Antifreeze proteins (AFPs) bind irreversibly to ice surfaces and prevent ice crystal growth at temperatures of up to 10 °C below the melting point. The remarkable ability of AFPs to adsorb onto the ice surface and stop further growth is not fully understood, and their structural diversity hinders efforts to find a universal ice-binding motif. Multivalent AFP assemblies have achieved better ice growth inhibition compared to the corresponding monomers, yet the mechanism of these improved inhibitors is poorly understood. The innovative approach of this study is to test the effect of multivalency on the AFP's adsorption rates to ice in addition to testing AFP activity. A monomer, dimer, and multimer (12-subunits) of type III AFP were tested for thermal hysteresis (TH) activity, and their adsorption rates were measured using fluorescence microscopy. To fit the experimental data, we developed a revised Langmuir adsorption model. As expected, the monomer achieved the lowest TH activity, and its adsorption rate was slowest, followed by the dimer, which achieved slightly higher TH activity. The multimer was the most active, and its adsorption rate was found to be 11-fold higher than that of the monomer. The newly developed model identifies cooperativity effects in the multimer but not in the monomer and dimer. The rate at which the ice surface is covered by multimeric AFPs increases with continuous binding, suggesting cooperative ice binding. These results suggest a mechanism for the AFP multimer binding. After the binding of its first subunit to ice, subsequent binding of a second subunit becomes faster, which in turn facilitates the binding of a third subunit. Thus, cooperative ice binding is key to the superior inhibition of ice growth.

Methane clathrates on continental margins contain the largest stores of hydrocarbons on Earth, yet the role of biomolecules in clathrate formation and stability remains almost completely unknown. Here, we report new methane clathrate-binding proteins (CbpAs) of bacterial origin discovered in metagenomes from gas clathrate-bearing ocean sediments. CbpAs show similar suppression of methane clathrate growth as the commercial gas clathrate inhibitor polyvinylpyrrolidone and inhibit clathrate growth at lower concentrations than antifreeze proteins (AFPs) previously tested. Unlike AFPs, CbpAs are selective for clathrate over ice. CbpA3 adopts a nonglobular, extended structure with an exposed hydrophobic surface, and, unexpectedly, its TxxxAxxxAxx motif common to AFPs is buried and not involved in clathrate binding. Instead, simulations and mutagenesis suggest a bipartite interaction of CbpAs with methane clathrate, with the pyrrolidine ring of a highly conserved proline residue mediating binding by filling empty clathrate cages. The discovery that CbpAs exert such potent control on methane clathrate properties implies that biomolecules from native sediment bacteria may be important for clathrate stability and habitability.

According to a USDA report, $161 billion worth of food products was not available for human consumption in 2010 due to food loss. One potential way to reduce food loss is to prevent damage during the freezing process. This study presents quantitative measurements of the two primary processes involved in freezing: ice nucleation and ice growth. Using a newly developed micro-thermography system, we measured in-situ rates of ice nucleation and growth. Our findings indicate that ice nucleation and ice growth are distinct and opposing processes. Specifically, ice nucleation rates in beef and zucchini were significantly higher than those in broccoli and potato, whereas ice growth was faster in broccoli and potato compared to beef and zucchini. Analyzing the chemical composition of these foods enables the application of established crystal growth principles on freezing of foods. Therefore, designing a customized freezing process for each food product will lead to improved quality.

Antifreeze proteins (AFPs) are biomolecules that can bind to ice and hinder its growth, thus holding significant potential for biotechnological and biomedical applications. AFPs are a subset of ice-binding proteins (IBPs) and are found in various organisms across different life kingdoms. This mini-review investigates the underlying mechanisms by which AFPs impede ice growth, emphasizing the disparities between hyperactive and moderate AFPs. Hyperactive AFPs exhibit heightened thermal hysteresis (TH) activity and can bind to both the basal and prism planes of ice crystals, enabling them to endure extremely cold temperatures. In contrast, moderate AFPs predominantly bind to the prism/pyramidal planes and demonstrate lower TH activity. The structural diversity of AFPs and the presence of ordered water molecules on their ice-binding sites (IBS) have been subjects of debate among researchers. Multiple hypotheses have been proposed concerning the significance of ordered water molecules in ice binding. Gaining insights into the binding dynamics and the factors influencing TH activity in AFPs is crucial for the development of efficient synthetic compounds and the establishment of comprehensive models to elucidate ice growth inhibition. Here we emphasize the necessity for further research to unravel the mechanisms of AFPs and presents a pathway for constructing models capable of comprehensively explaining their inhibitory effects on ice growth.

