Transformation of water clusters in wet starch under changing environmental conditions

Transformation of the water cluster distribution in wet potato starch (with a water content of 27 to 45%) at temperatures that ranged from–50 to +80°C was studied by differential scanning calorimetry. A significant difference was observed between the transformations in the temperature ranges below and above 0°C. Both cooling and heating at T < 0°C enabled a reorganization of the initial size distribution of water clusters characteristic for room temperature. These changes could lead to an increase of the average cluster size during both crystallization and melting. The transformation intensity depended on the water content and scanning rate and differed between the native and amorphous states of starch. In this case, the cluster-size distribution remained unimodal. However, heating of wet native starch to temperatures close to the point of transition into the amorphous state (75–80°C) induced a bimodal distribution due to the emergence of large water clusters; thus, the heterogeneity of the water distribution within the native granules increased.     

Influence of Microenvironment pH,Humidity, and Temperature on the Stability of Polymorphic and Amorphous Forms of Clopidogrel Bisulfate

The effect of microenvironment pH, humidity, and temperature was evaluated on the stability of polymorphic and amorphous forms of clopidogrel bisulfate, when present alone or in combinations. Oxalic acid and sodium carbonate were used as solid stressors to create acidic and alkaline pH, respectively. The samples without and with stressors were subjected for 3 months to (1) 0% RH, 25% RH, 75% RH, and 85% RH at 40°C and also to (2) 60°C, 80°C, and 100°C at 0% RH. In case of solid samples without stressors, the mixture of polymorphic and amorphous forms showed more degradation than the individual forms above critical relative humidity (85% RH). Similar higher degradation was observed between 75% RH and 85% RH in case of acid-stressed samples. In alkaline microenvironment, all the samples showed identical decomposition attributed to conversion of bisulfate salt to free base. Thermal studies indicated that polymorphic forms of clopidogrel bisulfate and also its glassy amorphous form were highly resistant to temperature, whereas the rubbery state of the drug degraded significantly at temperatures of ≥80°C.     

Influence of amylopectin structure and degree of phosphorylation on the molecular composition of potato starch lintners

Morphology, molecular structure, and thermal properties of potato starch granules with low to high phosphate content were studied as an effect of mild acid hydrolysis (lintnerization) to 80% solubilization at two temperatures (25 and 45°C). Light microscopy showed that the lintners contained apparently intact granules, which disintegrated into fragments upon dehydration. Transmission electron microscopy of rehydrated lintners revealed lacy networks of smaller subunits. The molecular composition of the lintners suggested that they largely consisted of remnants of crystalline lamellae. When lintnerization was performed at 45°C, the lintners contained more of branched dextrins compared to 25°C in both low and intermediate phosphate‐containing samples. High‐phosphate‐containing starch was, however, unaffected by temperature and this was probably due to an altered amylopectin structure rather than the phosphate content. After lintnerization, the melting endotherms were broad with decreased onset and increased peak melting temperatures. The relative crystallinity was lower in lintners prepared at 45°C. A hypothesis that combines the kinetics of lintnerization with the molecular and thermal characteristics of the lintners is presented. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 257–271, 2014.     

Thermal behavior of a pharmaceutical solid acetaminophen doped withp-aminophenol

Thermal behavior of a series of acetaminophen (APAP) doped withp-aminophenol (PANP) was studied by differential scanning calorimetry (DSC) to determine whether it exhibited a eutectic system. Within the temperature range of 120 to 200° C, accurately weighed (1–2 mg) samples sealed in hermetic pans were calorimetrically scanned with a low scanning rate of 1° C/min. The mixture formed a single eutectic with the composition ratio APAP/PANP of 0.6/0.4 at a temperature of 138° C, where it liquefied. Melting began as early as at the eutectic point, which was below the melting temperature of APAP (169° C). The melting point as well as heat of APAP fusion was depressed with the increase in doped PANP. It was postulated that there might be a deficit heat of APAP fusion in APAP doped with PANP, which was coincident with the heat consumed by early liquefaction. The deficit heat was used to correct fraction molten in the van’t Hoff law of purity determination. It was found that the purity determination of APAP doped with PANP was comparable to the UV-spectroscopic method up to the maximum doped PANP level of 8 mol percent. It was concluded that DSC was able to approach early heat of liquefaction of APAP doped with PANP. The van’t Hoff law may be applicable to the determination of APAP with the presence of PANP as a eutectic impurity.     

