State Analysis of a Bio-Based Hydrogel Subjected to Freeze-Thaw Processes by X-Ray Absorption Spectroscopy Using Cyclotron Radiation

State Analysis of a Bio-Based Hydrogel Subjected to Freeze-Thaw Processes by X-Ray Absorption Spectroscopy Using Cyclotron Radiation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article State Analysis of a Bio-Based Hydrogel Subjected to Freeze-Thaw Processes by X-Ray Absorption Spectroscopy Using Cyclotron Radiation Kana Monta, Masafumi Hidaka, Daitaro Ishikawa, Tomoyuki Fujii This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4661461/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Freezing hydrogels can compromise their network structures and modify their properties as a result of ice crystal formation. Therefore, understanding the internal structure, including ice crystals and the state of chemical components within hydrogels, is essential. In this study, we evaluated the elemental distribution in bio-based hydrogels subjected to freezing-thaw process using X-ray absorption spectroscopy with cyclotron radiation. A bio-based hydrogel, prepared from Alaska pollock, underwent both slow and rapid freezing processes. Tomographic images and linear X-ray absorption coefficient distributions of the rapidly frozen hydrogel displayed a uniform image with a mean absorption coefficient of 2.81 cm − 1 . Conversely, the slowly frozen sample exhibited distinct contrasts with peaks at 2.516 cm − 1 (dark) and 3.691 cm − 1 (bright), occupying 28% and 72% of the image, respectively. The mean absorption coefficient of the slowly frozen sample was comparable to that of the rapidly frozen sample, indicating no elemental loss. The elements within the hydrogel were categorized into organic elements, macrominerals, and trace elements. The bright areas in the images were attributed to the concentration of macrominerals. Notably, Cl and Na were the primary contributors to the absorption coefficients among the elements present, signifying salt migration during freezing. These findings suggest that the contrast observed in X-ray computed tomography images after freezing reflects the elemental distribution within the hydrogel and successfully demonstrates element localization due to cryoconcentration. Physical sciences/Chemistry/Analytical chemistry Physical sciences/Optics and photonics/Optical techniques linear X-ray absorption coefficient X-ray computed tomographic image cryoconcentration state analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Hydrogels are cross-linked polymers that form a three-dimensional network capable of absorbing solvents and swelling without dissolving. They are utilized in various applications [ 1 – 6 ] . Within hydrogels, water is confined in the network structure, which restricts the molecular mobility of the water. The structure and properties of water molecules in hydrogels are distinct from those in bulk water because of spatial constraints and interactions with other molecules at the interface. Given that the water retention properties of hydrogels are integral to the properties and biological functions of materials, assessing the water state in hydrogels is vital [ 7 – 10 ] . Typically, when hydrogels with high water content are subjected to freezing, ice crystals form, damaging the network structure [ 11 ] . The size and shape of these ice crystals significantly affect the texture of the final product, which has prompted extensive research on the topic [ 12 , 13 ] . For example, Watanabe et al. experimentally determined that the ice crystalline growth rate is inversely proportional to the average size of the ice crystals [ 14 ] . Furthermore, from a nucleation perspective, higher degrees of supersaturation are associated with the formation of smaller critical crystal nuclei. Simultaneously, when cellular biological materials, such as agricultural products, undergo freezing, osmotic dehydration occurs because of the concentration gradient between the cell interior and exterior, caused by the cryoconcentration (freeze concentration) of solutes in the extracellular space [ 15 ] . In biohydrogels, freeze concentration in the presence of electrolytes alters the gel properties. The mechanism of solute incorporation into the ice phase involves uptake into the interstices of ice crystal grains. Therefore, understanding the distribution of crystal grains, the liquid phase, and the elements within a hydrogel is imperative [ 16 , 17 ] . A previous study on the liquid phase distribution in a frozen sample involved dissolving a small amount of fluorescein in the sample, and the resulting fluorescence indicated the presence of a liquid pool at the ice crystal grain boundaries [ 18 ] . It was hypothesized that a minor liquid phase also resided in the interstices connecting the pools, although it remained undetected. The state of the liquid phase at the ice crystal grain boundaries was observed and assessed using X-ray fluorescence imaging [ 19 ] . X-ray computed tomography (CT) is a nondestructive method for observing the three-dimensional structures of hydrogel materials. In X-ray CT images, material components are discerned based on their differential X-ray absorption rates. With standard laboratory X-ray sources, observing the