According to a USDA report, 161 billion worth of food products was not available for human consumption in 2010 due to food loss. One potential way to reduce food loss is to prevent damage to the food product during the freezing process. This study presents quantitative measurements of the two primary processes involved in freezing of foods: ice nucleation and ice growth. Using a newly developed micro-thermography system, we measured in-situ ice nucleation and growth rates. We found that ice nucleation rates in beef and zucchini were significantly higher than those in broccoli and potato, whereas ice growth was faster in broccoli and potato compared to beef and zucchini. Thus, ice nucleation and ice growth in the foods tested here, were found to be opposing processes. By analyzing the chemical composition of these foods, we applied established crystal growth and nucleation principles to explain the reasons causing the inverted relationship between ice nucleation and ice growth. Therefore, designing a customized freezing process for each food product will lead to improved quality of the product, thereby limiting food loss.

Ice-binding proteins (IBPs) protect organisms living in sub-freezing conditions by inhibiting ice growth in fish and insects, limiting ice recrystallization in plants, and assisting bacteria to adhere to ice. The mechanisms by which these proteins bind to ice and inhibit its growth have been studied both experimentally and using molecular dynamic simulations. A unique experimental technique developed to test and characterize the interactions between IBPs and ice using a combination of a microfluidic device, cold stages with millikelvin temperature resolution, fluorescence-labeled IBPs, and fluorescence microscopy is described herein. The main advantage of this technique is the ability to exchange the solution around micron-sized ice crystals and characterize their binding to and inhibition of ice.

Ice crystal growth and nucleation rate measurements are usually done using visible light micros-copy in liquid and transparent samples. Yet, the understanding of important practical problems depends on monitoring ice growth inside solid materials. For example, how rapid ice growth leads to structural damage of food, or how the final structure of cementitious materials is affect-ed by ice during curing. Imaging crystal growth inside solid materials cannot be done with visi-ble light and is intrinsically more challenging than visible light imaging. Thermography is a technique that uses thermal (Infra-red) cameras to monitor temperature changes in a material, and it has been used to provide qualitative description of ice propagation and nucleation with a low spatial resolution. Here, we describe a method that uses a novel micro-thermography system to image ice nucleation, growth and melting inside non-transparent samples. This method relies on two major components: a cold stage with accurate temperature control (±0.001 ºC) and a thermal camera with high spatial and temperature resolution. Our experiments include imaging of ice formation and growth in pure water first, and inside plant leaves used as a model for a non-transparent material. Ice growth rate of 2.2 mm/s was measured inside a plant leaf at -12 ºC and ice nucleation in single plant cells was observed as a hot spot having a diameter of 160 µm. The results presented here provide experimental proof that high-quality imaging of ice growth is achievable, thus paving the way to quantitative measurements of ice growth kinetics and ice nu-cleation in solid materials.

Antifreeze proteins (AFPs) facilitate the survival of diverse organisms in frigid environments by adsorbing to ice crystals and suppressing their growth. The rate of AFP accumulation on ice is determined by an interplay between AFP diffusion from the bulk solution to the ice-water interface and the subsequent adsorption of AFPs to the interface. To interrogate the relative importance of these two processes, here we combine non-equilibrium fluorescence experiments with a reaction-diffusion model. We find that as diverse AFPs accumulate on ice, their concentration in the aqueous solution does not develop a gradient but remains equal to its bulk concentration throughout our experiments. These findings lead us to conclude that AFP accumulation on ice crystals, which are smaller than 100 microns in radius, is not limited by the diffusion of AFPs, but by the kinetics of AFP adsorption. Our results imply that mass transport limitations do not hinder AFPs from performing their biological function.

Ice nucleation and recrystallization plays a central role in our daily lives and understanding these processes will have great implications. The measurement of ice nucleation, growth and melting is usually done using light microscopy in liquid and transparent samples. However, crystal growth inside solid materials such as food products, cementitious materials and biological samples is more complex. Imaging ice inside these non-transparent materials would be beneficial in many ways since rapid ice growth leads to structural damage of food, and the weakening of the final structure of cementitious materials. Thermography is a technique that uses thermal (Infra-red) cameras to monitor temperature changes in a material, and it has been used to provide qualitative description of ice propagation and nucleation with a spatial resolution of millimeters. Here, the use of a novel micro-thermography system to image ice nucleation, growth and melting inside non-transparent samples with an emphasis on biological samples is described. This method relies on two major players; a cold stage with accurate temperature control (±0.001 ºC) coupled to a high-resolution (both temperature and spatial) thermal camera. The coupling of these instruments brings about the ability to measure the kinetics of ice growth and nucleation inside a variety of non-transparent samples. These experiments provide strong evidence for the high-quality imaging of ice growth, thus leading to quantitative measurements of ice growth velocity and ice nucleation in solid materials.

