which freezing takes place may be as high as 250 μs, which can be taken as an upper limit for the resolution time for rapid freezing.     

A simple pneumatic device for plunge-freezing cells grown on electron microscopy grids

A detailed design for a simple and inexpensive variable-speed (1.0–5.8 m s^?1) pneumatic plunge-freezing device is presented. Cultured cells, grown on Formvar-coated 75-mesh gold finder grids, are pneumatically driven into a stirring mixture of propane/isopentane (3:1) cooled by liquid nitrogen (LN₂). Premature freezing of the sample in the cryogenic vapors above the cryogen is prevented by plunging through an entry tube into an insulating box, to which a partial vacuum is applied. The cryogenic vapors are drafted into the box at the level of the liquid cryogen by the vacuum, thereby preventing a layer of cold gas from collecting above the cryogen. To prevent the sample from thawing during transfer from the cryogen to the substitution medium, the box top is removed and compressed air is forced through a corrugated tube running the length of the box. The resulting boiling LN₂ creates an atmosphere below ?120°C in which the transfer can be accomplished.     

Friction and wear of sintered cast iron products

The wear and frictional characteristics of five types of sintered cast iron swarf powder, or the same powder decarbonized by a mechanical procedure, were investigated using a pin-and-disk-type apparatus. The contact pressure was 19.6 N cm^?2 and the sliding velocity varied from 0.1 to 4.0 m s^?1. The wear rate exhibited a maximum at a velocity of 1.0 m s^?1. A slight improvement was found in specimens which were decarbonized and forged before sintering. However, the wear rate at lower velocities of these specimens was inferior to that of a specimen containing 5% graphite. The wear mechanism was investigated by scanning electron microscopy and electron probe X-ray microanalysis, and it was found that oxidation wear had an important effect.     

The tribological behavior of Ni–Cu–Ag-based PVD coatings for hybrid bearings under different lubrication conditions

In a cryogenic environment, components like bearings with interacting surfaces in relative motion (tribosystems) often generate undesired heat and experience high wear. Because the properties of conventional bearing materials like stainless steel cannot be applied to this temperature range, the PVD coating based on metal–metal pairs with better frictional properties must be employed. To test the suitability of the Ni–Cu–Ag-based PVD coatings of hybrid bearings for liquid rocket engine turbopumps and to obtain reliable coating material data in the extreme environment, the tribological behaviors of coatings under the cryogenic fluid (liquid oxygen and liquid nitrogen) and water lubricated conditions are studied, respectively. In the paper, the specimens are in a vibrocryotribometer with the ball-on-plane contact type, and various running conditions in terms of lubricants, contacting loading, and contacting velocity are examined. The simulated experiment for testing the actual tribological performance of Ni–Cu–Ag-based PVD coatings for hybrid bearings is tested. The results of the tests indicate that the coatings can be suitable for cryogenic tribosystems of turbopumps. In the cryogenic environment, the volume wear rate of coatings increases rapidly with the contacting loading, but 15 min later, the volume wear volume of coatings turns into 2.5–15×10⁻⁴ mm³. Besides, under the liquid oxygen condition in simulating the liquid rocket engine turbopumps environment, the friction coefficients are 0.03–0.1.     

Friction of Hexagonal Boron Nitride in Various Environments

The mechanisms of friction of hot-pressed h-BN sliding against itself in an AES/XPS analytical tribometer have been studied under high vacuum of 10^?8 Pa; under a low partial pressure of 10^?3 Pa of different gases, i.e., air, CO, C₃H₈ and H₂O; under a higher partial pressure of 10 Pa of N₂, O₂, air and C₃H₈; and in ambient air, 50 percent humidity, at an atmospheric pressure of 10⁵ Pa, respectively. Two crystallographic orientation combinations of basal plane alignments were tribotested, namely the flat/flat configuration and the edge/edge configuration, where the specimens were directionally fabricated from the anisotropic molds. The major finding is that h-BN basal plane slip appears to be the only deformation mode which is associated with low friction in the current test conditions. Generally, fracture, shear and amorphization of the BN crystallites give higher friction. The mechanism of friction-induced basal plane slip in h-BN is thought to be related mainly to the presence of condensed liquid water in crystal defects, leading to a decrease of the surface tension and, therefore, of the critical resolved shear strength. The mechanism of the friction-altering action of hydrocarbon adsorbates is different: a lubricating phase of carbon seems to be formed by a tribochemical reaction. The results suggest that hybrid combinations of h-BN and carbon interacting synergistically could act as a better tribomaterial in a wide-environmental regime.     

Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans , could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.     

Ultrastructure of Tracheal Surface Liquid: Low-Temperature Scanning Electron Microscopy

A layer of liquid lines the airways in the lung. Previous microscopic studies have suggested that it is in two phases, with a mucous gel lying above a periciliary sol. However, shrinkage artifacts due to chemical fixation, dehydration, and drying have prevented reliable estimates of the depth of these layers. To avoid such problems, we have studied the surface liquid of bovine trachea by low-temperature scanning electron microscopy (LTSEM). A polished copper probe cooled to liquid nitrogen temperature was applied to the mucosal surface of sheets of excised tracheal epithelium to effect rapid freezing of surface liquid. Tissue sheets were then mounted in an LTSEM (AMRay 1000A with Biochamber) which maintains samples at -180°C with a Joule-Thompson refrigerator built into the stage. Tissues were fractured at right angles to the epithelial surface, coated with gold, and viewed, all at 10^?5 to 10^?6 torr without transfer through air. The sample was stable under the electron beam at accelerating voltages up to 20 kV. Epithelial features (nuclei, cilia, microvilli, mucous granules) were well preserved. The mucosal surface of the cells was covered with material on the order of 8 μm in depth. The mucous gel and periciliary sol could be seen as distinct layers and could be distinguished by the size and pattern of ice crystal voids generated by radiant-etching of the fractured surface of the sample.     

Electron microscopy of frozen water and aqueous solutions

Thin layers of pure water or aqueous solutions are frozen in the vitreous state or with the water phase in the form of hexagonal or cubic crystals, either by using a spray-freezing method or by spreading the liquid on alkylamine treated films. The specimens are observed in a conventional and in a scanning transmission electron microscope at temperatures down to 25 K. In general, the formation of crystals and segregation of solutes during freezing, devitrification and evaporation upon warming, take place as foreseen by previous X-ray, thermal, optical and electron microscopical studies. Electron beam damage appears in three forms. The devitrification of vitreous ice. The slow loss of material for the specimen at a rate of about one molecule of pure water for every sixty electrons. The bubbling in solutions of organic material for doses in the range of thousands of e nm^?2. We propose a possible model for the mechanism of beam damage in aqueous solutions. The structural and thermal properties of pure frozen water important for electron microscopy are summarized in an appendix.     

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.     

Predicting BOG rate in cryogenic containers at different liquid levels based on a single test

As the boil-off gas (BOG) rate is tested to evaluate the thermal insulation performance of cryogenic containers at a 90 % liquid level in standard. This study aims to predict the BOG rate of cryogenic containers at different liquid levels based on a single test. If the BOGs at a 90 % liquid level can be evaluated based on the BOG test at a low liquid level, the large amount of working medium can be saved to reduce the test cost and the equilibration time can be shortened to improve the test efficiency. In this study, the QHALW (heat absorbed by the liquid from its corresponding outer wall) and QHTVL (heat transferred from the corresponding wall of the vapor to the liquid) are analysed to establish the energy equation of the liquid in the BOG test, indicating latent heat of BOG in cryogenic containers includes the QHALW and QHALW. Therefore, the equation between the latent heat of BOG and the two parts of heat isbuilt to predict BOGs at different liquid levels with the tested BOG. The maximum error of the experimental results is not more than 7.0 %, which shows that the BOG is successfully predicted at different liquid levels with a single-tested BOG.

     

Heat checking in the contact zone of a bearing seal (a two-dimensional model of a single moving asperity)

When two bodies are in sliding contact under heavy loads, local high temperature may occur as a result of excessive frictional heating near the contacting surfaces. Because of a combination of thermal heating and the mechanical load, the material may crack in the neighborhood of the contact zone. This phenomenon is called heat checking. It commonly occurs in mechanical seals and brakes. In recent years there has been increased emphasis on finding a solution. In this paper a simple model is proposed to determine those parameters which can be optimized to control the heat checking of the materials.Because of the size difference between the contact area and the seal, the mathematical model is represented by a half-space subjected to a fast-moving load which is distributed over a small area. The load comes from a combination of an arbitrarily distributed heat source and mechanical loads of pressure and friction. The general solutions are expressed in the form of integral equations with determined Green's functions. Numerical results for fracture criteria are then obtained using a computer program.For the current problem, a nominal pressure of 365 MPa (53000 Ibf in^?2) and a corresponding friction of 183 MPa (26500 Ibf in^?2) are used. The induced heat source is 1.5 × 10⁷ in Ibf in^?2 s^?1 (1.70 × 10⁶Jm^?2s^?1). Such a load results in fracture where the crack is first initiated immediately beneath the surface at the trailing edge of the moving load.     

