Ice Recrystallization Research Articles

Ice recrystallization inhibition behavior by wheat flour and its synergy effect with antifreeze protein III

Ice recrystallization (IR) is an undesirable phenomenon in which the size of an ice crystal grows in the frozen medium. Based on the Ostwald ripening theory, the IR kinetics of wheat flour system were investigated under isothermal conditions (−10 °C). The results showed that 30% (w/w) sucrose solution had an IR rate of 3.34 ± 0.29 μm3/min, however adding 5% (w/w) wheat flour reduced the rate to 0.56 ± 0.15 μm3/min. To understand the inhibitory effect of wheat flour, the size distribution was analyzed. The results indicated that wheat flour reduced both the size and size distribution of the ice crystals in the sucrose solution, suggesting that narrowing the size distribution is the candidate mechanism for IR inhibition. Further, combining antifreeze protein III with the wheat flour system decreased IR rate in a synergistic manner. In presence of wheat flour, only 0.1 μg/mL of AFP III was sufficient to lower IR rate significantly, while without it, 0.5 μg/mL of AFP III was required.

Advances in Antifreeze Molecules: From Design and Mechanisms to Applications

Freezing can cause serious hazards in various fields, such as cryoinjury in cell and organ cryopreservation, food quality deterioration, and ice accretion on surfaces including energy facilities (wind turbines, overhead transmission lines, and towers), aircraft, etc., causing serious equipment damages and economic losses. To alleviate these problems, antifreeze molecules have attracted significant attention and have been applied extensively in cryopreservation and anti-icing coatings. In this review, the progress on antifreeze molecules is summarized and discussed, including their classifications, mechanisms, and applications. The antifreeze molecules are divided into natural and artificial molecules, and their characteristics are introduced. Moreover, their antifreeze mechanisms are comprehensively outlined involving depression of freezing points, dynamic ice shaping, and ice recrystallization inhibition. Then, the representative applications in cryobiology, anti-icing surfaces, food technology, and agriculture are also summarized and discussed. Finally, the challenges and development prospects of antifreeze molecules are further provided.

Perturbation of Hydrogen Bonding Network Plays an Important Role in Determining Antifreeze Activity

In order to develop new antifreeze agents, it is crucial to understand the antifreeze activity. The adsorption-inhibition mechanism has been widely accepted, and most previous studies focus on ice-binding affinity of additive molecules but rarely discuss their influence on the hydrogen bonding network of ice. Herein, we investigate the ice recrystallization inhibition (IRI) activity of two structurally similar molecules, histidine (His) and phenylalanine (Phe). It is verified by experiment that Phe has excellent IRI activity, while His does not. Molecular dynamics simulation is conducted to study the process of ice growth with amino acids and to explore the origin of the difference between His and Phe. We find that His is prone to be swallowed up by ice and thus no longer hinders its growth, whereas Phe can remain unswallowed on the ice surface for a long period, which is attributed to their different abilities to perturb the hydrogen bonding network. Based on a statistical analysis of oxygen–hydrogen bond orientations, we propose a method to evaluate the order of the hydrogen bonding network. With this method, the IRI activity of His and Phe can be well interpreted, and the influence of different groups on the hydrogen bonding network can be quantified. This study takes the structural integrity of ice into consideration and provides a possibility to investigate the influence of different additive molecules on hydrogen bonding network, which is expected to improve the understanding of antifreeze mechanism.

Engineered Compounds to Control Ice Nucleation and Recrystallization.

One of the greatest concerns in the subzero storage of cells, tissues, and organs is the ability to control the nucleation or recrystallization of ice. In nature, evidence of these processes, which aid in sustaining internal temperatures below the physiologic freezing point for extended periods of time, is apparent in freeze-avoidant and freeze-tolerant organisms. After decades of studying these proteins, we now have easily accessible compounds and materials capable of recapitulating the mechanisms seen in nature for biopreser-vation applications. The output from this burgeoning area of research can interact synergistically with other novel developments in the field of cryobiology, making it an opportune time for a review on this topic.

Dimensional Reduction of Metal-Organic Frameworks for Enhanced Cryopreservation of Red Blood Cells.