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the formation of single microscopic crystals and a suitable microscopy system coupled to the cold stage. The method described herein adds another important feature that includes exchanging solutions around ice and clathrate hydrate crystals. The described system comprises a combination of unique and home-developed instruments, including microfluidics, high-resolution cold stages, and fluorescence microscopy. The cold stage was designed for microfluidic devices and allows for the formation of micron-sized ice/hydrate crystals inside microfluidic channels and the exchange of solutions around them. The temperature resolution and stability of the cold stage is one millikelvin, which is crucial for controlling the growth of these small crystals. This diverse system is used to study the different processes of ice and hydrate crystallization and the mechanism by which the growth of these crystals is inhibited. The protocol describes how to prepare microfluidic devices, how to grow and control microscopic crystals in the microfluidic channels, and how the utilization of the flow of liquids around ice/hydrate crystals affords new insights into the crystallization of water.

The mechanism by which safranine O (SFO), an ice growth inhibitor, halts the growth of single crystal tetrahydrofuran (THF) clathrate hydrates was explored using microfluidics coupled with cold stages and fluorescence microscopy. THF hydrates grown in SFO solutions exhibited morphology changes and were shaped as truncated octahedrons or hexagons. Fluorescence microscopy and microfluidics demonstrated that SFO binds to the surface of THF hydrates on specific crystal planes. Cryo-TEM experiments of aqueous solutions containing millimolar concentrations of SFO exhibited the formation of bilayered lamellae with an average thickness of 4.2±0.2 nm covering several μm2 . Altogether, these results indicate that SFO forms supramolecular lamellae in solution, which might bind to the surface of the hydrate and inhibit further growth. As an ice and hydrate inhibitor, SFO may bind to the surface of these crystals via ordered water molecules near its amine and methyl groups, similar to some antifreeze proteins.

Since some antifreeze proteins and glycoproteins(AF(G)Ps) cannot directly bind to all crystal planes, they change ice crystal morphologyby minimizing the area of the crystal planes to which they cannot bind until crystalgrowth is halted. Previous studies found that growth along the c-axis(perpendicular to the basal plane, the crystal plane to which these AF(G)Pscannot bind) is accelerated by some AF(G)Ps, while growth of other planes isinhibited. The effects of this growth acceleration on crystal morphology and onthe thermal hysteresis activity are unknown to date. Understanding theseeffects will elucidate the mechanism of ice growth inhibition by AF(G)Ps. Usingcold stages and an Infrared laser, ice growth velocities and crystalmorphologies in AF(G)P solutions were measured. Three types of effects ongrowth velocity were found: concentration-dependent acceleration,concentration-independent acceleration, and concentration-dependentdeceleration. Quantitative crystal morphology measurements in AF(G)P solutionsdemonstrated that adsorption rate of the proteins to ice plays a major role indetermining the morphology of the bipyramidal crystal. These results demonstratethat faster adsorption rates generate bipyramidal crystals with diminishedbasal surfaces at higher temperatures compared to slower adsorption rates. Theacceleration of growth along the c-axis generates crystals with smallerbasal surfaces at higher temperatures leading to increased growth inhibition ofthe entire crystal.

The growth of spontaneously twisted crystals is a common but poorly understood phenomenon. An analysis of the formation of twisted crystals of a metastable benzamide polymorph (form II) crystallizing from highly supersaturated aqueous and ethanol solutions is given here. Benzamide, the first polymorphic molecular crystal reported (1832), would have been the first helicoidal crystal observed had the original authors undertaken an analysis by light microscopy. Polymorphism and twisting frequently concur as they are both associated with high thermodynamic driving forces for crystallization. Optical and electron microscopies as well as electron and powder X-ray diffraction reveal a complex lamellar structure of benzamide form II needle-like crystals. The internal stress produced by the overgrowth of lamellae is shown to be able to create a twist moment that is responsible for the observed non-classical morphologies.

In some cold-adapted organisms, over a dozen isoforms of antifreeze (glyco)proteins or AF(G)Ps are present. Although these isoforms are structurally similar, their ability to inhibit ice growth varies significantly, and, in some fish, passive isoforms can be much more abundant than the active ones. Laboratory experiments demonstrated more than a decade ago that mixtures of AFP isoforms can exhibit synergistic enhancement of each other's activity. The mechanism of this synergy effect has remained obscure and is addressed here. Using cold-stages, microfluidics, and fluorescence microscopy, the activity of binary mixtures of structurally distinct AF(G)Ps from different fish and plant species was measured. While several mixtures exhibited enhancement, some mixtures exhibited antagonism. These latter mixtures included AF(G)Ps that bind to the same crystal planes, thereby exhibiting competition. Fluorescence microscopy experiments with a synergistic mixture of two isoform types labeled with different dyes showed they bound to different crystal planes. These results helped develop a kinetic description of the mechanism by which AF(G)Ps achieve synergy. The requirements of an active isoform include high adsorption rates, and prism plane binding, while passive isoforms usually bind to a pyramidal plane at slower rates. For synergy to occur, an active isoform first binds to the faster growing prism plane. This binding slows the advancement of the prism plane and creates more pyramidal surfaces to which a passive isoform bind. These results, in part, explain the biological observation of isoform distribution in fish, and the physical chemistry of the synergistic crystal growth inhibition by two inhibitors.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) inhibit ice growth via an adsorption-inhibition mechanism that assumes irreversible binding of AF(G)Ps to embryonic ice crystals and the inhibition of further growth. The irreversible binding of antifreeze glycoproteins (AFGPs) to ice has been questioned and remains poorly understood. Here, we used microfluidics and fluorescence microscopy to investigate the nature of the binding of small and large AFGP isoforms. We found that both AFGP isoforms bind irreversibly to ice, as evidenced by microfluidic solution exchange experiments. We measured the adsorption rate of the large AFGP isoform and found it to be 50% faster than that of AFP type III. We also found that the AFGP adsorption rate decreased by 65% in the presence of borate, a well-known inhibitor of AFGP activity. Our results demonstrate that the adsorption rate of AFGPs to ice is crucial for their ice growth inhibition capability.

Significance Freezing and melting of ice are one of the most common events on Earth. The dynamics of ice crystallization are relevant to climate research, mitigating frost damage in agriculture and construction, glacier dynamics, tissue and food preservation, and transportation. We describe the use of microfluidic devices, accompanied by precise temperature control, to examine the effect of H/D isotope exchange between liquid light water and solid heavy water on ice growth dynamics. These studies revealed unusual morphologies at the ice surface in contact with the liquid, including curious unsteady morphological features that give the appearance of oscillation due to complex interplay of H/D exchange, thermal gradients, and local surface curvature.

The growth dynamics of D2O ice in liquid H2O in a microfluidic device were investigated between the melting points of D2O ice (3.8 °C) and H2O ice (0 °C). As the temperature was decreased at rates between 0.002 °C/s and 0.1 °C/s, the ice front advanced but retreated immediately upon cessation of cooling, regardless of the temperature. This is a consequence of the competition between diffusion of H2O into the D2O ice, which favors melting of the interface, and the driving force for growth supplied by cooling. Raman microscopy tracked H/D exchange across the solid H2O-solid D2O interface, with diffusion coefficients consistent with transport of intact H2O molecules at the D2O ice interface. At fixed temperatures below 3 °C, the D2O ice front melted continuously, but at temperatures near 0 °C a scalloped interface morphology appeared with convex and concave sections that cycled between growth and retreat. This behavior, not observed for D2O ice in contact with D2O liquid or H2O ice in contact with H2O liquid, reflects a complex set of cooperative phenomena, including H/D exchange across the solid-liquid interface, latent heat exchange, local thermal gradients, and the Gibbs-Thomson effect on the melting points of the convex and concave features.

The growth dynamics of D2O ice in liquid H2O in a microfluidic device were investigated between the melting points of D2O ice (3.8 ◦C) and H2O ice (0 ◦C). As the temperature was decreased at rates between 0.002 ◦C/s and 0.1 ◦C/s, the ice front advanced but retreated immediately upon cessation of cooling, regardless of the temperature. This is a consequence of the competition between diffusion of H2O into the D2O ice, which favors melting of the interface, and the driving force for growth supplied by cooling. Raman microscopy tracked H/D exchange across the solid H2O– solid D2O interface, with diffusion coefficients consistent with transport of intact H2O molecules at the D2O ice interface. At fixed temperatures below 3 ◦C, the D2O ice front melted continuously, but at temperatures near 0 ◦C a scalloped interface morphology appeared with convex and concave sections that cycled between growth and retreat. This behavior, not observed for D2O ice in contact with D2O liquid or H2O ice in contact with H2O liquid, reflects a complex set of cooperative phenomena, including H/D exchange across the solid–liquid interface, latent heat exchange, local thermal gradients, and the Gibbs–Thomson effect on the melting points of the convex and concave features.