An examination of the moisture sorption characteristics of commercial magnesium stearate

The objective of this study was to characterize the moisture sorption of magnesium stearate and the morphological changes, if any, resulting from moisture sorption. Six samples of commercial magnesium stearate USP were examined. Moisture sorption isotherms were obtained at 25°C and 5% to 98% relative humidity (RH) using a moisture balance. Changes in crystal form resulting from moisture sorption were determined by x-ray diffraction. There were differences in the shape of the isotherm, reversibility of moisture uptake, and shape of the hysteresis loop in the isotherms of crystalline and amorphous magnesium stearates. The isotherm of crystalline magnesium stearate was almost parallel to the pressure axis until and RH of ∼80%. The isotherm of the amorphous sample was characterized by continuous uptake of water over the entire range of RH. Exposure of amorphous magnesium stearate to RH greater than 70% resulted in the formation of the trihydrate. The trihydrate was converted into the anhydrous form when heated to a temperature of 100°C to 105°C. The trihydrate could be generated by exposing the anhydrate to RH higher than 70%.     

Investigation of molecular motion in collagen using the spin-probe technique.

The spin-probe technique was employed to study molecular motion in collagen and modified collagen samples in the ?160° to +200°C region. The effect of water content in the 0–30-wt. % range, relative to vacuum dried samples, on the electron spin resonance spectrum of the probe was also investigated. The spectra at the lowest temperatures consisted of a broad asymmetric triplet. Narrowing of this triplet above ?40° to ?70°C and the appearance of additional lines in the spectrum, interpreted as due to a narrow triplet, at a temperature dependent on the water content were observed. For samples with low water contents [(0–0.4)%] the broad triplet was that expected for a glassy system up to 150–190°C; for these samples the narrow triplet appears at a temperature above 50°C, its intensity increasing with increasing temperature up to 70–100°C, then decreasing with a further temperature increase. For samples with water contents near 30%, the narrow triplet first completely appears at about 0°C, and reaches relative intensities of 35% at 30°C. The motion taking place in collagen and related samples is discussed in terms of these results.     

Glass transition temperatures and water sorption isotherms of cassava starch

The effect of water content on the glass transition temperatures of cassava starch was determined by differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMTA). Samples were transformed to the amorphous state by compression molding at high temperature (as demonstrated by wide angle X-ray diffraction, WAXS), and then the samples were moisture conditioned. Both DSC and DMTA showed that water anti-plasticized cassava starch at lower moisture contents, and plasticized it at higher water contents. Samples with higher moisture contents stored at room temperature, 45 °C and 80 °C underwent retrogradation as indicated by WAXS. Sorption isotherms of cassava starch showed that for a_w values lower than around 0.85, the sorption capacity decreased with increasing temperature; while the opposite behavior was observed at a_w > 0.85. This inversion point (a_w = 0.85) was attributed to the fact that more active sites were exposed to the adsorption processes, due to the enhanced molecular mobility promoted in the amorphous regions by starch crystallization.     

Effect of temperature and pH on the kinetics of methane production, organic nitrogen and phosphorus removal in the batch anaerobic digestion process of cattle manure

A study of the effect of temperature and pH on the kinetics of methane production and organic nitrogen and phosphorus degradation in the anaerobic digestion process of cattle manure was carried out. Two laboratory-scale batch completely mixed reactors, operating at 35v°C (mesophilic temperature), and other two, operating at 60v°C (thermophilic temperature) were used. For each temperature selected, the influent pH values were 7.6 (initial pH of the waste used) and 7.0. The apparent kinetic constants of the biomethanization process increased 2.3 times when the initial pH of the influent was increased from 7.0 to 7.6 at mesophilic temperature. The values found at thermophilic temperature were similar. The kinetic constants of methane production decreased 2.6 and 7.2 times when the operating temperature increased from 35 °C to 60v°C for the experiments carried out at initial pH of 7.0 and 7.6, respectively. The methane yield coefficient (l CH₄ STP/g VS removed) also decreased when the temperature increased from 35v°C to 60v°C for the two initial pH values studied. This behaviour agreed with the major inhibition level observed at thermophilic temperature as a result of the higher organic nitrogen removal and ammonia nitrogen production observed at 60v°C. Specifically, the specific rate constants for organic nitrogen removal and ammonia nitrogen production increased 3.6 and 12 times when the temperature was increased from 35v°C to 60v°C for the experiments carried out at initial pH values of 7.0 and 7.6, respectively. In the same way, the values of the kinetic constant for phosphorus removal were 44% and 80% higher than those obtained at 35v°C for the two initial pH values above-mentioned, respectively. Finally, the experimental values of organic nitrogen and phosphorus concentrations were reproduced with deviations equal to or less than 10% and 15% in every case, respectively.     

The Physical and Morphological Characteristics of Mango Powder (<Emphasis Type="Italic">Mangifera indica</Emphasis> L. cv Tommy Atkins) Produced by Foam Mat Drying

The thermal behavior, moisture adsorption properties and structural and morphological characteristics of mango powders were evaluated. The powders were obtained by foam mat drying methodology using albumin (ALB), mixture (EB) of monoglycerides of fatty acids, sorbitan monostearate and polyoxyethylene sorbitan monostearate and a combination of the two (EB-ALB) as foaming agents. The evaluation was done by differential scanning calorimetry (DSC), X-ray diffraction (XRD) and scanning electron microscopy (SEM), and the moisture adsorption isotherms were also determined. The powder with EB had a glass transition temperature (T_g) of ?4.2 °C. The denaturation temperature of pure albumin (82.2 °C) increased in the powders with ALB to 117 °C and in those with EB-ALB to 102 °C, due to the thermal stability provided by the pulp sugars. The moisture sorption isotherm of the EB-powder showed a higher water equilibrium content than the other powders. All the powders were in the amorphous state. The morphology of the powder with EB showed corrugated particles, whereas those with ALB and EB-ALB showed particles with a less porous aspect and more compact surfaces than the powders with EB.     

Melting curves on less than 1 μg of nucleic acid

Two types of microcuvettes were developed for the registration of melting curves in normal uv-spectrophotometers. Nucleic acid (0.5–1 μg) is required to determine changes in absorption with an accuracy of 0.1 mA. The quartz cuvette allows the registration of equilibrium melting curves up to 100°C. The other type of cuvette made from gilded brass allows to measure slow temperature jumps up to 80°C and equilibrium melting curves up to 70°C. The time range of ～3 s to several hours is accessible complementing the Eigen/De Mayer temperature-jump technique (1963, in Techniques of Organic Chemistry, Vol. VIII/2, pp. 895–1054, Wiley, New York). Temperature jumps of high amplitude as well as jumps to lower temperatures are possible. Examples of measurements on viroids and double-stranded viral RNA are presented.     