To increase the red blood cell (RBC) cryopreservation efficiency by metal-organic frameworks (MOFs), a dimensional reduction approach has been proposed. Namely, 3D MOF nanoparticles are progressively reduced to 2D ultra-thin metal-organic layers (MOLs). We found that 2D MOLs are beneficial for enhanced interactions of the interfacial hydrogen-bonded water network and increased utilization of inner ordered structures, due to the higher surface-to-volume ratio. Specifically, a series of hafnium (Hf)-based 2D MOLs with different thicknesses (monolayer to stacked multilayers) and densities of hydrogen bonding sites have been synthesized. Both ice recrystallization inhibition activity (IRI) and RBCs cryopreservation assay confirm the pronounced better IRI activity and excellent cell recovery efficiency (up to ≈63 % at a very low concentration of 0.7 mg mL-1 ) of thin-layered Hf-MOLs compared to their 3D counterparts, thereby verifying the dimensional reduction strategy to improved cryoprotectant behaviors.

Dimensional Reduction of Metal–Organic Frameworks for Enhanced Cryopreservation of Red Blood Cells

AbstractTo increase the red blood cell (RBC) cryopreservation efficiency by metal–organic frameworks (MOFs), a dimensional reduction approach has been proposed. Namely, 3D MOF nanoparticles are progressively reduced to 2D ultra‐thin metal–organic layers (MOLs). We found that 2D MOLs are beneficial for enhanced interactions of the interfacial hydrogen‐bonded water network and increased utilization of inner ordered structures, due to the higher surface‐to‐volume ratio. Specifically, a series of hafnium (Hf)‐based 2D MOLs with different thicknesses (monolayer to stacked multilayers) and densities of hydrogen bonding sites have been synthesized. Both ice recrystallization inhibition activity (IRI) and RBCs cryopreservation assay confirm the pronounced better IRI activity and excellent cell recovery efficiency (up to ≈63 % at a very low concentration of 0.7 mg mL−1) of thin‐layered Hf‐MOLs compared to their 3D counterparts, thereby verifying the dimensional reduction strategy to improved cryoprotectant behaviors.

Development and Implementation of Microbial Antifreeze Protein Based Coating for Anti‐Icing

AbstractIce formation on a solid surface is a major challenge in industrial applications, it causes higher energy consumption and performance deterioration and may lead to catastrophic results. The preparation of anti‐icing surfaces to prohibit ice accumulation on a surface is crucial to reduce operational costs and to extend the surface's lifetime. The utilization of cryoprotectants to obtain anti‐icing surfaces is an effective method and is applicable in multiple fields. Antifreeze proteins (AFPs) are natural cryoprotectants to obtain anti‐icing surfaces, which have the ability to decrease the freezing point and to prevent ice‐crystal growth via thermal hysteresis (TH) and ice recrystallization inhibition (IRI). This study reports the molecular cloning, expression, and production of AFP protein from Escherichia coli (E. Coli). This wok also demonstrates the activity of coated AFP on aluminum surfaces. The expressed AFP is immobilized on aluminum surfaces treated by oxygen plasma. The coated AFP exhibits promising antifreeze activity with a high anti‐icing ability on aluminum surfaces in the size of evaporator fins (40 × 40 cm). The outcome of this study provides new insights into the biotechnological implementation of AFPs to various industrial applications for energy‐saving and higher performance.

Oligonucleotides as Inhibitors of Ice Recrystallization.

Oligonucleotides of adenine (A20), guanine (G20), cytosine (C20), thymine (T20), cytosine-guanine ((CG)20), and adenine-thymine ((AT)20) were investigated as model compounds for ice recrystallization inhibition (IRI). Dehydroxy uracil (dU20), U20, and T20 were also compared to investigate the effect of minute changes in the hydrophobicity of the oligonucleotides on the IRI activity. Among the oligonucleotides considered in this study, T20 exhibited the best performance for IRI. In addition, the degree of polymerization of oligothymines varied over 5, 10, 20, 30, 50, and 100, and T20 was found to be the most effective for IRI. The IRI mechanism was investigated by comparing U20 and T20, which exhibited the lowest and highest IRI activity, respectively, among the oligonucleotides for their dynamic ice-shaping, thermal hysteresis, and ice nucleation inhibition. Little or no dynamic ice-shaping activity and small thermal hysteresis were observed for both nucleotides. All of the findings suggest that not the ice-polymer adhesion but the hydrophobic interactions of T20 in the interface layer might interfere with the water deposition on the ice crystal surfaces and contribute to the IRI activity of the T20 oligonucleotide.