concentration differences between ice crystals and other substances, including element distribution, is challenging. Hence, evaluations employing monochromatic light from synchrotron radiation are beneficial. Sato et al. explored the potential for understanding ice crystals by assessing the X-ray absorption coefficient during the freezing process of tuna and soybean curd [ 20 ] . Nonetheless, the significance of the contrast in the linear X-ray absorption coefficient remains to be fully elucidated. The linear X-ray absorption coefficient for each element is calculated as the product of the mass absorption coefficient, which is dependent on the X-ray wavelength, and the elemental density. For Cl, which has a mass absorption coefficient of 30, the linear X-ray absorption coefficient for a material with 0.01 g cm − 3 of Cl ions is approximately 0.3 cm − 1 , enabling the visualization of Cl localization at this concentration as a contrast in X-ray CT. This study aims to investigate the distribution of linear X-ray absorption coefficients in bio-based hydrogels subjected to freeze-thaw cycles, to elucidate the cryoconcentration phenomena occurring during the process. Methods Preparation of a Bio-Based Hydrogel The surimi paste of Alaska pollock ( Gadus chalcogrammus ) was used to fabricate the bio-based hydrogel. The Japan Food Research Laboratories measured the weights of the paste components, as presented in Supplementary Table 1. The paste was vacuum-sealed and heated to a central temperature exceeding 95°C for 20 min to form the hydrogel, followed by gradual cooling to room temperature (25°C). The sample underwent slow and rapid freezing at − 25°C using a freezer and liquid nitrogen, respectively, with the cooling rates duly recorded. The frozen samples were stored overnight at − 25°C and subsequently thawed at room temperature (25°C) the next day. This freeze-thaw cycle was repeated four times. Measurement of CT Images Based on X-Ray Absorption Spectroscopy Gel samples, preserved in an icebox with dry ice, were transported to SPring-8. All samples were handled at low temperatures. Upon opening the box in a − 80°C freezer, the sample was sectioned with a saw and shaped into dimensions of 5 × 5 × 20 mm 3 using a chisel, all while encased in dry ice. X-ray CT measurements were conducted at BL14B2 of SPring-8 with X-rays of 1 Å wavelength. The three-dimensional composition of the images was assembled using a beamline. Data comprised 1200 TIFF tomographic images with a resolution of 2.92 µm/px, each pixel featuring a 32-bit gradation. To enhance the signal-to-noise ratio, average binning processing utilized 4 × 4 × 4 voxels. Subsequently, the data was analyzed as image data with a resolution of 11.68 µm/px. Calculation of the Linear X-Ray Absorption Coefficient The linear X-ray absorption coefficient was calculated to assess element concentration. Absorption was conceptualized as the diminution of primary X-ray intensity traversing matter. The intensity, I 0 , of the monochromatic beam diminished exponentially according to the equation: I = I 0 exp(- µx ), (1) where the linear absorption coefficient, µ (cm − 1 ), signifies the mean number of absorption and scattering events encountered by a single photon passing through an absorber of thickness x cm. The linear X-ray absorption coefficient µ was defined by the equation: µ = dΣ pi ( u/p ) I , (2) where d represents the density (g cm − 3 ) of the material, p i is the fractional part (by weight) of the constituent elements of the compound, and the summation extends over all elements. Results and Discussion Tomography of Bio―Based Hydrogels Subjected to Rapid and Slow Freezing Processes Two-dimensional tomographic images and the distribution of linear X-ray absorption coefficients in a hydrogel sample subjected to rapid freezing are shown in Fig. 1 . The image contrast reflects the distribution of linear X-ray absorption coefficients corresponding to each element within the gel samples. A uniform X-ray CT image was obtained from the rapidly frozen sample, characterized by a Gaussian distribution with a singular peak. Insert Fig. 1 The mean distribution of the linear X-ray absorption coefficient, represented by pixel intensity, was 2.81 cm − 1 . The X-ray CT image and histogram of the linear X-ray absorption coefficient are depicted in Fig. 2 . Distinct contrast is observed in the X-ray CT image of the slow-frozen sample. The distribution of the linear X-ray absorption coefficient for the slow-frozen sample separated into two components with peaks at 2.516 and 3.691 cm − 1 from the dark and bright parts of the image, respectively, occupying 28% and 72% of the image. The mean linear X-ray absorption coefficient for the entire sample was 2.848 cm − 1 . Consequently, the mean values of the linear X-ray absorption coefficients for the slow- and quick-frozen samples were similar, at 2.81 cm − 1 , indicating no elemental loss and unchanged total element content. Figure 3 shows the CT image of the hydrogel during thawing, where the X-ray absorption coefficients of the samples subjected to freeze-thaw cycles were uniformly restored. Insert Figs. 2 and 3 Estimation of the Linear X-ray Absorption Coefficient from Elements in the Hydrogel In this study, the elements comprising the hydrogel were categorized for analysis into three groups: “organic and bulk elements” (H, C, N, and O), “macrominerals” (Na, K, Mg, Ca, Cl, P, and S), and “trace elements” (Mn, Fe, Cu, Zn, Se, Co, Mo, and I), following the current classification for elements [ 22 ] . The estimation of the X-ray absorption coefficient for each element in hydrogel samples can be calculated from Eq. (2). Here, the linear X-ray absorption coefficient for the entire sample was estimated to be 1.001 × 2.81 = 2.81 cm − 1 , with 1.001 representing the density ( d ) from Eq. (2). Based on the components of the hydrogel sample in Supplemental Table 2, the occupied volume of each component was calculated using the formula of Choi et al. [ 23 ] . And as shown in Supplemental Table 2, d was calculated as 1.001 g cm − 1 in this study. The estimated linear X-ray absorption coefficient of 2.81 cm − 1 is consistent with that calculated from the histogram of tomography (Figs. 1 and 2 ). Thus, the method of calculating the X-ray absorption coefficient based on elemental analysis was validated. The results in Table 1 indicate that when the element distribution was homogeneous, the linear X-ray absorption coefficients for organic elements and macrominerals were 2.46 and 0.355 cm -1 , respectively. As illustrated in Fig. 2 , in the hydrogel subjected to the slow freezing process, the dark and bright parts of the image accounted for 72% and 28% of the total, respectively. Considering the cryoconcentration of organic elements in the dark and bright parts due to the freezing process, the linear X-ray absorption coefficients are calculated as 2.46/0.28 = 8.8 and 2.46/0.72 = 3.42 cm -1 , respectively. For macrominerals, the estimated linear X-ray absorption coefficients are 0.355/0.28 = 1.27 and 0.355/0.72 = 0.49 cm -1 . Therefore, as shown in Fig. 3 , the contrast in the slow-frozen sample is explained by the distribution of organic elements throughout the sample, while macrominerals are cryoconcentrated, forming the bright part of the image. Consequently, the linear X-ray absorption coefficients estimated by the elements in the hydrogel were 2.46 + 1.27 = 3.73 and 2.46 cm -1 , respectively. These values correspond to the peaks of the linear X-ray absorption coefficient of 3.691 cm -1 for the bright part and 2.516 cm -1 for the dark part of the image. Insert Table 1 It is highly probable that the contrast observed in X-ray CT images of hydrogels subjected to freezing processes visualizes the distribution of macrominerals. As indicated in Table 2 , the predominant contributions to the estimated linear X-ray absorption coefficients were from Cl and Na. Consequently, this contrast is attributed to the migration of salts within the hydrogel during the freeze-concentration process. Furthermore, as depicted in Fig. 4 , the linear X-ray absorption coefficient becomes uniformly distributed after thawing. Thus, if NaCl significantly contributes to this experiment, it is evident that NaCl reverts to a homogeneous state post-thawing. Declarations Acknowledgements This study was financially supported by JSPS KAKENHI [D. Ishikawa, Grant-in-Aid for Scientific Research (C),23K05461]. Author Contributions Kana Monta: Methodology, Research Work, Writing-original draft. Masahumi Hidaka:Research proposal and planning of research work, Research work Daitaro Ishikawa: Methodology, Research Work, Writing draft. Tomoyuki Fujii: Conceptualization, Methodology, Writing, Reviewing. Competing Interests There are no conflicts to declare. Data Availability The data underlying this article will be shared upon reasonable request to the corresponding author. Ethics Declarations This article does not contain any studies with human or animal subjects performed by any of the authors References Maitra, J. A Brief review on cross-linking in hydrogel. Res. Aspects Chem. Mater. Sci. 3 17–31 (2022). Varaprasad, K., Raghavendra, G. M., Jayaramudu, T., Yallapu, M. M. & Sadiku, R. A mini review on hydrogels classification and recent developments in miscellaneous applications. Mater. Sci. Eng. C 79 , 958–971 (2017). Huang, J., Liang, Y., Huang, Z., Xiong, J. & Wang, D. 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FTIR spectroscopy characterization of poly (vinyl alcohol) hydrogel with different hydrolysis degree and chemically crosslinked with glutaraldehyde. Mater. Sci. Eng. C 28 , 539–548 (2008). Miyawaki, O. Analysis and control of ice crystal structure in frozen food and their application to food processing. Food Sci. Technol. Res. 7 , 1–7 (2001). Hagiwara, T., Hayashi, R., Suzuki, T. & Takai, R. Fractal analysis of ice crystals in frozen fish meat. Japan J. Food Eng. 4 , 11–17 (2003). Grenier, J. et al. Mechanisms of pore formation in hydrogel scaffolds textured by freeze-drying. Acta Biomater. 94 , 195–203 (2019). Shi, Y., Geng, J.-T., Yoshida, Y., Jiang, J. & Osako, K. Mechanism study of the gel-forming ability of heat-induced gel from Peruvian hake ( Merluccius gayi peruanus ) surimi. Food Chem. 413 , 135635 (2023). Watanabe, A., Miyawaki, O., Watanabe, M. & Suzuki, T. Mechanism of solute incorporation into ice phase in progressive freeze-concentration. Japan J. Food Eng. 14 , 163–168 (2013). Ohnishi, S. & Miyawaki, O. Osmotic Dehydrofreezing for Protection of Rheological Properties of Agricultural Products from Freezing-Injury. Food Sci. Technol. Res. 11 , 52–58 (2005). Aider, M. & Ounis, W. Ben. Skim milk cryoconcentration as affected by the thawing mode: Gravitational vs. microwave‐assisted. Int. J. Food Sci. & Technol. 47 , 195–202 (2011). Arsiccio, A. & Pisano, R. The ice-water interface and protein stability: A review. J. Pharm. Sci. 109 , 2116–2130 (2020). Hashimoto, T., Tasaki, Y., Harada, M. & Okada, T. Electrolyte-doped ice as a platform for atto- to femtoliter reactor enabling zeptomol detection. Anal. Chem. 83 , 3950–3956 (2011). Tokumasu, K., Harada, M. & Okada, T. X-ray fluorescence imaging of frozen aqueous NaCl solutions. Langmuir 32 , 527–533 (2016). Sato, M., Kajiwara, K. & Sano, N. Non-destructive Three-dimensional Observation of Structure of Ice Grains in Frozen Food by X-ray Computed Tomography Using Synchrotron Radiation. Japan J. Food Eng. 17 , 83–88 (2016). Hubbell, J. H. & Seltzer, S. M. Tables of X-Ray Mass Attenuation Coefficients and Mass Energy-Absorption Coefficients 1 KeV to 20 MeV for Elements Z = 1 to 92 and 48 Additional Substances of Dosimetry Interest . http://dx.doi.org/10.6028/nist.ir.5632 (1995) doi:10.6028/nist.ir.5632. Zoroddu, M. A. et al. The essential metals for humans: A brief overview. J. Inorg. Biochem. 195 , 120–129 (2019). Choi, Y. & Okos, M. R. The thermal properties of tomato juice concentrates. Trans. ASAE 26 , 305–311 (1983). Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementalTables.docx Tables.docx Cite Share Download PDF Status: Published Journal Publication published 05 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 29 Jul, 2024 Reviews received at journal 22 Jul, 2024 Reviews received at journal 20 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviewers agreed at journal 10 Jul, 2024 Reviewers invited by journal 08 Jul, 2024 Editor assigned by journal 08 Jul, 2024 Editor invited by journal 04 Jul, 2024 Submission checks completed at journal 03 Jul, 2024 First submitted to journal 30 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4661461","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":330015312,"identity":"59f2ff03-d044-4ba9-abb2-91a100b83568","order_by":0,"name":"Kana Monta","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Kana","middleName":"","lastName":"Monta","suffix":""},{"id":330015314,"identity":"f9d24e93-2bfa-4c6a-af75-3ca654cfcd33","order_by":1,"name":"Masafumi Hidaka","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Masafumi","middleName":"","lastName":"Hidaka","suffix":""},{"id":330015316,"identity":"4af89c9b-63f0-457f-b497-c00bfe069d9e","order_by":2,"name":"Daitaro Ishikawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIie2RsWoCQRCGZ4ncNRttb0liXuHkKlHwVe6a8zU2zXbpfRKxXBnQZsVWQdAjYKWgTVC4QEaNsXLZMpD9YJdh4WPm3wHweP4imklIQfNXQA1UX4hclMbbKHVVrjea+KbYqI5RwqpcPDNlPqPjoF2HkCZsDu4rwmQSMrXmIZ/0xbvJE+B5Ss/3lVifFImc9Sb96FFhJiMeg1AWZVpQ/BI5LDdr8eWkzKhLGpCiTfDk1EXMilMW5A05SlovKk8CyqJtWarT7oodSuzQKov5VrXrtRCHH8LyY0R4+Ckq5wUGdFBIq/LLw+5asb2j4vF4PP+Cb8WdWa4dQKB8AAAAAElFTkSuQmCC","orcid":"","institution":"Tohoku University","correspondingAuthor":true,"prefix":"","firstName":"Daitaro","middleName":"","lastName":"Ishikawa","suffix":""},{"id":330015317,"identity":"b20307b5-eec4-4734-80d9-e1e7527f08b9","order_by":3,"name":"Tomoyuki Fujii","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Tomoyuki","middleName":"","lastName":"Fujii","suffix":""}],"badges":[],"createdAt":"2024-06-30 06:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4661461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4661461/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-76152-z","type":"published","date":"2024-11-05T15:58:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61197418,"identity":"f835a8dd-9f7e-40bf-9c2d-e94306bc597d","added_by":"auto","created_at":"2024-07-27 00:31:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94271,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Tomography of a hydrogel sample after rapid freezing; (b) Distribution of linear X-ray absorption coefficients (cm\u003csup\u003e-1\u003c/sup\u003e) from panel (a).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4661461/v1/66f9f95ea933633439be308b.png"},{"id":61197421,"identity":"70d30cfd-7152-4414-a813-0438b2a41f63","added_by":"auto","created_at":"2024-07-27 00:31:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":142085,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Tomography of a hydrogel sample after slow freezing; (b) Distribution of linear X-ray absorption coefficients (cm\u003csup\u003e-1\u003c/sup\u003e) from panel (a).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4661461/v1/fd39be9c28303a3daa671c5f.png"},{"id":61198523,"identity":"9f19258b-6666-4b49-ae3f-f0b3ca7413e6","added_by":"auto","created_at":"2024-07-27 00:47:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187980,"visible":true,"origin":"","legend":"\u003cp\u003eTomography of a hydrogel sample post-thaw.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4661461/v1/87419918d7a149c0a054ec01.png"},{"id":61197419,"identity":"96fa11e9-7229-492e-9d08-7fa0af8be9b5","added_by":"auto","created_at":"2024-07-27 00:31:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52481,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the concentrations of organic elements and macrominerals, and the linear X-ray absorption coefficients foreach element.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4661461/v1/09f06cbcc73a287a4684a601.png"},{"id":68750051,"identity":"8a4d6ae7-6475-44f8-916f-f157303be678","added_by":"auto","created_at":"2024-11-11 16:08:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":874541,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4661461/v1/03d5d6f6-a676-4add-b8c1-ddc97d87624c.pdf"},{"id":61198252,"identity":"a41e05f5-ced6-4008-a46d-9bed9f786ca9","added_by":"auto","created_at":"2024-07-27 00:39:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":99114,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4661461/v1/abb549d24bcb8a27868797d9.docx"},{"id":61198253,"identity":"8b4dd56a-d01b-4aac-826b-d7c0a0fc762e","added_by":"auto","created_at":"2024-07-27 00:39:32","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":377881,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4661461/v1/168198def1defc6876d25e89.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"State Analysis of a Bio-Based Hydrogel Subjected to Freeze-Thaw Processes by X-Ray Absorption Spectroscopy Using Cyclotron Radiation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydrogels are cross-linked polymers that form a three-dimensional network capable of absorbing solvents and swelling without dissolving. They are utilized in various applications\u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Within hydrogels, water is confined in the network structure, which restricts the molecular mobility of the water. The structure and properties of water molecules in hydrogels are distinct from those in bulk water because of spatial constraints and interactions with other molecules at the interface. Given that the water retention properties of hydrogels are integral to the properties and biological functions of materials, assessing the water state in hydrogels is vital\u003csup\u003e[\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTypically, when hydrogels with high water content are subjected to freezing, ice crystals form, damaging the network structure\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The size and shape of these ice crystals significantly affect the texture of the final product, which has prompted extensive research on the topic\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. For example, Watanabe et al. experimentally determined that the ice crystalline growth rate is inversely proportional to the average size of the ice crystals\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Furthermore, from a nucleation perspective, higher degrees of supersaturation are associated with the formation of smaller critical crystal nuclei.\u003c/p\u003e \u003cp\u003eSimultaneously, when cellular biological materials, such as agricultural products, undergo freezing, osmotic dehydration occurs because of the concentration gradient between the cell interior and exterior, caused by the cryoconcentration (freeze concentration) of solutes in the extracellular space\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In biohydrogels, freeze concentration in the presence of electrolytes alters the gel properties. The mechanism of solute incorporation into the ice phase involves uptake into the interstices of ice crystal grains. Therefore, understanding the distribution of crystal grains, the liquid phase, and the elements within a hydrogel is imperative\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. A previous study on the liquid phase distribution in a frozen sample involved dissolving a small amount of fluorescein in the sample, and the resulting fluorescence indicated the presence of a liquid pool at the ice crystal grain boundaries\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. It was hypothesized that a minor liquid phase also resided in the interstices connecting the pools, although it remained undetected. The state of the liquid phase at the ice crystal grain boundaries was observed and assessed using X-ray fluorescence imaging\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eX-ray computed tomography (CT) is a nondestructive method for observing the three-dimensional structures of hydrogel materials. In X-ray CT images, material components are discerned based on their differential X-ray absorption rates. With standard laboratory X-ray sources, observing the concentration differences between ice crystals and other substances, including element distribution, is challenging. Hence, evaluations employing monochromatic light from synchrotron radiation are beneficial. Sato et al. explored the potential for understanding ice crystals by assessing the X-ray absorption coefficient during the freezing process of tuna and soybean curd\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Nonetheless, the significance of the contrast in the linear X-ray absorption coefficient remains to be fully elucidated. The linear X-ray absorption coefficient for each element is calculated as the product of the mass absorption coefficient, which is dependent on the X-ray wavelength, and the elemental density. For Cl, which has a mass absorption coefficient of 30, the linear X-ray absorption coefficient for a material with 0.01 g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of Cl ions is approximately 0.3 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, enabling the visualization of Cl localization at this concentration as a contrast in X-ray CT. This study aims to investigate the distribution of linear X-ray absorption coefficients in bio-based hydrogels subjected to freeze-thaw cycles, to elucidate the cryoconcentration phenomena occurring during the process.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of a Bio-Based Hydrogel\u003c/h2\u003e \u003cp\u003eThe surimi paste of Alaska pollock (\u003cem\u003eGadus chalcogrammus\u003c/em\u003e) was used to fabricate the bio-based hydrogel. The Japan Food Research Laboratories measured the weights of the paste components, as presented in Supplementary Table\u0026nbsp;1. The paste was vacuum-sealed and heated to a central temperature exceeding 95\u0026deg;C for 20 min to form the hydrogel, followed by gradual cooling to room temperature (25\u0026deg;C). The sample underwent slow and rapid freezing at \u0026minus;\u0026thinsp;25\u0026deg;C using a freezer and liquid nitrogen, respectively, with the cooling rates duly recorded. The frozen samples were stored overnight at \u0026minus;\u0026thinsp;25\u0026deg;C and subsequently thawed at room temperature (25\u0026deg;C) the next day. This freeze-thaw cycle was repeated four times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of CT Images Based on X-Ray Absorption Spectroscopy\u003c/h2\u003e \u003cp\u003eGel samples, preserved in an icebox with dry ice, were transported to SPring-8. All samples were handled at low temperatures. Upon opening the box in a \u0026minus;\u0026thinsp;80\u0026deg;C freezer, the sample was sectioned with a saw and shaped into dimensions of 5 \u0026times; 5 \u0026times; 20 mm\u003csup\u003e3\u003c/sup\u003e using a chisel, all while encased in dry ice. X-ray CT measurements were conducted at BL14B2 of SPring-8 with X-rays of 1 \u0026Aring; wavelength. The three-dimensional composition of the images was assembled using a beamline. Data comprised 1200 TIFF tomographic images with a resolution of 2.92 \u0026micro;m/px, each pixel featuring a 32-bit gradation. To enhance the signal-to-noise ratio, average binning processing utilized 4 \u0026times; 4 \u0026times; 4 voxels. Subsequently, the data was analyzed as image data with a resolution of 11.68 \u0026micro;m/px.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCalculation of the Linear X-Ray Absorption Coefficient\u003c/h2\u003e \u003cp\u003eThe linear X-ray absorption coefficient was calculated to assess element concentration. Absorption was conceptualized as the diminution of primary X-ray intensity traversing matter. The intensity, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e, of the monochromatic beam diminished exponentially according to the equation:\u003c/p\u003e \u003cp\u003e \u003cem\u003eI\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003eexp(-\u003cem\u003e\u0026micro;x\u003c/em\u003e), (1)\u003c/p\u003e \u003cp\u003ewhere the linear absorption coefficient, \u0026micro; (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), signifies the mean number of absorption and scattering events encountered by a single photon passing through an absorber of thickness x cm. The linear X-ray absorption coefficient \u0026micro; was defined by the equation:\u003c/p\u003e \u003cp\u003e \u003cem\u003e\u0026micro;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003edΣ\u003c/em\u003e\u003csub\u003e\u003cem\u003epi\u003c/em\u003e\u003c/sub\u003e(\u003cem\u003eu/p\u003c/em\u003e)\u003csub\u003e\u003cem\u003eI\u003c/em\u003e\u003c/sub\u003e, (2)\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ed\u003c/em\u003e represents the density (g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) of the material, \u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the fractional part (by weight) of the constituent elements of the compound, and the summation extends over all elements.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTomography of Bio―Based Hydrogels Subjected to Rapid and Slow Freezing Processes\u003c/h2\u003e \u003cp\u003eTwo-dimensional tomographic images and the distribution of linear X-ray absorption coefficients in a hydrogel sample subjected to rapid freezing are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The image contrast reflects the distribution of linear X-ray absorption coefficients corresponding to each element within the gel samples. A uniform X-ray CT image was obtained from the rapidly frozen sample, characterized by a Gaussian distribution with a singular peak.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eThe mean distribution of the linear X-ray absorption coefficient, represented by pixel intensity, was 2.81 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The X-ray CT image and histogram of the linear X-ray absorption coefficient are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Distinct contrast is observed in the X-ray CT image of the slow-frozen sample. The distribution of the linear X-ray absorption coefficient for the slow-frozen sample separated into two components with peaks at 2.516 and 3.691 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from the dark and bright parts of the image, respectively, occupying 28% and 72% of the image. The mean linear X-ray absorption coefficient for the entire sample was 2.848 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Consequently, the mean values of the linear X-ray absorption coefficients for the slow- and quick-frozen samples were similar, at 2.81 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating no elemental loss and unchanged total element content. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the CT image of the hydrogel during thawing, where the X-ray absorption coefficients of the samples subjected to freeze-thaw cycles were uniformly restored.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eInsert Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eEstimation of the Linear X-ray Absorption Coefficient from Elements in the Hydrogel\u003c/h2\u003e \u003cp\u003eIn this study, the elements comprising the hydrogel were categorized for analysis into three groups: \u0026ldquo;organic and bulk elements\u0026rdquo; (H, C, N, and O), \u0026ldquo;macrominerals\u0026rdquo; (Na, K, Mg, Ca, Cl, P, and S), and \u0026ldquo;trace elements\u0026rdquo; (Mn, Fe, Cu, Zn, Se, Co, Mo, and I), following the current classification for elements\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe estimation of the X-ray absorption coefficient for each element in hydrogel samples can be calculated from Eq.\u0026nbsp;(2). Here, the linear X-ray absorption coefficient for the entire sample was estimated to be 1.001 \u0026times; 2.81\u0026thinsp;=\u0026thinsp;2.81 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with 1.001 representing the density (\u003cem\u003ed\u003c/em\u003e) from Eq.\u0026nbsp;(2). Based on the components of the hydrogel sample in Supplemental Table\u0026nbsp;2, the occupied volume of each component was calculated using the formula of Choi et al.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. And as shown in Supplemental Table\u0026nbsp;2, d was calculated as 1.001 g cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in this study. The estimated linear X-ray absorption coefficient of 2.81 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is consistent with that calculated from the histogram of tomography (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, the method of calculating the X-ray absorption coefficient based on elemental analysis was validated.\u003c/p\u003e \u003cp\u003eThe results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicate that when the element distribution was homogeneous, the linear X-ray absorption coefficients for organic elements and macrominerals were 2.46 and 0.355 cm\u003csup\u003e-1\u003c/sup\u003e, respectively. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, in the hydrogel subjected to the slow freezing process, the dark and bright parts of the image accounted for 72% and 28% of the total, respectively. Considering the cryoconcentration of organic elements in the dark and bright parts due to the freezing process, the linear X-ray absorption coefficients are calculated as 2.46/0.28\u0026thinsp;=\u0026thinsp;8.8 and 2.46/0.72\u0026thinsp;=\u0026thinsp;3.42 cm\u003csup\u003e-1\u003c/sup\u003e, respectively. For macrominerals, the estimated linear X-ray absorption coefficients are 0.355/0.28\u0026thinsp;=\u0026thinsp;1.27 and 0.355/0.72\u0026thinsp;=\u0026thinsp;0.49 cm\u003csup\u003e-1\u003c/sup\u003e. Therefore, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the contrast in the slow-frozen sample is explained by the distribution of organic elements throughout the sample, while macrominerals are cryoconcentrated, forming the bright part of the image. Consequently, the linear X-ray absorption coefficients estimated by the elements in the hydrogel were 2.46\u0026thinsp;+\u0026thinsp;1.27\u0026thinsp;=\u0026thinsp;3.73 and 2.46 cm\u003csup\u003e-1\u003c/sup\u003e, respectively. These values correspond to the peaks of the linear X-ray absorption coefficient of 3.691 cm\u003csup\u003e-1\u003c/sup\u003e for the bright part and 2.516 cm\u003csup\u003e-1\u003c/sup\u003e for the dark part of the image.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInsert Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eIt is highly probable that the contrast observed in X-ray CT images of hydrogels subjected to freezing processes visualizes the distribution of macrominerals. As indicated in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the predominant contributions to the estimated linear X-ray absorption coefficients were from Cl and Na. Consequently, this contrast is attributed to the migration of salts within the hydrogel during the freeze-concentration process. Furthermore, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the linear X-ray absorption coefficient becomes uniformly distributed after thawing. Thus, if NaCl significantly contributes to this experiment, it is evident that NaCl reverts to a homogeneous state post-thawing.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by JSPS KAKENHI [D. Ishikawa, \u0026nbsp; \u0026nbsp;\u0026nbsp;Grant-in-Aid for Scientific Research (C),23K05461].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKana Monta: Methodology, Research Work, Writing-original draft. Masahumi Hidaka:Research proposal and planning of research work, Research work Daitaro Ishikawa:\u0026nbsp;Methodology, Research Work,\u0026nbsp;Writing\u0026nbsp;draft. Tomoyuki Fujii: Conceptualization, Methodology, Writing, Reviewing. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data underlying this article will be shared upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human or animal subjects performed by any of the authors\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaitra, J. A Brief review on cross-linking in hydrogel. \u003cem\u003eRes. Aspects Chem. 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A. \u003cem\u003eet al.\u003c/em\u003e The essential metals for humans: A brief overview. \u003cem\u003eJ. Inorg. Biochem.\u003c/em\u003e \u003cstrong\u003e195\u003c/strong\u003e, 120\u0026ndash;129 (2019).\u003c/li\u003e\n\u003cli\u003eChoi, Y. \u0026amp; Okos, M. R. The thermal properties of tomato juice concentrates. \u003cem\u003eTrans.\u003c/em\u003e\u003cem\u003e ASAE\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 305\u0026ndash;311 (1983).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"linear X-ray absorption coefficient, X-ray computed tomographic image, cryoconcentration, state analysis","lastPublishedDoi":"10.21203/rs.3.rs-4661461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4661461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFreezing hydrogels can compromise their network structures and modify their properties as a result of ice crystal formation. Therefore, understanding the internal structure, including ice crystals and the state of chemical components within hydrogels, is essential. In this study, we evaluated the elemental distribution in bio-based hydrogels subjected to freezing-thaw process using X-ray absorption spectroscopy with cyclotron radiation. A bio-based hydrogel, prepared from Alaska pollock, underwent both slow and rapid freezing processes. Tomographic images and linear X-ray absorption coefficient distributions of the rapidly frozen hydrogel displayed a uniform image with a mean absorption coefficient of 2.81 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Conversely, the slowly frozen sample exhibited distinct contrasts with peaks at 2.516 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (dark) and 3.691 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (bright), occupying 28% and 72% of the image, respectively. The mean absorption coefficient of the slowly frozen sample was comparable to that of the rapidly frozen sample, indicating no elemental loss. The elements within the hydrogel were categorized into organic elements, macrominerals, and trace elements. The bright areas in the images were attributed to the concentration of macrominerals. Notably, Cl and Na were the primary contributors to the absorption coefficients among the elements present, signifying salt migration during freezing. These findings suggest that the contrast observed in X-ray computed tomography images after freezing reflects the elemental distribution within the hydrogel and successfully demonstrates element localization due to cryoconcentration.\u003c/p\u003e","manuscriptTitle":"State Analysis of a Bio-Based Hydrogel Subjected to Freeze-Thaw Processes by X-Ray Absorption Spectroscopy Using Cyclotron Radiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-27 00:31:27","doi":"10.21203/rs.3.rs-4661461/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-29T11:38:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-22T07:59:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-20T16:54:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59667503317416649929444711897934599073","date":"2024-07-19T10:40:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135823573039263188203817905911444247163","date":"2024-07-10T09:57:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-08T09:50:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-08T08:17:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-04T21:08:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-03T05:29:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-30T06:21:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"578f3e20-d6b2-4abc-9242-c332dc29a0dc","owner":[],"postedDate":"July 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34930580,"name":"Physical sciences/Chemistry/Analytical chemistry"},{"id":34930581,"name":"Physical sciences/Optics and photonics/Optical techniques"}],"tags":[],"updatedAt":"2024-11-11T16:03:57+00:00","versionOfRecord":{"articleIdentity":"rs-4661461","link":"https://doi.org/10.1038/s41598-024-76152-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-05 15:58:17","publishedOnDateReadable":"November 5th, 2024"},"versionCreatedAt":"2024-07-27 00:31:27","video":"","vorDoi":"10.1038/s41598-024-76152-z","vorDoiUrl":"https://doi.org/10.1038/s41598-024-76152-z","workflowStages":[]},"version":"v1","identity":"rs-4661461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4661461","identity":"rs-4661461","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}