New instruments which facilitate rapid freezing at 83 K and 6 K

We have developed three instruments to freeze different biological specimens for various purposes in the easiest and most economical way: a punching device, a rapid immersion device and a device to freeze the specimens against a copper block cooled by liquid helium. Cooling rate measurements are made by a thin flat thermocouple. Freezing against a copper block cooled down to 4 K gives cooling rates 3–5 times higher than freezing by immersion into propane at 85 K.     

Countercurrent plunge cooling: A new approach to increase reproducibility in the quick freezing of biological tissue

Conditions fulfilling all the accepted criteria for optimum cryoquenching are realized for the first time in a plunge freezing device. Samples are plunged down through a smoothly rising cryogen stream that is maintained within ± 0.25 K of a preselected temperature. Dry nitrogen gas at ambient temperature removes evaporated cryogen molecules from the plunging path immediately above the liquid coolant to prevent pre-cooling of the sample. Symmetrical instrument design and conditions of flow ensure symmetrical freezing under optimum conditions in a totally repeatable fashion. The device has been used with halocarbons, propane and ethane. It provides consistent cooling rates comparable with the best so far obtained by any other plunging method.     

Validation of convection-limited cooling of samples for freeze-fracture electron microscopy

Rapid freezing is the most important step in sample preparation for freeze-fracture and other cryotechniques for electron microscopy. A simple heat transfer model is experimentally validated to show that convection from the cryogen to the specimen is the limiting step in rapid freezing of small samples [Biot modulus, (hd/k) < 1] by measuring cooling rates in a variety of samples, materials, and cryogens. In comparison to the commonly accepted conduction-limited model, the convection-limited model predicts, and our experiments show, that cooling rates are proportional to the surface area to volume ratio, independent of the sample thermal conductivity, and inversely proportional to the product of sample density and heat capacity. We show that almost any material can be frozen at similar rates if the sample thickness, the cryogen, and the method and velocity of contact with cryogen are similar. Liquid ethane or propane cooled to liquid nitrogen temperature are shown to give the best results.     

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.     

High-pressure freezing causes structural alterations in phospholipid model membranes

The influence of high-pressure freezing (HPF) on the lipid arrangement in phospholipid model membranes has been investigated. Liposomes consisting of pure dipalmitoylphosphatidylcholine (DPPC) and of DPPC mixed with a branched-chain phosphocholine (1,2-di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine) have been analysed by freeze-fracture electron microscopy. The liposomes were frozen either by plunging into liquid propane or by HPF. The characteristic macroripple-phase of the two-component liposome system is drastically changed in its morphology when frozen under high-pressure conditions. The influence of ethanol which acts as pressure transfer medium was ruled out by control experiments. In contrast, no high-pressure alterations of the pure DPPC bilayer membrane have been observed. We assume that the modification of the binary system is due to a pressure-induced relaxation of a stressed and unstable lipid molecule packing configuration. HPF was performed with a newly designed sample holder for using sandwiched copper platelets with the high-pressure freezing machine Balzers HPM010. The sandwich construction turned out to be superior to the original holder system with regard to freeze-fracturing of fluid samples. By inserting a spacer between the supports samples with a thickness of 20–100 μm can be high-pressure frozen. The sandwich holder is provided with a thermocouple to monitor cooling rates and allows exact sample temperature control. Despite a two-fold mass reduction compared to the original holder no HPF cooling rate improvement has been achieved (4000 °C s⁻¹). We conclude that the cooling process in high-pressure freezing is determined mainly by cryogen velocity.     