Dimethylglycine Can Enhance the Cryopreservation of Red Blood Cells by Reducing Ice Formation and Oxidative Damage

The cryopreservation of red blood cells (RBCs) holds great potential for ensuring timely blood transfusions and maintaining an adequate RBC inventory. The conventional cryoprotectants (CPAs) have a lot of limitations, and there is an obvious need for novel, efficient, and biocompatible CPAs. Here, it is shown for the first time that the addition of dimethylglycine (DMG) improved the thawed RBC recovery from 11.55 ± 1.40% to 72.15 ± 1.22%. We found that DMG could reduce the mechanical damage by inhibiting ice formation and recrystallization during cryopreservation. DMG can also scavenge reactive oxygen species (ROS) and maintain endogenous antioxidant enzyme activities to decrease oxidative damage during cryopreservation. Furthermore, the properties of thawed RBCs were found to be similar to the fresh RBCs in the control. Finally, the technique for order performance by similarity to ideal solution (TOPSIS) was used to compare the performance of glycerol (Gly), hydroxyethyl starch (HES), and DMG in cryopreservation, and DMG exhibited the best efficiency. This work confirms the use of DMG as a novel CPA for cryopreservation of RBCs and may promote clinical transfusion therapy.

A Synergistic Combination of AuNRs and C Dots as a Multifunctional Material for Ice Recrystallization Inhibition and Rapid Rewarming.

Robust platforms and advanced biocompatible materials having diverse performances are in tremendous demand for cryopreservation of biocells, which are greatly limited by the crystallization, formation, and growth of ice crystals. The fickle structure and the arduous extraction process of modern attainable antifreezing proteins cause fatal cryoinjury of the cells making it challenging to develop anti-icing materials. Thus, designing Au colloids is an effective way to combat cell-damaging concerns during the ice freezing-thawing process. Herein, we propose an emerging biomimetic hybrid nanomaterial (AuNR@SiO2-CDs) prepared by combining the photoheating and rewarming controlling characteristics of carbon dots (CDs) and gold nanorods (AuNRs), respectively, via a SiO2 scaffold that has an optimal aspect ratio of ∼4.4. The performance of the material is applied in the freezing and resuscitation of Hela cells. The typical linkage between the AuNR and CDs not only retains the stable structure but also possesses the symmetric functional characteristics of affirmative cryoprotectant materials and sustained low cytotoxicity of cell viability >90%. The cell recovery rate of the Hela cell is significantly improved to ∼60%, which is propped up to >4% higher by the laser irradiation dose. The above hybrid material is paving a path toward novel bionic antifreezing proteins and is envisioned for ice recrystallization inhibition and rapid rewarming.

The Atomistic Understanding of the Ice Recrystallization Inhibition Activity of Antifreeze Glycoproteins

As the most potent ice recrystallization inhibitors, antifreeze glycoproteins (AFGPs) have been extensively studied since their discovery. However, the molecular mechanism of how they inhibit ice growth remains controversial—notably, which group directly contributes to the binding of AFGPs to ice is hotly debated. Here, we use molecular dynamics simulations to investigate the atomistic details of the binding of AFGP8 to ice. We show that the binding of AFGP8 to ice can be divided into three cases: backbone dominant binding (BDB), disaccharide dominant binding (DDB) and weak binding (WB). Hydrogen-bonding and hydrophobic groups contribute equally to the binding of AFGP8 to ice and synergistically promote the binding. The –CH3 groups promote the contacting of AFGP8 to ice via hydrophobic effect, and the hydrogen-bonding groups anchor AFGP8 to ice surfaces through direct hydrogen bonding with ice. Specially, we verify that the -CONH- groups anchor the backbone of AFGP8 to ice by forming hydrogen bonds with ice surfaces while the –OH groups not only anchor the disaccharide to ice but also slow down the dynamics of the surrounding water. In addition, we reveal that both the backbone and the disaccharide can bind to ice surfaces while the latter is more flexible, which also perturbs the hydrogen bond network of potential ice-like water molecules by swaying in the solution to further enhance its antifreeze activity. This work provides the atomistic details of the ice growth inhibition mechanism of AFGP8, which is helpful for the design of high-efficacy cryoprotectants.

