Supercooling and nucleation of ice in single cells

An emulsion droplet formation procedure was employed to isolate yeast cells and, in separate experiments, human red blood cells, one from another in individual droplets, and to segregate extraneous materials catalyzing the formation of ice. Emulsification succeeded in isolating the cells and permitted the observation of the supercooling of droplets containing cells whereby each droplet was observed to nucleate ice at a temperature that depended only upon the components of the droplet. The droplet formation procedures were characterized. It was shown that the surface coatings and the carrier fluids used in the preparation of the emulsions did not act as ice nucleators. It was, in this manner, possible to study the nucleation of ice brought about by supercooling and homogeneous nucleation in the volume of the droplet or by the catalysis of nucleation on or in the cells contained in the droplets. It was shown that yeast cells and red blood cells could each be supercooled to about ?40 °C in short-term experiments. The results also revealed that yeast cells did not store for infinite times at temperatures above the observed upper limit of homogeneous nucleation. The yeast cells died at rates that were exponential functions of time at ?20, ?22.5, ?25, ?29 and ?33 °C. The temperature dependence of the death rate did not correspond to a process with a normal Arrhenius activation energy. The temperature dependence did, however, suggest a potentiated heterogeneous catalysis of ice resulting in the death of the yeast cells.     

Effect of hydrophobic environments on the hypothesized liquid-liquid critical point of water

The complex behavior of liquid water, along with its anomalies and their crucial role in the existence of life, continue to attract the attention of researchers. The anomalous behavior of water is more pronounced at subfreezing temperatures and numerous theoretical and experimental studies are directed towards developing a coherent thermodynamic and dynamic framework for understanding supercooled water. The existence of a liquid–liquid critical point in the deep supercooled region has been related to the anomalous behavior of water. However, the experimental study of supercooled water at very low temperatures is hampered by the homogeneous nucleation of the crystal. Recently, water confined in nanoscopic structures or in solutions has attracted interest because nucleation can be delayed. These systems have a tremendous relevance also for current biological advances; e.g., supercooled water is often confined in cell membranes and acts as a solvent for biological molecules. In particular, considerable attention has been recently devoted to understanding hydrophobic interactions or the behavior of water in the presence of apolar interfaces due to their fundamental role in self-assembly of micelles, membrane formation and protein folding. This article reviews and compares two very recent computational works aimed at elucidating the changes in the thermodynamic behavior in the supercooled region and the liquid–liquid critical point phenomenon for water in contact with hydrophobic environments. The results are also compared to previous reports for water in hydrophobic environments.     

A ferromagnetic model for the action of electric and magnetic fields in cryopreservation

Recent discussions in the literature have questioned the ability of electromagnetic exposure to inhibit ice crystal formation in supercooled water. Here we note that strong electric fields are able to disrupt the surface boundary layer of inert air on the surface of materials, promoting higher rates of heat transport. We also note that most biological tissues contain ferromagnetic materials, both biologically precipitated magnetite (Fe₃O₄) as well as environmental contaminants that get accidentally incorporated into living systems. Although present at trace levels, the number density of these particulates is high, and they have extraordinarily strong interactions with weak, low-frequency magnetic fields of the sort involved in claims of electromagnetic cryopreservation. Magnetically-induced mechanical oscillation of these particles provides a plausible mechanism for the disruption of ice-crystal nucleation in supercooled water.     

Dynamic light scattering studies of the aggregation of lysozyme under crystallization conditions.

The intensity autocorrelation functions of light scattered by lysozyme solutions under pre-crystallization conditions in NaCl-containing media were recorded at scattering angles from 20 degrees to 90 degrees. The measurements, conducted on freshly prepared protein solutions supersaturated more than 3-fold, indicate the simultaneous presence of two scatterer populations which can be assigned to individual protein molecules and to large particles. When solutions are undersaturated, or slightly supersaturated, light scattering only reveals the presence of the small scatterers. In the supersaturated medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are correlated with the nucleation step during protein crystallization.     

Ice nucleation in nature: supercooling point (SCP) measurements and the role of heterogeneous nucleation

In biological systems, nucleation of ice from a supercooled aqueous solution is a stochastic process and always heterogeneous. The average time any solution may remain supercooled is determined only by the degree of supercooling and heterogeneous nucleation sites it encounters. Here we summarize the many and varied definitions of the so-called "supercooling point," also called the "temperature of crystallization" and the "nucleation temperature," and exhibit the natural, inherent width associated with this quantity. We describe a new method for accurate determination of the supercooling point, which takes into account the inherent statistical fluctuations of the value. We show further that many measurements on a single unchanging sample are required to make a statistically valid measure of the supercooling point. This raises an interesting difference in circumstances where such repeat measurements are inconvenient, or impossible, for example for live organism experiments. We also discuss the effect of solutes on this temperature of nucleation. Existing data appear to show that various solute species decrease the nucleation temperature somewhat more than the equivalent melting point depression. For non-ionic solutes the species appears not to be a significant factor whereas for ions the species does affect the level of decrease of the nucleation temperature.     

Cold hardiness and deep supercooling in xylem of shagbark hickory

Differential thermal analysis, differential scanning calorimetry, pulsed nuclear magnetic resonance spectroscopy, and low temperature microscopy are utilized to investigate low temperature freezing points or exotherms which occur near −40 C in the xylem of cold-acclimated shagbark hickory (Carya ovata L.). Experiments using these methods demonstrate that the low temperature exotherm results from the freezing of cellular water in a manner predicted for supercooled dilute aqueous solutions. Heat release on freezing, nuclear magnetic resonance relaxation times, and freezing and thawing curves for hickory twigs all point to a supercooled fraction in the xylem at subfreezing temperatures. Calorimetric and low temperature microscopic analyses indicate that freezing occurs intracellularly in the xylem ray parenchyma. The supercooled fraction is found to be extremely stable, even at temperatures only slightly above the homogeneous nucleation temperature for water (−38 C). Xylem water is also observed to be resistant to dehydration when exposed to 80% relative humidity at 20 C. D₂O exchange experiments find that only a weak kinetic barrier to water transport exists in the xylem rays of shagbark hickory.     

Factors affecting ice nucleation in plant tissues

Factors affecting the ice nucleation temperature of plants and plant tissues were examined. The mass of a sample had a marked effect on ice nucleation temperature. Small tissue samples supercooled to −10°C and were not accurate predictors of the nucleation temperature of intact plants in either laboratory or field experiments. This effect was not unique to plant tissues and was observed in autoclaved and control soil samples. Ice nucleation temperatures of bean, corn, cotton, and soybean seedlings were influenced by the length of subzero exposure, presence of ice nucleation active bacteria, and leaf surface wetness. The number of factors influencing ice nucleation temperature suggested that predicting the freezing behavior of plants in the field will be complex.     

Slow dynamics in glass-forming materials

Dynamical properties of glass-formers such as glassy crystals, molecular liquids and model atomic liquids have been investigated in the pico–nanosecond (ps–ns) regime with dl_poly. The change in nature of translation and rotational dynamics are investigated in the supercooled state. Some predictions of the mode-coupling theory and the coupling model are checked. The microscopic origin of the fragility, i.e. the characteristic parameter involved in the liquid–glass transition, is also highlighted: the interaction potential, especially its anharmonicity and capacity for intermolecular coupling, is the key parameter controlling both the long time dynamics in supercooled systems and the short time dynamics in their glassy states.     

Freezing avoidance by supercooling in Olea europaea cultivars: the role of apoplastic water,solute content and cell wall rigidity

Plants can avoid freezing damage by preventing extracellular ice formation below the equilibrium freezing temperature (supercooling). We used Olea europaea cultivars to assess which traits contribute to avoid ice nucleation at sub‐zero temperatures. Seasonal leaf water relations, non‐structural carbohydrates, nitrogen and tissue damage and ice nucleation temperatures in different plant parts were determined in five cultivars growing in the Patagonian cold desert. Ice seeding in roots occurred at higher temperatures than in stems and leaves. Leaves of cold acclimated cultivars supercooled down to ?13 °C, substantially lower than the minimum air temperatures observed in the study site. During winter, leaf ice nucleation and leaf freezing damage (LT₅₀) occurred at similar temperatures, typical of plant tissues that supercool. Higher leaf density and cell wall rigidity were observed during winter, consistent with a substantial acclimation to sub‐zero temperatures. Larger supercooling capacity and lower LT₅₀ were observed in cold‐acclimated cultivars with higher osmotically active solute content, higher tissue elastic adjustments and lower apoplastic water. Irreversible leaf damage was only observed in laboratory experiments at very low temperatures, but not in the field. A comparative analysis of closely related plants avoids phylogenetic independence bias in a comparative study of adaptations to survive low temperatures.     

Quantitative analysis of cholesterol nucleation with time in supersaturated model bile

The amount of cholesterol (Ch) crystals formed in supersaturated taurochenodeoxycholate (TCDC) - lecithin (L) solutions of the same Ch saturation index (CSI) but at different Ch thermodynamic activities (Ch A_T) was quantified at different time intervals. The initial Ch nucleation rate (i.e., amount of Ch crystals formed with respect to time) in a Ch A_T = 1.73 and TCDC to L molar ratio (TCDC:L) = 5.1 system was faster than that in a Ch A_T = 1.42 and TCDC:L = 3.4 system. Shaking could enhance the early appearance of Ch crystals and cause the fast initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. The final Ch nucleation rates were faster than the initial Ch nucleation rates for the TCDC:L = 5.1 and the TCDC:L = 3.4 systems. According to a light scattering analysis of vesicle concentration in supersaturated TCDC–L solutions, vesicles provide nucleation sites only in the Ch nucleation process and the vesicle concentration may not be an important factor for the Ch nucleation rate. A model of a mixed TCDC–L micelle releasing Ch molecules together with the surface area of Ch crystals formed was used in the interpretation of the Ch nucleation.     

The Role of Molecular Dynamics Simulations for the Study of Slow Dynamics

Abstract

A two step strategy is proposed to study dynamical properties of a physical system much slower than the time scales accessible by molecular dynamics simulations. The strategy is applied to investigate the slow dynamics of supercooled liquids.     

CaCO3 nucleation by cyanobacteria: laboratory evidence for a passive, surface-induced mechanism

Calcite nucleation on the surface of cyanobacteria of the Synechococcus leopoliensis strain PCC 7942 was investigated to assess the influence of photosynthetic uptake of inorganic carbon and active ion exchange processes across the cell membrane on the nucleation and precipitation mechanisms. We performed long-term precipitation experiments at a constant CO₂ level in ambient air by adding suspensions of previously washed cyanobacteria to solutions of NaHCO₃/CaCl₂ which were supersaturated with respect to calcite. Induction times between 4 and 110 h were measured over a range of saturation states, Ω, between 8 and 4. The kinetics of CaCO₃ nucleation was compared between experiments: (i) with ongoing photosynthesis, (ii) with cells metabolizing but not undergoing photosynthetic uptake of inorganic carbon and (iii) in darkness without photosynthesis. No significant differences were observed between the three treatments. The results reveal that under low nutrient concentrations and permanent CO₂ supply, photosynthetic uptake of inorganic carbon predominantly uses CO₂ and consequently does not directly influence the nucleation process of CaCO₃ at the surface of S. leopoliensis. Furthermore, ion exchange processes did not affect the kinetics, indicating a passive nucleation process wherein the cell surface or extracellular polymers provided preferential sites for mineral nucleation. The catalyzing effect of the cyanobacteria on calcite nucleation was equivalent to a ∼18% reduction in the specific interfacial free energy of the calcite nuclei. This result and the ubiquitous abundance of cyanobacteria suggest that this process may have an impact on local and global carbon cycling.     

Injection of highly supersaturated oxygen solutions without nucleation

It is possible to inject highly supersaturated aqueous solutions of gas through a small capillary into an aqueous environment without the formation of significant gas bubbles. Such a technique has considerable potential therapeutic value in the treatment, for example, of heart attacks and strokes. The present paper is the second in a series (see Brereton et al. [1]) investigating the basic phenomenon behind this surprising effect. Recent experiments clearly demonstrate that the nucleation, when it does occur, results from heterogeneous nucleation on the interior surface of the distal end of the capillary. This paper describes the effects of the treatment of this interior surface on the nucleation processes and the results of high speed video observations of the phenomena. A heterogeneous nucleation model is presented which is in accord with the experimental observations.     

Endosperm and pericarp involvement in the supercooling of imbibed lettuce seeds

Fully hydrated lettuce (Lactuca sativa L.) seeds showed dual freezing exotherms (−9 and −18°C), even after 10 hours imbibition. Only the −9°C exotherm was observed in seeds imbibed for 20 hours, but without external nucleation, all water in the embryo supercooled. Results indicate that the endosperm acts as a barrier to ice propagation. Other experiments suggest that the pericarp may also protect the embryo under certain freezing conditions.     

Depression of the ice-nucleation temperature of rapidly cooled mouse embryos by glycerol and dimethyl sulfoxide.

The temperature at which ice formation occurs in supercooled cytoplasm is an important element in predicting the likelihood of intracellular freezing of cells cooled by various procedures to subzero temperatures. We have confirmed and extended prior indications that permeating cryoprotective additives decrease the ice nucleation temperature of cells, and have determined some possible mechanisms for the decrease. Our experiments were carried out on eight-cell mouse embryos equilibrated with various concentrations (0-2.0 M) of dimethyl sulfoxide or glycerol and then cooled rapidly. Two methods were used to assess the nucleation temperature. The first, indirect, method was to determine the in vitro survival of the rapidly cooled embryos as a function of temperature. The temperatures over which an abrupt drop in survival occurs are generally diagnostic of the temperature range for intracellular freezing. The second, direct, method was to observe the microscopic appearance during rapid cooling and note the temperature at which nucleation occurred. Both methods showed that the nucleation temperature decreased from - 10 to - 15 degrees C in saline alone to between - 38 degrees and - 44 degrees C in 1.0-2.0 M glycerol and dimethyl sulfoxide. The latter two temperatures are close to the homogeneous nucleation temperatures of the solutions in the embryo cytoplasm, and suggest that embryos equilibrated in these solutions do not contain heterogeneous nucleating agents and are not accessible to any extracellular nucleating agents, such as extracellular ice. The much higher freezing temperatures of cells in saline or in low concentrations of additive indicate that they are being nucleated by heterogeneous agents or, more likely, by extracellular ice.     

Supercooling characteristics of isolated peach flower bud primordia

The amount of unfrozen water in dormant peach (Prunus persica [L.] Batsch, cv Redhaven) flower buds, isolated primordia, and bud axes was determined during freezing using pulse nuclear magnetic resonance methods. Differential thermal analysis studies were conducted on whole buds and isolated primordia in the presence of ice nucleation. The results showed that some of the water in isolated primordia remained supercooled in the presence of ice nucleation. Although most tissue water froze (57.5%) following ice nucleation at −2.5°C, a considerable amount of water was found to supercool. In the presence of ice nucleation, increased hydration of isolated primordia resulted in the elimination of the supercooling characteristic. The structural integrity of isolated primordia appeared to be essential for supercooling.     

Numerical Calculation of the Bridge Function for Soft-Sphere Supercooled Fluids via Molecular Dynamics Simulations

Abstract

Isokinetic molecular dynamics simulations have been performed for 13,500 soft-spheres interacting through the inverse-power potential, ε([sgrave]/r)ⁿ , near and below the freezing temperature. The bridge function for the integral equation of the theory of liquids is extracted from the pair distribution function (PDF) obtained by the computer simulations for n = 6 and 12. The result is compared with that of approximate theories, i.e., the Rogers-Young (RY) approximation and a modified hypernetted-chain approximation for supercooled soft-sphere fluids (MHNCS approximation). Below the freezing temperature, the bridge function obtained by the computer simulation begins to oscillate around zero at intermediate distances where the second peak of the PDF appears. Such oscillatory behavior of the bridge function is well reproduced by the MHNCS approximation which includes correlations given by the leading elementary diagram, in remarkable contrast to that of the RY approximation. The present result suggests that the split second peak of the PDF for highly supercooled liquids is essentially dominated by the intermediate-distance-range correlation of the leading elementary diagram.     

Cholesterol crystallite nucleation in supersaturated model biles from a thermodynamic standpoint

A rapid, silicone polymer film uptake method was used to determine the cholesterol (Ch) thermodynamic activity (A(T)) in taurocholate (TC)-lecithin (L) and taurochenodeoxycholate (TCDC)-L model biles supersaturated with Ch. Also, time-dependent quasielastic light scattering (QLS) measurements and microscopic observations were made to determine the nature of particle species and the Ch nucleation times. In all cases in which Ch-L vesicles were present, a linear relationship between the logarithm of Ch nucleation times and Ch A(T) was found. These findings support that Ch A(T) is the appropriate parameter that represents the Ch nucleation tendency and that vesicles are catalytic sites in the Ch nucleation process. When Ca2+, a nucleation promoter ion, was present in the supersaturated model biles, the increased values of Ch A(T) quantitatively correlated with shorter Ch nucleation times. These latter findings further demonstrate that Ch A(T) is the dominant factor in explaining the Ch nucleation tendencies in supersaturated model biles.     

Physiological and biochemical analysis of overwintering and cold tolerance in two Central European populations of the spruce bark beetle, Ips typographus

Overwintering success is one of the key aspects affecting the development and outbreaks of the spruce bark beetle, Ips typographus (L.) populations. This paper brings detailed analysis of cold tolerance, and its influence on overwintering success, in two Central European populations of I. typographus during two cold seasons. Evidence for a supercooling strategy in overwintering adults is provided. The lower lethal temperature corresponds well to the supercooling point that ranges between −20 and −22 °C during winter months. The supercooled state is stabilized by the absence of internal ice nucleators and by seasonal accumulation of a mixture of sugars and polyols up to the sum concentration of 900 mM. The cryoprotective function of accumulated metabolites is probably based on increasing the osmolality and viscosity of supercooled body fluids and decreasing the relative proportion of water molecules available for lethal formation of ice nuclei. No activity of thermal hysteresis factors (stabilizers of supercooled state) was detected in hemolymph. Lethal times for 50% mortality (Lts50) in the supercooled state at −5, −10 or −15 °C are weeks (autumn, spring) or even months (winter), suggesting relatively little mortality caused by chill injury. Lts50 at −15 °C are significantly shorter in moist (6.9 days) than in dry (>42 days) microenvironment because there is higher probability of external ice nucleation and occurrence of lethal freezing in the moist situation.     

Bovine gallbladder mucin promotes cholesterol crystal nucleation from cholesterol-transporting vesicles in supersaturated model bile

This study examined the ability of purified gallbladder mucin to accelerate the nucleation of cholesterol monohydrate crystals from the cholesterol-transporting particles in supersaturated model bile. Mixed lipid micelles and cholesterol-phosphatidylcholine vesicles in supersaturated model bile were separated by Sephadex G-200 column chromatography. Mixed lipid micelles prepared by column chromatography had a low cholesterol-phosphatidylcholine ratio (0.30) and did not spontaneously nucleate cholesterol monohydrate crystals. In contrast, vesicles prepared by column chromatography had a cholesterol-phosphatidylcholine ratio of 1.00 and nucleated cholesterol crystals rapidly (P less than 0.001). Nucleation of cholesterol crystals was significantly accelerated in a concentration- and time-dependent manner by purified bovine gallbladder mucin in cholesterol containing vesicles, but not in mixed lipid micelles (P less than 0.001). A rapid filtration binding assay demonstrated significant binding of cholesterol and phosphatidylcholine in vesicles to gallbladder mucin but only minimal binding of cholesterol and phosphatidylcholine in mixed micelles. These data indicate that gallbladder mucin binds cholesterol and phosphatidylcholine in vesicles and accelerates the nucleation of cholesterol monohydrate crystals from these cholesterol-transporting particles in supersaturated model bile.