Cooling rate and ice-crystal measurement in biological specimens plunged into liquid ethane,propane, and Freon 22

Specimens sandwiched between copper planchettes were plunged up to a depth of 430 mm into coolants used for cryofixation. Hydrated gelatin containing a miniature thermocouple was used to mimic the behaviour of tissue during freezing. Gelatin and red blood cells were used for ice-crystal analysis. Ethane produced the fastest cooling rates and the smallest ice-crystal profiles, and Freon 22 produced the slowest cooling rates and the largest crystal profiles. Smaller crystal profiles were often seen in the centre of the specimens than in subsurface zones. The results show that ethane, rather than propane, should be used for freezing metal-sandwiched freeze-fracture specimens by the plunging method, and probably also in the jet-cooling method. They further suggest that good cryofixation could occur at the centre of thin specimens rather than only at their surfaces. Comparison between theoretical and experimental ice-crystal sizes was satisfactory, indicating that where the experimental parameters can be defined then realistic predictions can be made regarding cryofixation results.     

On crystal size and cooling rate

A theoretical model is proposed which is used to derive a quantitative relationship between the critical cooling rate and average crystal size at any location within a biological specimen of given shape subject to rapid freezing. The model is applicable to the slamming, plunging or spraying methods of cryofixation provided the ice crystal size is at least 5 times greater than the size of the critical nucleus. Complete vitrification of pure water or aqueous solutions is shown to take place at cooling rates in excess of about 3 × 10⁶ K/s.     

The relative efficiency of cryogens used for plunge-cooling biological specimens

Coolants used for freezing biological specimens were tested for cooling performance in the continuous plunge mode. Results from bare thermocouples showed that ethane cooled faster than propane or a propane: pentane mixture, even when warmed to 25 K above its freezing point. Propane coolants were more efficient than Freon 22 and the slowest cooling occurred in boiling liquid nitrogen. Hydrated gelatin specimens showed similar results with ethane cooling about 33% faster than propane. Epoxy resin specimens cooled faster than hydrated gelatin specimens of similar size. Hydrated and resin specimens cooled over increasing distances as plunge velocity increased. A bare thermocouple, however, cooled over a constant distance when plunged above a critical velocity. This phenomenon may reflect vapour formation and its suppression at high plunge velocities. The rate of cooling in hydrated specimens is shown to have an absolute limit and cannot be modelled by bare thermocouples or resin specimens.     

Vitrification of articular cartilage by high-pressure freezing

For more than 20 years, high-pressure freezing has been used to cryofix bulk biological specimens and reports are available in which the potential and limits of this method have been evaluated mostly based on morphological criteria. By evaluating the presence or absence of segregation patterns, it was postulated that biological samples of up to 600 μm in thickness could be vitrified by high-pressure freezing. The cooling rates necessary to achieve this result under high-pressure conditions were estimated to be of the order of several hundred degrees kelvin per second. Recent results suggest that the thickness of biological samples which can be vitrified may be much less than previously believed. It was the aim of this study to explore the potential and limits of high-pressure freezing using theoretical and experimental methods. A new high-pressure freezing apparatus (Lei?a EM HPF), which can generate higher cooling rates at the sample surface than previously possible, was used. Using bovine articular cartilage as a model tissue system, we were able to vitrify 150-μm-thick tissue samples. Vitrification was proven by subjecting frozen-hydrated cryosections to electron diffraction analysis and was found to be dependent on the proteoglycan concentration and water content of the cartilage. Only the lower radical zone (with a high proteoglycan concentration and a low water content compared to the other zones) could be fully vitrified. Our theoretical calculations indicated that applied surface cooling rates in excess of 5000 K/s can be propagated into specimen centres only if samples are relatively thin (<200 μm). These calculations, taken together with our zone-dependent attainment of vitrification in 150-μm-thick cartilage samples, suggest that the critical cooling rates necessary to achieve vitrification of biological samples under high-pressure freezing conditions are significantly higher (1000–100 000 K/s) than previously proposed, but are reduced by about a factor of 100 when compared to cooling rates necessary to vitrify biological samples at ambient pressure.     