High Molecular Weight Polyproline as a Potential Biosourced Ice Growth Inhibitor: Synthesis, Ice Recrystallization Inhibition, and Specific Ice Face Binding.

Ice-binding proteins (IBPs) from extremophile organisms can modulate ice formation and growth. There are many (bio)technological applications of IBPs, from cryopreservation to mitigating freeze-thaw damage in concrete to frozen food texture modifiers. Extraction or expression of IBPs can be challenging to scale up, and hence polymeric biomimetics have emerged. It is, however, desirable to use biosourced monomers and heteroatom-containing backbones in polymers for in vivo or environmental applications to allow degradation. Here we investigate high molecular weight polyproline as an ice recrystallization inhibitor (IRI). Low molecular weight polyproline is known to be a weak IRI. Its activity is hypothesized to be due to the unique PPI helix it adopts, but it has not been thoroughly investigated. Here an open-to-air aqueous N-carboxyanhydride polymerization is employed to obtain polyproline with molecular weights of up to 50000 g mol-1. These polymers were found to have IRI activity down to 5 mg mL-1, unlike a control peptide of polysarcosine, which did not inhibit all ice growth at up to 40 mg mL-1. The polyprolines exhibited lower critical solution temperature behavior and assembly/aggregation observed at room temperature, which may contribute to its activity. Single ice crystal assays with polyproline led to faceting, consistent with specific ice-face binding. This work shows that non-vinyl-based polymers can be designed to inhibit ice recrystallization and may offer a more sustainable or environmentally acceptable, while synthetically scalable, route to large-scale applications.

Amyloid protein fibrils show enhanced ice recrystallization inhibition activity when serve as pickering emulsion stabilizer

Ice recrystallization adversely affects the quality of frozen foods. Therefore, food-grade materials with ice recrystallization inhibition (IRI) activity are attracting growing attention. Our previous study has found IRI activity of amyloid protein fibrils (APFs); however, as with most other food-grade IRI active materials, the IRI activity of APFs is moderate and needed to be enhanced. Here, we report that IRI activity is greatly enhanced when APFs serve as Pickering emulsion stabilizer. Adsorption of APFs to the oil-water interface resulted in the formation of microsized Pickering emulsion droplets. The IRI activity resulted from the adsorbed APFs rather than steric hindrance of emulsion droplets because self-induced emulsion droplets without APFs on the surface had no IRI activity. The oil core chemistry does not affect the IRI activity of adsorbed APFs. A structure−activity relationship analysis revealed that IRI activity of adsorbed APFs increases with larger particle size and emulsion droplet number (total number of droplets per unit volume). The enhancement of IRI activity caused by oil-water interface adsorption is reasonably ascribed to enlarged size, crowding of APFs on the surface of emulsion droplets, and enhanced ice adsorption of APFs. Compared with free APFs, adsorbed APFs were more IRI active at higher annealing temperatures and higher unfrozen water contents. Given the amphiphilic nature of most IRI-active materials, the findings of this study shed light on the application of IRI-active materials as emulsifiers to improve the quality of frozen food containing emulsions, such as ice cream and frozen meat paste.

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.

Depletion interaction may reduce ice recrystallization inhibition activity of cellulose nanocrystals (CNCs) at high concentrations

In ice cream, food hydrocolloids are commonly used as stabilizers for controlling ice recrystallization. Recent studies suggest that some stabilizers might bind to ice crystal surfaces and inhibit recrystallization. However, this ice-binding mechanism is challenged by the fact that stabilizers at high concentrations sometimes have reduced activity or even accelerate ice recrystallization. In this work, we determined the ice recrystallization inhibition (IRI) activity of cellulose nanocrystals (CNCs) at different concentrations in the 3.0% sucrose solution by the splat assay and in the 25.0% and 40.0% sucrose solutions by the sandwich assay. Reduced activity at high CNCs concentrations was observed in the sandwich assay. It was not linked to the phase separation or liquid crystal phase formation of CNCs but was caused by the accretion of nearby ice crystals and the forming of large ice crystals with irregular morphology and high aspect ratios. A new explanation based on the depletion-interaction-induced accretion of ice crystals was proposed. Our findings might resolve the contradiction between the ice-binding mechanism and the stabilizer concentration effect and add more support to the interfacial mechanism of ice recrystallization inhibition. They also pointed out the importance of selecting suitable stabilizer concentrations for screening IRI active materials and manufacturing frozen desserts.