The influence of specimen geometry and sliding mode on the friction and wear of iron-chromium alloys in controlled environments

The friction and wear behaviour of iron-chromium alloys containing 5–20% Cr has been studied in unidirectional and reciprocating motions under conditions where frictional heating should be minimal. These experiments were performed in an ultrahigh vacuum chamber in which the oxygen partial pressure could be maintained from 10^?6 Pa to atmospheric pressure. At 10^?6 Pa the friction behaviour and the pattern of wear were essentially similar for both types of motion, although slightly lower wear was observed under reciprocating motion. At higher oxygen partial pressures the containment of debris within the track led to the formation of compacted oxide “islands”. Hemispherical upper specimens seemed more effective than the conical ones in trapping this debris.     

Effect of atmospheric pressure on friction and wear of 0.45% C steel in fretting

Oxidative wear is significant in fretting wear when sufficient oxygen is supplied. In vacuum, however, oxide does not form readily. In this paper friction and wear behaviours were studied at various atmospheric pressures in order to clarify the effect of ambient pressure on them.Experiments were conducted with 0.45% C steel at ambient pressures from 1.0 × 10⁵ to 1.3 × 10^?3 Pa. The load was 14 N, the peak-to-peak slip amplitudes were 35 and 110 μm and the frequency was usually 8.3 Hz.Friction behaviours are characterized into three types according to the ambient pressure: 1.0 × 10⁵ ? 10 Pa, 10 ? 10^?1 Pa and below 10^?1 Pa. The coefficient of friction increases with a decrease in ambient pressure below 1 Pa. The critical pressure in fretting is found to be 10 Pa, above which the oxidation rate is independent of the ambient pressure and α-Fe₂O₃ is formed. Wear decreases with ambient pressure below the critical pressure where Fe₃O₄ is formed. Adhesive transfer of metallic debris occurs below 10^?1 Pa.The relationship between the coefficient of friction and oxide thickness is obtained analytically, and the effect of frequency on the oxidation rate is considered.     

Flow instability due to cryogenic cavitation in the downstream of orifice

Flow instability in LRE (liquid rocket engine) occurs due to various reasons such as flow interactions with valve, orifice and venturi, etc. The inception of cavitation, especially in the propellant feeding system, is the primary cause of mass and pressure oscillations because of the cyclic formation and depletion of cavitation. Meanwhile, the main propellant in a liquid rocket engine is the cryogenic fluid, which properties are very sensitive to temperature variation. And the change of propellant properties to temperature variation by thermodynamic effect needs to be properly taken into account in the flow analysis in order to understand basic mechanisms for cryogenic cavitation. The present study focuses on the formation of cryogenic cavitation by using the IDM model suggested by Shyy and coworkers. The flow instability was also numerically investigated in the downstream of orifice with a developed numerical code. Calculation results show that cryogenic cavitation can be a primary source of flow instability, leading to mass fluctuations accompanied by pressure oscillations. The prediction of cavitation in cryogenic fluid is of vital importance in designing a feeding system of an LRE. This paper was recommended for publication in revised form by Associate Editor Jun Sang Park Changjin Lee received his B.S. and M.S. degrees in Aeronautical Engineering from Seoul National University in 1983 and 1985. He then went on to receive his Ph.D. degree from University of Illinois at Urbana- Champaign in 1992. Dr. Lee is currently a Professor at the department of Aerospace Engineering at Konkuk University in SEOUL, Korea. His research interests are in the area of combustion instabilities of hybrid, liquid rocket and jet propulsions. Tae-Seong Roh received his B.S. and M.S. degrees in Aeronautical Engineering from Seoul National University in 1984 and 1986. He then went on to receive his Ph.D. degree from Pennsylvania State University in 1995. Dr. Roh is currently a Professor at the department of Aerospace Engineering at Inha University in Incheon, Korea. His research interests are in the area of combustion instabilities, rocket and jet propulsions, interior ballistics, and gas turbine engine defect diagnostics.     

Spectroscopy of single InAs quantum dots

Ensembles of InAs quantum dots with a very low density (～10⁶ cm^?2) are grown by molecular beam epitaxy, which allows the spectral characteristics of emission of single quantum dots to be studied by the method of cryogenic microphotoluminescence. With increasing quantum dot size, the splitting of exciton states is demonstrated to increase steadily to ～10² µeV. In the exciton energy range of 1.3–1.4 eV, the magnitude of this splitting is comparable with the natural width of the exciton lines. This result is important for the development of emitters of entangled photon pairs based on InAs quantum dots.     

A thin-film resistive sensor for measuring atomic hydrogen flux density

An eight-channel thin-film resistive atomic hydrogen (AH) sensor is described. It is intended to measure the AH flux density in an atomic-molecular mixture at a reduced gas pressure (10^?2–10^?4 Pa), particularly under the action of infrared and visible radiation noise, in a computer-aided mode. The sensor can be used for measuring a distribution of the AH flux density of the large cross-section beam. The range of AH flux density measurements is 5 × 10¹³ ? 10¹⁶ atoms/(cm² s), the measurement time is 1–10 min, and the measurement error is 10%. The sensitive element of the sensor is made using planar technology, which offers a chance to attain a high resolution in spatial distribution mesurements.     

A sectioned spectrometer of fast neutrons

Development of a capture gated spectrometer on the basis of a liquid organic scintillator doped with enriched ⁶Li is discussed. Particular interest is evoked by the good pulse height resolution of the spectrometer for 14-MeV neutrons, which is expected to be very high, ～10–15%. This resolution is attained by compensating for the nonlinearity of the light yield in the scintillator owing to the use of separate optically isolated sections, which independently detect scintillations from each recoil proton. The detector is sensitive to fluence rates ranging from 10^?4 to 10² cm^?2 s^?1 above a threshold of 500 keV under conditions of uncorrelated γ-ray background at a level of up to 10² s^?1 (E > 100 keV). A pilot model of the detector based on a scintillator without a lithium dopant has been produced and tested. The detector efficiency is governed by the scintillator volume (～1.2 l); for 3-MeV neutrons, its value is 0.2–0.5%. The response of the pilot detector to neutrons from a Pu-α-Be source with energies of up to 10 MeV has been measured. Initial testing indicates a low threshold at an ～600-keV energy of a recoil proton. A good spectral response is obtained using the criterion that three optical sections of the detector operate at a time. This spectrometer can find application in low-background experiments in basic physics research, as well as in space research and nuclear medicine for measuring the parameters of the neutron flux.     

A field-emission source of charges based on nanotubes for low-temperature experiments

Methods for the production of field-emission sources of charges on the basis of carbon nanotubes are described. These sources can be used to study the properties of injected charges in cryogenic liquids and crystals (the area of the source surface is about several square millimeters, the dissipated power is <10^?6 W). The first and second batches of sources were produced by means of the deposition of nanotubes from an arc discharge on a flat copper substrate and via mechanical rubbing of nanotubes into a porous metal, respectively. Tests of sources from the first batch in a diode with a gap of 0.5 mm showed that in superfluid He-II, the current of negative charges at a level of 10^?12 A arises when the voltage at the cathode is U = ?140 V and increases to 10^?9 A, when U rises to 170 V. When the voltage polarity changes, the current of positive charges arises in the diode for the voltage U ≥ 240 V. A source from the first batch was used to observe the movement of negative and positive charges in solid-helium samples at T < 75 mK. For second-batch sources, the current of negative charges at a level of 10^?12 A in superfluid He-II arises at U = ?260 V.     

Low Friction Carbide-Derived Carbon Coating on SiC in Vacuum Achieved by Microstructure Formulation and Solid–Liquid Lubrication

Carbide-derived carbon (CDC) coatings with dimple (CDC@GSiC coating) and loosely dispersive particles structures (CDC@RBSiC coating) were prepared on two kinds of SiC substrates by using chlorination at 1,000 °C in a 5 vol.% Cl₂–Ar gas. Microstructural effect makes the two CDC coatings exhibit different frictional behavior in ambient pressure and in vacuum. For the CDC@RBSiC coating, the friction coefficient was from 0.08 to 0.12 at ambient pressure and is sensitive to evacuation from ambient pressure to 10³ Pa while it was as high as 0.42 up to a pressure of 10^?4 Pa. Progressive evacuation does not vary the friction coefficient of the CDC@GSiC coating up to 10^?3 Pa. The wear of the CDC@GSiC coating was low with a maximum depth of 8 μm and much lower than that of the CDC@RBSiC coating (70 μm). The dimples on the surface and pores in the CDC@GSiC coating are reservoirs for ion liquid (IL), and the IL impregnated CDC@GSiC coating shows very low friction and wear at ambient pressure and in vacuum.     

An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation

The metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N₂. The microwave irradiation, together with two technical improvements (a light-mass plunger and a recently developed β-gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 μm, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.