The direct measurement of temperature changes within freeze-fracture specimens during rapid quenching in liquid coolants

We have evaluated the cooling rates of specimens mounted in a variety of freeze-fracture holders when plunged into a series of liquid coolants. These rates were measured using miniature thermocouples placed within the mounted specimens. The most rapid cooling rates were obtained using propane at 83 K as the coolant. When mounted on a newly devised ‘copper sandwich’ holder, specimen cooling rates in excess of 4500 K/s have been recorded. A simple guillotine-like device for quenching freeze-fracture specimens under reproducible conditions is presented.     

Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21°C)

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethyl-sulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configurations at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0·3 K min^?1 or 2 K min^?1 were related to the distribution of the ice phase within the tissue.     

Numerical analysis for prediction of flow rate of the motor cooling fan

In this study, we analyzed the three dimensional unsteady flow around a motor cooling fan using the vortex panel method. For a popular type of motor cooling fan that has thin blades, we predicted the flow rate through numerical analysis without experimental data, such as the free stream velocity, which is a boundary condition of the flow field. We also calculated the flow rate for various cooling fan geometries and rotating speeds. For these fans, the numerical results showed flow rates within 3% of the experimental results. This paper was presented at the 9^th Asian International Conference on Fluid Machinery (AICFM9), Jeju, Korea, October 16–19, 2007.     

A low-temperature capacitive dilatometer

A dilatometer with a capacitive displacement sensor intended for measuring the thermal expansion of solid samples in a temperature range of 4–300 K is described. The sensor of the instrument was mounted inside of industrial insert VTI SIV with controlled circulation of liquid helium encased in portable cryostat and was successfully used jointly with a simple commercial temperature control system. The dilatometer allows studies of the thermal expansion of samples with a sensitivity of ∼1.4 ? in consecutive cooling and warming cycles with a rate of ∼10⁻³ K/min. The results of measuring the thermal expansion of a CoS₂ sample near the phase-transition point are presented.     

Interfacial reactions in PZT/Pd/PZT sandwich structures

The interfacial reactions of palladium foil and lead zirconate-titanate (PZT) were studied using samples with a sandwich structure in the temperature range 1373–1523 K and under conditions where no lead is lost to the environment. The interfacial reactions were analysed using scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction and wavelength-dispersive X-ray spectroscopy analysis. The density of the PZT powder phase increased with increasing temperature and, when sintered above 1373 K, reached over 95% of the theoretical density of PZT. The weight loss of pellets was less than 0·8% when sintered below 1523 K. The degree of interfacial reactions became more severe with increasing temperature, as indicated by an expanding reacted region. The reaction at the PZT side of the Pd/PZT interface involved the decomposition of PZT into a monoclinic ZrO₂ phase, PbO and a lower x-value Pb(Zr_xTi₁__x)O₃ composition. Three distinguishable microstructures exist on the Pd side when sintered below 1473 K: a thin layer of PbPd₃ phase, a Pd–Pb solid solution zone and an unreacted region. Only the cubic PbPd₃ eutectic structure was found when sintered above 1473 K. The oxidation of palladium occurred during interfacial reactions, expedited by increasing temperature and resulting in the formation of the tetragonal PdO phase and the hexagonal PbPdO₂ phase. A model for the overall reaction is proposed involving decomposition of the PZT, migration of PbO and diffusion of Pb into Pd foil.     

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.     

Characteristics of vertically injected buoyant jets of highly diluted propane

In coflow jets with the nozzle diameter of O (1 cm) and the fuel jet velocity of O (10 cm/s), the buoyancy induced by the density difference between the fuel and air influences the jet structure appreciably. The present study investigated the behavior of such a buoyant jet numerically and experimentally, especially when the fuel stream had higher density than air. When the fuel jet was composed of propane highly diluted with nitrogen, the fuel jet was decelerated and formed a stagnation region. Consequently, the fuel was carried downstream by the coflow having a circular cone shape. When the fuel was moderately diluted or as the jet velocity increased, numerical results showed the Kelvin-Helmholtz type instability along the mixing layer of the jet. When the fuel jet velocity was relatively high, the stagnation height increased nonlinearly with fuel jet velocity having the power of approximately 1.62. In the relatively high Reynolds number regime of Re > 80, the stagnation height can be correlated to Re^0.62Ri^−0.5, indicating the combined effects of buoyancy and jet momentum. As the Reynolds number becomes small, the stagnation height was affected by the streamwise diffusion due to fuel concentration gradient and by the wake behavior near the nozzle tip. Accordingly, the stagnation heights approach to none-zero values, which were found to be relatively insensitive to fuel dilution.     