Rediscovery of poly(ethylene glycol)s as a cryoprotectant for mesenchymal stem cells

BackgroundA medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at –196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required.MethodAs biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at –196 °C for 7 days. Then, cell recovery was assayed.ResultsWe found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants.ConclusionsPEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system.Graphical

Inhibition of Chitosan Ice Coating on the Quality Deterioration of Quick-Frozen Fish Balls during Repeated Freeze-Thaw Cycles.

Chitosan ice coating's properties and its inhibitory effect on the quality deterioration of quick-frozen fish balls during repeated freeze-thaw cycles were investigated. When the chitosan (CH) coating concentration increased, the viscosity and ice coating rate increased, while water vapor permeability (WVP), water solubility, and transmittance decreased, and 1.5% CH was regarded as the excellent coating to apply to freeze-thaw quick-frozen fish balls. As the freeze-thaw cycles increased, the frost production, total volatile base nitrogen (TVB-N) values, and free water content of all of the samples increased significantly (p < 0.05), and the whiteness values, textural properties, and water-holding capacity (WHC) decreased. Freeze-thaw cycles expanded the aperture between the muscle fibers and the occurrence of crystallization and recrystallization between cells increased, damaging the original intact tissue structure, which were confirmed by SEM and optical microscopy. Compared with the untreated ones, the frost production, free water, and TVB-N of the samples with 1.5% CH decreased during 1, 3, 5, and 7 cycles, and were reduced by 23.80%, 32.21%, 30.33%, and 52.10% by the 7th cycle. The WHC and texture properties showed an increasing trend during the freeze-thaw cycles. Therefore, the chitosan ice coating effectively inhibited the quality deterioration by reducing water loss, the occurrence of ice crystallization and recrystallization, and the pores of the samples.

Ice Nucleation Promotion Impact on the Ice Recrystallization Inhibition Activity of Polyols.

Heterogeneous ice nucleation occurs vis-à-vis nucleating agents already present in solution yet can occur within a rather broad range of temperatures (0 to ca. -38 °C). Controlling this temperature and the subsequent growth of resulting ice crystals is crucial for the survival of biological organisms (certain insects, fish, and plants that endure subzero temperatures), as well as in the context of medical cryopreservation and food science. In these environments, uncontrolled crystal shape and size can rupture the cell membrane causing irreversible and catastrophic damage. Antifreeze (AF) proteins and synthetic AF analogs address this issue to restrict crystal growth and to shape ice crystals. Yet, if the nucleation temperature is not controlled and occurs in a lower temperature range, nascent ice crystals will have grown to a significantly larger size before the AF agents can be active on their surface to halt or slow the Ostwald ripening process during recrystallization. At a higher nucleation temperature, diffusion of AF macromolecules is enhanced, and dynamic crystal shaping can start earlier, producing smaller crystals overall. While antifreeze proteins, the inspiration for these synthetic analogs, are always applied in a salt buffer aqueous environment (most typically phosphate-buffered saline (PBS) buffer), the heterogeneous nucleation events are stochastic and occur within a wide temperature range. Silver iodide (AgI), however, is a highly effective ice nucleation promoter as its crystal lattice structure is a 98% lattice match to the basal plane of hexagonal ice (Ih) crystals acting as a template for water molecule orientation and decreasing the interfacial free energy. Here, we expose the advantage of purposely seeding such nascent ice crystals with AgI at a defined and higher temperature (-7 °C) in ultrapure water (UPW) such that nucleation can only come from AgI (and also in AgI/PBS), resulting in the most potent synthetic IRI observed to date (at concentrations as low as 0.001 mg·mL-1).

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis.

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in -omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50μg dry TMT per channel was used to label 6-12μg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.

Nanoscopy of single antifreeze proteins reveals that reversible ice binding is sufficient for ice recrystallization inhibition but not thermal hysteresis

Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity.