Over the last twenty years, technological and digital developments such as smartphones and high-speed internet have transformed our daily life. Specifically, fast-paced lifestyle is very common in developed countries, which has increased the demand for the convenience of frozen pre-cooked food. $53 billion exchanged hands in the purchase of frozen food products in the US in 2018, and in 2020, that number grew to $65.1 billion, which was affected by the COVID-19 pandemic, as people accumulated frozen food products. As the demand for frozen foods keeps growing, obtaining high quality food and nutritional benefits drives and will continue driving the development and improvement of frozen food products and the conditions of storage. Understanding the process of freezing (ice formation or nucleation) and ice recrystallization (IR) in food products are of utmost importance to improve the quality of frozen foods. When the temperature of the food product is lowered below the equilibrium melting point of water (0 °C), the water molecules become supercooled, i.e., exist as liquid below the melting point. As the temperature continues to decrease, a small ice nucleus is formed followed by rapid ice growth (freezing). This process leads to the concentration of solutes in the food, structural damage to the food and aggregation of proteins. Upon thawing, the food product may lose its structural integrity and drip loss occurs. IR is a temperature-dependent process driven by Ostwald-ripening in which large crystals grow on the expense of smaller crystals, while the total ice volume is constant. The changes of the ice crystals' size in the food product destroys the structural integrity of the food and its texture. Importantly, IR is affected by temperature fluctuations that might occur in transit, which typically increase the rate of IRand lead to the deterioration of the food quality. Another important factor is the temperature at which frozen food is stored, which is currently -20 °C. Increasing this storage temperature by a few degrees will save energy, limit difficulties in frozen food transportation and even might have, given the large food market, the positive side effect of contributing to reducing global warming.Thus, understanding and elucidating the mechanism by which IR inhibition (IRI) operates is an important task to limit the damages to frozen foods during storage and transit. IRI was demonstrated by natural and synthetic compounds that presumably bind to the ice crystal surface and limit its growth during IR. However, the mechanism of IRI is still unclear. For example, the Davies group found no convincing correlation between the ability of antifreeze proteins (AFPs) to inhibit IRI and their structure or their thermal hysteresis (TH) activity (a quantitative measurement of AFPs' activity that monitors the gap between the melting and freezing temperatures). Gibson's group, who specializes in IRI-active polymer synthesis, suggested that binding to ice may not be needed for IRI. Some IRI active polymers, such as polyvinyl alcohol (PVA), were found to inhibit IRI, but have almost non-existent TH activity. Mechanistic studies have largely focused on the structure-activity relationship of AFPs and synthetic polymers, rather than developing new ways to monitor and image IRI. One possible approach is to label the potential IR inhibitors with fluorescence dyes and monitor the binding process to ice during IR. The PI has pioneered this approach studying the binding mechanism of AFPs to ice. Hagiwara's group recently labeled corn starch molecules with a fluorescence dye and measured IR in the presence of an AFP. However, they were focusing on the aggregation of the labeled starch, rather than the binding of the labeled molecules to the ice surface. The first aim of this proposal is to develop a novel fluorescence IRI measurement that will reveal the binding process of IR inhibitors to ice during the IR process. These insights will finally explain why some IR inhibitors are better than others, and if binding to ice is a requirement of IRI. Next, this project will focus on monitoring IR and ice growth in-situ, i.e., in frozen food products. Direct and real time measurement of ice growth in thick and non-transparent samples is very challenging, and current methodology uses indirect spectroscopy methodsor direct methods such as SEM (scanning electron microscopy)and histology. However, these methods are limited in their capability to quantify the kinetic processes of freezing, such as ice growth rates, ice nucleation rates and IR rates.The second aim of this proposal is to develop a novel approach to measure IR, ice nucleation and ice growth rates in-situ using micro-thermography, which is a technique that uses Infra-red cameras to capture temperature changes in the sample. This approach relies on the release of heat from ice crystallization, which is captured by the Infra-red camera. This method was used previously to study ice growth in plants and in food. However, currently both the temperature control of the system and the spatial and temperature resolution of the Infra-red camera are limited. Overcoming these limitations is possible with the right tools and expertise. The PI is particularly well situated to tackle these problems and, more generally accomplish the stated goals, as his expertise is building cold stages with accurate temperature control (±0.001 ºC, which is an order of magnitude higher compared to the commercially available stages) combined with fluorescence and light microscopy. Using these cold stages (described below) coupled to an Infra-red camera with spatial resolution of 17 μm and temperature resolution of 0.03 ºC, the PI will measure the kinetics of ice growth and nucleation in addition to IR rates in food products. The PI's laboratory is in an undergraduate and female only institution (Stern College), thus if this proposal is funded, it will also promote women in science by providing more students with research opportunities during the semester and summer.The approach of this proposal is to measure and understand the kinetics of IRI in aqueous solutions using fluorescence microscopy, and the kinetics of IR, ice nucleation and growth in frozen food products using micro-thermography (Infra-red based imaging).Understanding the mechanism of IRI and the kinetics of ice growth and nucleation in frozen food will lead to the development of new manufacturing standards of food freezing rates, storage temperatures and the addition of IR inhibitors. These steps address NIFA's Program Area Priority as they will improve the quality of life by limiting the deterioration of frozen food quality during storage, thereby reducing food waste, and will increase storage periods of frozen food products.Objectives: 1. To understand and characterize IR inhibition by various additives.2. To develop a new and reliable imaging method based on micro-thermography that will capture ice growth kinetics in food products.3. To control and limit IR in food products by using additives and/or by changing the conditions in which frozen food is stored.Specific aims: To characterize and understand the effect of adsorption rates on IR inhibition using fluorescence microscopy.To quantify and control IR rates, ice nucleation and ice growth in frozen foods with/without additives using micro-thermography.