液滴低温气体雾化射流冷却换热理论分析与实验研究

论述低温气体雾化射流冷却原理及特点,以池内膜态沸腾为基础,将喷雾颗粒的冲击作为一种扰动,建立雾滴射流进入切削区冷却高温壁面的模型,讨论雾滴冷却高温壁面的换热系数,着重探讨工艺参数水流密度对喷雾冷却换热系数的影响.进行了不同水剂量的低温气体雾化射流冷却钛合金高温壁面的瞬态实验,获得了钛合金试件表面温度分别为100℃、150℃、200℃和250℃时,低温气体雾化射流冷却达到最佳冷却效果时水剂量,分析了水剂量对冷却效果的理论依据,结果证实该模型对实际应用具有一定的指导作用.     

A dilution microcryostat-insert

An autonomous dilution microcryostat in which ³He circulates due to its condensation in the volume cooled by sorption evacuation of ³He from a separate bath. A specific feature of this apparatus is the position of the sample holder in its upper part, which facilitates access to it. The holder is connected to the mixer through a heat conductor made from annealed copper wire with a length of ∼0.5 m. The cryostat operates while inserted into a nitrogen-free portable (35 l) helium-filled vessel. The operating cycle of the cryostat includes the procedures of desorption, condensation, and cooling of the ³He-filled bath to 0.35–0.40 K and the mixer to 0.05–0.10 K (lasting ∼1.5–2.0 h) and also a period of maintaining the temperature below 0.1 K (12–14 h). The amount of liquid helium in the portable cryostat is sufficient for operating for 6 days. The lowest reached temperature of the holder is 0.04 K. When the power dissipated in the holder is 0.5 μW, its temperature does not increase above 0.1 K. The instrument is mainly designed for cooling sensitive radiation detectors. Original Russian Text ? V.S. Edelman, 2009, published in Pribory i Tekhnika Eksperimenta, 2009, No. 2, pp. 159–165.     

A setup for measuring the temperature dependence of the refractive indices of solids

A setup for measuring the temperature dependences of refractive indices n(T) of semiconductors and dielectrics in the temperature range 300–700 K at the wavelengths of a He—Ne laser λ=0.63, 1.15, and 3.39 μm is described. Samples in the form of plane—parallel plates serve as Fabry—Perot etalons the optical thickness of which depends on the temperature. Upon heating and subsequent cooling of a sample, interference oscillations of the refiected-light intensity are recorded and used to determine the dependence n(T). The values of the refractive index at room temperature and the thermal expansion coefficient used in calculations are taken from the literature. Comparing the interferograms obtained for a heated and cooled sample allows evaluation of the temperature difference between the sample’s probed area and a measuring thermocouple. The relative error in determining thermooptical coefficient dn/dT in the range 300–700 K is within 1%.     

A new heat transfer model to predict cooling rates for rapid freezing fixation

A simple heat transfer model is presented to show that convection from the cryogen to the specimen is the limiting step in rapid freezing of small samples for electron microscopy when the Biot modulus, which measures the ratio of convective to conductive heat transfer, (hd/k) < 1. In comparison to the commonly accepted conduction-limited model, the convection-limited model predicts that cooling rates are proportional to the surface area/volume ratio, independent of the sample thermal conductivity, and inversely proportional to the product of sample density and heat capacity. Literature values of experimentally measured cooling rates fit the convection limited model. Simple analogies to predict the heat transfer coefficient as a function of cryogen properties, specimen geometry and cryogen velocity are presented.