Step-bunching instability of growing interfaces between ice and supercooled water.

SignificanceStep-bunching instability (SBI) is one of the interfacial instabilities driven by self-organization of elementary step flow associated with crystal-growth dynamics, which has been observed in diverse crystalline materials. However, despite theoretical suggestions of its presence, no direct observations of SBI for simple melt growth have been achieved so far. Here, with the aid of a type of optical microscope and its combination with a two-beam interferometer, we realized quantitative in situ observations of the spatiotemporal dynamics of the SBI. This enables us to examine the origin of the SBI at the level of the step-step interaction. We also found that the SBI spontaneously induces a highly stable spiral growth mode, governing the late stage of the growth process.

Reversible Stacking and Delamination-Regulation of MXene via Controlled Freezing.

MXene's configuration, whether it is aggregated or dispersed in a monolayer, determines the specific application areas and even greatly influences the intrinsic properties of MXene. However, how to desirably control MXene's configuration is challenging. Here, a simple, additive-free, chemical reaction-free, and scalable strategy to optionally and reversibly regulate MXene's ordered stacking and delamination of MXene aggregates (AM) is reported. Just by controlled freezing of MXene aqueous dispersions, the aggregation percentage, delamination percentage, and interlayer spacing of AM can be finely tuned. Experimental results reveal that the freezing-induced aggregation and delamination effects can be explained by the squeezing action of growing ice grains on the MXene excluded/concentrated between ice grains and the expanding action caused by the ice formation between AM lamellae, respectively. The dominance between them depends on the freezing parameter-influenced ice nucleation sites, numbers, and ice grain sizes. This work not only contributes to the preparation, storage, and practical applications of MXene, but also opens a new and green avenue for controlling materials' assembly structures.

Effect of different freezing conditions on ice crystal formation behavior and ice-growth inhibition by cryoprotectants.

BACKGROUND: The formation of ice crystals will have adverse effects on aquatic products, and the key to ensure the long-term preservation and better quality preservations of the product is to evaluate the intercellular ice crystal formation to find suitable refrigeration conditions and cryoprotectants. RESULTS: The ice crystal formation was successfully captured by using an inverted microscope cryomicroscopic system equipped with a low-temperature stage, the ice crystals formed under different freezing methods between tuna muscle cells were observed directly, the deformation degree of muscle tissue pores during crystallization was evaluated, and the effect of freeze-thaw times on tuna samples was analyzed. The effects of the use of cryoprotectant such as cellobiose and carboxylated cellulose nanofibers on ice-growth inhibition were investigated, and the reliability of the ice crystal observation results was further verified by the determination of physical properties. The results showed that carboxylated cellulose nanofibers had the best ice-growth inhibition effect, they prevented about 50% cell deformation compared with the control group, and also reduced the minimum size of ice crystal formation. In addition, the addition of cellobiose and sodium tripolyphosphate gave the ice crystals a more uniform size and roundness. CONCLUSION: The experiment proposed a stable and clear observation method for the process of intercellular ice crystal formation, and the accuracy of the observation method was further verified by some physical indicators. This may help in the selection of suitable measurement methods to directly observe ice crystal formation behavior and screen cryoprotectants. © 2024 Society of Chemical Industry.

A novel automated protocol for ice crystal segmentation analysis using Cellpose and Fiji.

Accurate measurement of ice crystal size is an essential step in quantitative ice recrystallization inhibition (IRI) analysis using the sucrose sandwiching assay (SSA) and splat assay (SA). Here, we introduce a novel method of measuring ice crystal size and shape using Fiji and Cellpose, an anatomical segmentation algorithm, to address the time-consuming and limited number of ice particle determination associated with the mean largest grain size measurement. This new automated approach, displaying rapid segmentation of ∼70 s per image, measures every ice crystal in an image field of view, consequently reducing bias introduced by subjectively selecting the largest crystals in an image. Consistent in determining a diverse set of crystal sizes and shapes, this method allows for the evaluation of ice crystals using Feret's diameter, a parameter that better accounts for irregular particle shape. This method provides new outputs such as standard deviation, particle size distributions of a population of ice crystals, and circularity to characterize and further provide insight into an analyte's IRI ability. Applicable to the SSA, the "shape descriptor" measurement can be used to quantify ice binding. This work presents a novel and accurate approach for ice crystal quantitative analysis.

Observation on the ice crystal formation process of large yellow croaker (Pseudosciaena crocea) and the effect of multiple cryoprotectants pre-soaking treatments on frozen quality.

By observing the formation behavior of ice crystals, the quality of food products under different freezing conditions can be intuitively judged. In this paper, large yellow croaker was taken as the research object, and a novel cryomicroscopic system was developed to directly observe the structure of ice crystals during the freezing process. The cryoprotective effects of 4% sucrose +4% sorbitol (SU + SO), 4% xylo-oligosaccharide (XO), 4% xylo-oligosaccharide + 0.3% tetrasodium pyrophosphate (XO + TSPP) and 0.2% antifreeze protein (AFP) at different freezing temperatures were investigated. And the evaluation indicators, such as cell deformation degree, equivalent diameters, roundness, elongation and fractal dimension were introduced to quantify the damage of ice crystals to muscle tissues and fibers. The results indicate that reducing the freezing temperature and adding cryoprotectants can improve the quality of large yellow croaker. AFP has the best cryoprotective effect, with a reduction in cell deformation degree of 54.78% and 67.83% compared to the Control group at -5 °C and -20 °C, respectively. SU + SO and XO have the equivalent antifreeze effect, which is slightly inferior to XO + TSPP. In addition, physical parameters of large yellow croaker samples were measured to verify the influence of ice crystal structure on product quality. Therefore, direct observation of the ice crystal formation process and evaluation of ice crystal structure can accurately reflect the quality of frozen products, which is of great significance for the development of refrigeration and preservation technology.

Cellulose Nanocrystals Facilitate Needle-like Ice Crystal Growth and Modulate Molecular Targeted Ice Crystal Nucleation.

Ice nucleators are of crucial and important implications in various fields including chemistry, climate, agriculture, and cryobiology. However, the complicated extract and biocompatibility of ice nucleators remain unresolved, and the mechanism of ice nucleation remains largely unknown. Herein, we show that natural nanocrystalline cellulose materials possess special properties to enhance ice nucleation and facilitate needle-like ice crystal growth. We reveal the molecular level mechanism that the efficient exposure of cellulose hydroxyl groups on (-110) surface leads to faster nucleation of water. We further design chitosan-decorated cellulose nanocrystals to accomplish molecular cryoablation in CD 44 high-expression cells; the cell viability shows more than ∼10 times decrease compared to cryoablation alone and does not show evident systematic toxicity. Collectively, our findings also offer improved knowledge in molecular level ice nucleation, which may benefit multiple research communities and disciplines.

Control of ice crystal nucleation and growth during the food freezing process.

Freezing can maintain a low-temperature environment inside food, reducing water activity and preventing microorganism growth. However, when ice crystals are large, present in high amounts, and/or irregularly distributed, irreversible damage to food can occur. Therefore, ice growth is a vital parameter that needs to be controlled during frozen food processing and storage. In this review, ice growth theory and control are described. Macroscopic heat and mass transfer processes, the relationship between the growth of ice crystals and macroscopic heat transfer factors, and nucleation theory are reviewed based on the reported theoretical and experimental approaches. The issues addressed include how heat transfer occurs inside samples, variations in thermal properties with temperature, boundary conditions, and the functional relationship between ice crystal growth and freezing parameters. Quick freezing (e.g., cryogenic freezing) and unavoidable temperature fluctuations (e.g., multiple freeze-thaw cycles) are both taken into consideration. The approaches for controlling ice crystal growth based on the ice morphology and content are discussed. The characteristics and initial mechanisms of ice growth inhibitors (e.g., antifreeze proteins (AFPs), polysaccharides, and phenols) and ice nucleation agents (INAs) are complex, especially when considering their molecular structures, the ice-binding interface, and the dose. Although the market share for nonthermal processing technology is low, there will be more work on freezing technologies and their theoretical basis. Superchilling technology (partial freezing) is also mentioned here.

Ice-binding proteins: a remarkable ice crystal regulator for frozen foods.

Ice crystal growth during cold storage presents a quality problem in frozen foods. The development of appropriate technical conditions and ingredient formulations is an effective method for frozen food manufacturers to inhibit ice crystals generated during storage and distribution. Ice-binding proteins (IBPs) have great application potential as ice crystal growth inhibitors. The ability of IBPs to retard the growth of ice crystals suggests that IBPs can be used as a natural ice conditioner for a variety of frozen products. In this review, we first discussed the damage caused by ice crystals in frozen foods during freezing and frozen storage. Next, the methods and technologies for production, purification and evaluation of IBPs were summarized. Importantly, the present review focused on the characteristics, structural diversity and mechanisms of IBPs, and the application advances of IBPs in food industry. Finally, the challenges and future perspectives of IBPs are also discussed. This review may provide a better understanding of IBPs and their applications in frozen products, providing some valuable information for further research and application of IBPs.

Ice recrystallization inhibition activity varies with ice-binding protein type and does not correlate with thermal hysteresis.

Ice-binding proteins (IBPs) inhibit the growth of ice through surface adsorption. In some freeze-resistant fishes and insects, circulating IBPs serve as antifreeze proteins to stop ice growth by lowering the freezing point. Plants are less able to avoid freezing and some use IBPs to minimize the damage caused in the frozen state by ice recrystallization, which is the growth of large ice grains at the expense of small ones. Here we have accurately and reproducibly measured the ice recrystallization inhibition (IRI) activity of over a dozen naturally occurring IBPs from fishes, insects, plants, and microorganisms using a modified 'splat' method on serial dilutions of IBPs whose concentrations were determined by amino acid analysis. The endpoint of IRI, which was scored as the lowest protein concentration at which no recrystallization was observed, varied for the different IBPs over two orders of magnitude from 1000 nM to 5 nM. Moreover, there was no apparent correlation between their IRI levels and reported antifreeze activities. IBPs from insects and fishes had similar IRI activity, even though the insect IBPs are typically 10x more active in freezing point depression. Plant IBPs had weak antifreeze activity but were more effective at IRI. Bacterial IBPs involved in ice adhesion showed both strong freezing point depression and IRI. Two trends did emerge, including that basal plane binding IBPs correlated with stronger IRI activity and larger IBPs had higher IRI activity.

Magnetic control of heterogeneous ice nucleation with nanophase magnetite: Biophysical and agricultural implications.

In supercooled water, ice nucleation is a stochastic process that requires ∼250-300 molecules to transiently achieve structural ordering before an embryonic seed crystal can nucleate. This happens most easily on crystalline surfaces, in a process termed heterogeneous nucleation; without such surfaces, water droplets will supercool to below -30 °C before eventually freezing homogeneously. A variety of fundamental processes depends on heterogeneous ice nucleation, ranging from desert-blown dust inducing precipitation in clouds to frost resistance in plants. Recent experiments have shown that crystals of nanophase magnetite (Fe3O4) are powerful nucleation sites for this heterogeneous crystallization of ice, comparable to other materials like silver iodide and some cryobacterial peptides. In natural materials containing magnetite, its ferromagnetism offers the possibility that magneto-mechanical motion induced by external oscillating magnetic fields could act to disrupt the water-crystal interface, inhibiting the heterogeneous nucleation process in subfreezing water and promoting supercooling. For this to act, the magneto-mechanical rotation of the particles should be higher than the magnitude of Brownian motions. We report here that 10-Hz precessing magnetic fields, at strengths of 1 mT and above, on ∼50-nm magnetite crystals dispersed in ultrapure water, meet these criteria and do indeed produce highly significant supercooling. Using these rotating magnetic fields, we were able to elicit supercooling in two representative plant and animal tissues (celery and bovine muscle), both of which have detectable, natural levels of ferromagnetic material. Tailoring magnetic oscillations for the magnetite particle size distribution in different tissues could maximize this supercooling effect.

Atomic Steps Induce the Aligned Growth of Ice Crystals on Graphite Surfaces.

Heterogeneous ice nucleation on atmospheric aerosols strongly affects the earth's climate, and at the microscopic level, surface-irregularity-induced ice crystallization behaviors are common but crucial. Because of the lack of visual evidence and effective experimental methods, the mechanism of atomic-structure-dependent ice formation on aerosol surfaces is poorly understood. Here we chose highly oriented pyrolytic graphite (HOPG) to represent soot (a primary aerosol), and environmental scanning electron microscopy (ESEM) was performed for in situ observations of ice formation. We found that hexagonal ice crystals show an aligned growth pattern via a two-stage pathway with one a axis coinciding with the direction of atomic step edges on the HOPG surface. Additionally, the ice crystals grow at a noticeably higher speed along this direction. This study reveals the role of atomic surface defects in heterogeneous ice nucleation and may pave the way to control icing-related processes in practical applications.

Production, structure-function relationships, mechanisms, and applications of antifreeze peptides.

Growth of ice crystals can cause serious problems, such as frozen products deterioration, road damage, energy losses, and safety risks of human beings. Antifreeze peptides (AFPs), a healthy and effective cryoprotectant, have great potential as ice crystal growth inhibitors for a variety of frozen products. In this review, methods and technologies for the production, purification, evaluation, and characterization of AFPs are comprehensively summarized. First, this review describes the preparation of AFPs, including the methods of enzymatic hydrolysis, chemical synthesis, and microbial fermentation. Next, this review introduces the major methods by which to evaluate AFPs' antifreeze activity, including nanoliter osmometer, differential scanning calorimetry, splat-cooling, the biovaluation model, and novel technology. Moreover, this review presents an overview of the molecular characteristics, structure-function relationships, and action mechanisms of AFPs. Furthermore, advances in the application of AFPs to frozen food, including frozen dough, meat products, fruits, vegetable products, and dairy, are summarized and holistically analyzed. Finally, challenges of AFPs and future perspectives on their use are also discussed. An understanding of the production, structure-function relationships, mechanisms and applications of AFPs provides inspiration for further research into the use of AFPs in food science and food nutrition applications.

Distinct ice patterns on solid surfaces with various wettabilities.

No relationship has been established between surface wettability and ice growth patterns, although ice often forms on top of solid surfaces. Here, we report experimental observations obtained using a process specially designed to avoid the influence of nucleation and describe the wettability-dependent ice morphology on solid surfaces under atmospheric conditions and the discovery of two growth modes of ice crystals: along-surface and off-surface growth modes. Using atomistic molecular dynamics simulation analysis, we show that these distinct ice growth phenomena are attributable to the presence (or absence) of bilayer ice on solid surfaces with different wettability; that is, the formation of bilayer ice on hydrophilic surface can dictate the along-surface growth mode due to the structural match between the bilayer hexagonal ice and the basal face of hexagonal ice (ice Ih), thereby promoting rapid growth of nonbasal faces along the hydrophilic surface. The dramatically different growth patterns of ice on solid surfaces are of crucial relevance to ice repellency surfaces.

Superchilling of muscle foods: Potential alternative for chilling and freezing.

Superchilling is an attractive technique for preservation of muscle foods which freezes part of the water and insulate the food products from temperature fluctuations thereby enhancing the shelf-life during storage, transportation and retailing. Superchilling process synergistically improves the product shelf-life when used in combination with vacuum or modified atmospheric packaging. The shelf-life of muscle foods was reported to be increased by 1.5 to 4.0 times relative to traditional chilling technique. Advantages of superchilling and its ability to maintain the freshness of muscle foods over freezing has been discussed and its potential for Industrial application is highlighted. Present review also unravel the mechanistic bases for ice-crystal formation during superchilling and measures to ameliorate the drip loss. The future challenges especially automation in superchilling process for large scale Industrial application is presented.

Crystal-plane-dependent effects of antifreeze glycoprotein impurity for ice growth dynamics.

An impurity effect on ice crystal growth in supercooled water is an important subject in relation to ice crystal formation in various conditions in the Earth's cryosphere regions. In this review, we consider antifreeze glycoprotein molecules as an impurity. These molecules are well known as functional molecules for controlling ice crystal growth by their adsorption on growing ice/water interfaces. Experiments on free growth of ice crystals in supercooled water containing an antifreeze protein were conducted on the ground and in the International Space Station, and the normal growth rates for the main crystallographic faces of ice, namely, basal and prismatic faces, were precisely measured as functions of growth conditions and time. The crystal-plane-dependent functions of AFGP molecules for ice crystal growth were clearly shown. Based on the magnitude relationship for normal growth rates among basal, prismatic and pyramidal faces, we explain the formation of a dodecahedral external shape of an ice crystal in relation to the key principle governing the growth of polyhedral crystals. Finally, we emphasize that the crystal-plane dependence of the function of antifreeze proteins on ice crystal growth relates to the freezing prevention of living organisms in sub-zero temperature conditions. This article is part of the theme issue 'The physics and chemistry of ice: scaffolding across scales, from the viability of life to the formation of planets'.

Natural cryoprotectants combinations of l-proline and trehalose for red blood cells cryopreservation.

Cryopreservation of red blood cells (RBCs) holds great potential benefits for supplying transfusion timely in emergencies. Currently, glycerol is the main cryoprotectant permitted in clinical therapy for RBCs cryopreservation, but its broad application is limited by the toxicity and complex deglycerolization process. Successful cryopreservation of RBCs using more effective materials should be studied to reduce freezing damage, increase biocompatibility, and save processing time. Herein, a simple protocol using natural cryoprotectants combinations of l-proline and trehalose attains a low degree of hemolysis (11.2 ±â€¯2.73%) after thawing compared to glycerol. Furthermore, the morphology of RBCs and the activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase maintain well. Further mechanism study shows that l-proline plays an important role in decreasing the freezing points and inhibiting the growth of ice crystal by permeating into cells during the freezing process. While trehalose works as an inhibitor of ice growth in the freezing process and ice recrystallization in the thawing process. This simple l-proline & trehalose combinations protocol is a promising method to replace current time-consuming and labor-intensive cryopreservation methods of RBCs.

Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions.

XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the arrangement of basic and acidic amino acids is conserved between dicotyledonous and monocotyledonous species. Its gene expression was observed at the same level in roots and shoots under long-day conditions, but highly induced under short-day conditions and at low temperatures in roots, especially in endodermis and xylem parenchyma in the root hair region of Populus trichocarpa, and its protein level was high in dormant buds, but not in roots or branches. Addition of recombinant PtXSP25 protein mitigated the denaturation of lactate dehydrogenase by drying, but showed only a slight effect on that caused by freeze-thaw cycling. Recombinant PtXSP25 protein also showed ice recrystallization inhibition activity to reduce the size of ice crystals, but had no antifreezing activity. We suggest that PtXSP25 protein produced in shoots and/or in roots under short-day conditions and at non-freezing low temperatures followed by translocation via xylem sap to shoot apoplast may protect the integrity of the plasma membrane and cell wall functions from freezing and drying damage in winter environmental conditions.

Nanoparticle-mediated cryosurgery for tumor therapy.

Cryosurgery is an energy-based surgical technique. It is minimally invasive and has fewer side effects than surgical resection. However, its insufficient freezing to target tumor and unavoidable injury to healthy tissue have restricted its success. Nano-cryosurgery is the combination of cryogenic biomedicine and nanotechnology. Its principle is to introduce a nanoparticle solution into target tissues to maximize heat transfer, lower the end temperature, increase ice ball formation, and prevent healthy tissues from being frozen. This review covers common nanoplatforms for nano-cryosurgery. The characteristics, advantages, potential challenges, future prospects of applying nano-cryosurgery are discussed in detail.

Effect of Controlled Nucleation of Ice Crystals on the Primary Drying Stage during Lyophilization.

The freezing stage cannot be directly controlled, which leads to variation in product quality and low productivity during the lyophilization process. Our objective was to establish a robust design space for the primary drying stage using ice nucleation control based on the pressurization and depressurization technique. We evaluated the specific surface area (SSA), water content, scanning electron microscopy (SEM) images, and water vapor transfer resistance of the dried layer (Rp) of the products. The ice nucleation control resulted in a reduction of the SSA value and in an increase in water content. SEM observation suggested that the ice nucleation control enabled formation of large ice crystals, which was consistent with the reduction in the Rp value. As a result, the generation of collapsed cakes was inhibited, whereas 18% of the collapsed cakes were observed without ice nucleation control. Finally, this technique succeeded in determining a robust design space for the primary drying stage to produce uniform products of higher productivity. It was considered, from the present findings, that controlling the formation of large ice crystals impacted the product quality and productivity.

A first test of the hypothesis of biogenic magnetite-based heterogeneous ice-crystal nucleation in cryopreservation.

An outstanding biophysical puzzle is focused on the apparent ability of weak, extremely low-frequency oscillating magnetic fields to enhance cryopreservation of many biological tissues. A recent theory holds that these weak magnetic fields could be inhibiting ice-crystal nucleation on the nanocrystals of biological magnetite (Fe3O4, an inverse cubic spinel) that are present in many plant and animal tissues by causing them to oscillate. In this theory, magnetically-induced mechanical oscillations disrupt the ability of water molecules to nucleate on the surface of the magnetite nanocrystals. However, the ability of the magnetite crystal lattice to serve as a template for heterogeneous ice crystal nucleation is as yet unknown, particularly for particles in the 10-100 nm size range. Here we report that the addition of trace-amounts of finely-dispersed magnetite into ultrapure water samples reduces strongly the incidence of supercooling, as measured in experiments conducted using a controlled freezing apparatus with multiple thermocouples. SQUID magnetometry was used to quantify nanogram levels of magnetite in the water samples. We also report a relationship between the volume change of ice, and the degree of supercooling, that may indicate lower degassing during the crystallization of supercooled water. In addition to supporting the role of ice-crystal nucleation by biogenic magnetite in many tissues, magnetite nanocrystals could provide inexpensive, non-toxic, and non-pathogenic ice nucleating agents needed in a variety of industrial processes, as well as influencing the dynamics of ice crystal nucleation in many natural environments.