Comparative differential scanning calorimetric analysis of vegetable oils: I. Effects of heating rate variation

The melting curves of 11 vegetable oils have been characterised. Vegetable oil samples that were cooled at a constant rate (5 degrees C/min) from the melt showed between one and seven melting endotherms upon heating at four different heating rates (1, 5, 10 and 20 degrees C/min) in a differential scanning calorimeter (DSC). Triacylglycerol (TAG) profiles and iodine value analyses were used to complement the DSC data. Generally, the melting transition temperature shifted to higher values with increased rates of heating. The breadth of the melting endotherm and the area under the melting peak also increased with increasing heating rate. Although the number of endothermic peaks was dependent on heating rate, the melting curves of the oil samples were not straightforward in that there was no correlation between the number of endothermic peaks and heating rates. Multiple melting behaviour in DSC experiments with different heating rates could be explained by: (1) the melting of TAG populations with different melting points; and (2) TAG crystal reorganisation effects. On the basis of the corollary results obtained, vegetable oils and fats may be distinguished from their offset-temperature (Toff) values in the DSC melting curves. The results showed that Toff values of all oil samples were significantly (p < 0.01) different in the melting curves scanned at four different scanning rates. These calorimetric results indicate that DSC is a valuable technique for studying vegetable oils.     

Melting, glass transition, and apparent heat capacity of α-d-glucose by thermal analysis

The thermal behaviors of α-d-glucose in the melting and glass transition regions were examined utilizing the calorimetric methods of standard differential scanning calorimetry (DSC), standard temperature-modulated differential scanning calorimetry (TMDSC), quasi-isothermal temperature-modulated differential scanning calorimetry (quasi-TMDSC), and thermogravimetric analysis (TGA). The quantitative thermal analyses of experimental data of crystalline and amorphous α-d-glucose were performed based on heat capacities. The total, apparent and reversing heat capacities, and phase transitions were evaluated on heating and cooling. The melting temperature (T_m) of a crystalline carbohydrate such as α-d-glucose, shows a heating rate dependence, with the melting peak shifted to lower temperature for a lower heating rate, and with superheating of around 25 K. The superheating of crystalline α-d-glucose is observed as shifting the melting peak for higher heating rates, above the equilibrium melting temperature due to of the slow melting process. The equilibrium melting temperature and heat of fusion of crystalline α-d-glucose were estimated. Changes of reversing heat capacity evaluated by TMDSC at glass transition (T_g) of amorphous and melting process at T_m of fully crystalline α-d-glucose are similar. In both, the amorphous and crystalline phases, the same origin of heat capacity changes, in the T_g and T_m area, are attributable to molecular rotational motion. Degradation occurs simultaneously with the melting process of the crystalline phase. The stability of crystalline α-d-glucose was examined by TGA and TMDSC in the melting region, with the degradation shown to be resulting from changes of mass with temperature and time. The experimental heat capacities of fully crystalline and amorphous α-d-glucose were analyzed in reference to the solid, vibrational, and liquid heat capacities, which were approximated based on the ATHAS scheme and Data Bank.     

Melting, glass transition, and apparent heat capacity of α-D-glucose by thermal analysis

The thermal behaviors of α-D-glucose in the melting and glass transition regions were examined utilizing the calorimetric methods of standard differential scanning calorimetry (DSC), standard temperature-modulated differential scanning calorimetry (TMDSC), quasi-isothermal temperature-modulated differential scanning calorimetry (quasi-TMDSC), and thermogravimetric analysis (TGA). The quantitative thermal analyses of experimental data of crystalline and amorphous α-D-glucose were performed based on heat capacities. The total, apparent and reversingheat capacities, and phase transitions were evaluated on heating and cooling. The melting temperature (T(m)) of a crystalline carbohydrate such as α-D-glucose, shows a heating rate dependence, with the melting peak shifted to lower temperature for a lower heating rate, and with superheating of around 25K. The superheating of crystalline α-D-glucose is observed as shifting the melting peak for higher heating rates, above the equilibrium melting temperature due to of the slow melting process. The equilibrium melting temperature and heat of fusion of crystalline α-D-glucose were estimated. Changes of reversing heat capacity evaluated by TMDSC at glass transition (T(g)) of amorphous and melting process at T(m) of fully crystalline α-D-glucose are similar. In both, the amorphous and crystalline phases, the same origin of heat capacity changes, in the T(g) and T(m) area, are attributable to molecular rotational motion. Degradation occurs simultaneously with the melting process of the crystalline phase. The stability of crystalline α-D-glucose was examined by TGA and TMDSC in the melting region, with the degradation shown to be resulting from changes of mass with temperature and time. The experimental heat capacities of fully crystalline and amorphous α-D-glucose were analyzed in reference to the solid, vibrational, and liquid heat capacities, which were approximated based on the ATHAS scheme and Data Bank.     

Optimization of the spray drying of a Paenibacillus polymyxa-based biopesticide on pilot plant and production scales

Spore survival and moisture content are two important properties of biopesticides, and both are related to field biocontrol efficacy and storage shelf life. In this study, Paenibacillus polymyxa (HY96-2) was spray-dried on both pilot plant and production scales, and the effects of inlet and outlet temperatures on spore survival and moisture content were investigated. The results showed that inlet temperatures ranging from 170 to 230 °C (at an outlet temperature of 80 °C) had no obvious effect on the two properties during pilot scale processing, although an inlet temperature of 230 °C resulted in higher feed speed. When the outlet temperature on the pilot scale was reduced from 100 to 80 °C, no obvious variations in spore survival and moisture content were found, while a further reduction from 80 to 65 °C resulted in a decline in spore survival from 81.0 to 67.0% and an increase in moisture content from 2.3 to 31.7%. These results indicate that both outlet temperature and moisture content have an effect on spore survival. Optimum inlet and outlet temperatures for P. polymyxa processing were 230 °C and 85–90 °C on a production scale. Under these conditions, spore survival and moisture content were 83.5–86.6% and 2.73––4.12%, respectively.     

Physicochemical stability of cimetidine amorphous forms estimated by isothermal microcalorimetry

The effect of humidity on the physicochemical properties of amorphous forms of cimetidine was investigated using differential scanning calorimetry, isothermal microcalorimetry, and x-ray diffraction analysis. Amorphous forms were obtained by the melting (amorphous form M [AM]) and the cotton candy (amorphous form C [AC]) methods. Thermal behaviors of AM and AC with or without seed crystals were measured using an isothermal microcalorimeter under various conditions of relative humidity (RH) and temperature, respectively. The crystallization kinetics of amorphous solids was analyzed based on 10 kinds of solid-state reaction models. AM transformed into form A at 11% RH, 50°C but transformed into a mixture of form A and monohydrate at 51% and 75% RH at 25°C. The mean crystallization times (MCTs) of the heat flow curve of AM and AC at 11% RH, 50°C were 47.82 and 32.00 hours, respectively, but at 11% RH, 25°C both were more than 4320 hours. In contrast, AC transformed into form A under all storage conditions. The MCTs of AC at 51% and 75% RH were 29.61 and 11.81 hours, respectively; whereas the MCTs of AM were 46.79 and 15.52 hours, respectively. The crystallization of amorphous solids followed the three-dimensional growth of nuclei (Avrami equation) with an induction period (IP). The IP for AM at 11% RH, 50°C was more than 2 times that for AC, but the difference in the crystal growth rate constant (CR) between AC and AM was within 10%. The IP for AM at 75% RH, 25°C was reduced to only 10% of the IP at 51% RH with increasing humidity, but the CR did not change significantly. In contrast, the IP for AC was slightly reduced at 75% RH compared with 51% RH, but the CR was about 5 times greater. At 75% RH, 25°C, the IP and CR of AM were about one-fourth the values of AC. This result suggests that the crystallization process consists of an initial stage during which the nuclei are formed and a final stage of growth.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

Amorphous Solid Dispersions of Sulfonamide/Soluplus® and Sulfonamide/PVP Prepared by Ball Milling

The aim of this paper is to investigate the physicochemical properties of binary amorphous dispersions of poorly soluble sulfonamide/polymeric excipient prepared by ball milling. The sulfonamides selected were sulfathiazole (STZ), sulfadimidine (SDM), sulfamerazine (SMZ) and sulfadiazine (SDZ). The excipients were polyvinylpyrrolidone (PVP) and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft co-polymer, commercially known as Soluplus®. Co-milled systems were characterised by powder X-ray diffraction and differential scanning calorimetry. PVP was shown to form amorphous dispersions over a wider composition range than Soluplus® for the four sulfonamides tested. Moreover, amorphous dispersions made with PVP were homogeneous [single glass transition (Tg)], while amorphous dispersions made from Soluplus® were heterogeneous (two Tgs). This behaviour is consistent with the fact that all the sulfonamides tested presented a lower solubility in Soluplus® than in PVP, as evidenced by Flory–Huggins parameters determined. Amorphous dispersions of SDM with Soluplus® could be produced even though SDM does not amorphise alone upon milling and Soluplus® presents Tg at a lower temperature than SDM. Amorphous dispersions of SMZ could be prepared with a lower excipient concentration compared to STZ, SDM and SDZ, which may reflect the one-dimensional H-bonding network in SMZ compared to the 2D or 3D H-bonding network found in the other sulfonamides. Stability tests (60% RH/25°C) revealed that dispersions made with Soluplus® remained dry and powdery compared to those made with PVP that formed a sticky paste in less than 2 weeks, indicating a possible advantage of using Soluplus® in terms of increased physical stability under high humidity storage conditions.     

Temperature-induced transitions in the structure and interfacial rheology of human meibum

Meibomian lipids are the primary component of the lipid layer of the tear film. Composed primarily of a mixture of lipids, meibum exhibits a range of melt temperatures. Compositional changes that occur with disease may alter the temperature at which meibum melts. Here we explore how the mechanical properties and structure of meibum from healthy subjects depend on temperature. Interfacial films of meibum were highly viscoelastic at 17°C, but as the films were heated to 30°C the surface moduli decreased by more than two orders of magnitude. Brewster angle microscopy revealed the presence of micron-scale inhomogeneities in meibum films at higher temperatures. Crystalline structure was probed by small angle x-ray scattering of bulk meibum, which showed evidence of a majority crystalline structure in all samples with lamellar spacing of 49 Å that melted at 34°C. A minority structure was observed in some samples with d-spacing at 110 Å that persisted up to 40°C. The melting of crystalline phases accompanied by a reduction in interfacial viscosity and elasticity has implications in meibum behavior in the tear film. If the melt temperature of meibum was altered significantly from disease-induced compositional changes, the resultant change in viscosity could alter secretion of lipids from meibomian glands, or tear-film stabilization properties of the lipid layer.     

Devitrification of amorphous celecoxib

The purpose of this research was to analyze the devitrification of amorphous celecoxib (CEL) in the presence of different stressors (temperature, pressure, and/or humidity) encountered during processing of solid dosage forms. Amorphous CEL was prepared in situ in the analytical instruments, as well as in laboratory, by quench-cooling of melt process, and analyzed by dynamic mechanical thermal analysis, differential scanning calorimetry, microscopy, and Fourier-transform infrared spectroscopy. Amorphous CEL prepared in situ in the analytical instruments was resistant to crystallization under the influence of temperature and/or pressure, because of its protection from the external environment during preparation. These samples exhibited structural relaxation during annealing at 25°C/0% relative humidity (RH) for 16 hours. Generation of amorphous CEL in the laboratory resulted in partially crystalline samples, because of exposure to environmental temperature and humidity, resulting in incomplete vitrification. Subjection to thermal stress favored crystallization of amorphous CEL into metastable polymorphic forms, which were not obtained by solvent recrystallization approach. Temperature and humidity were identified as the major factors promoting devitrification of amorphous CEL, leading to loss of solubility advantage. Exposure to International Conference on Harmonization-specified accelerated stability storage conditions (40°C/75% RH) resulted in complete devitrification of amorphous CEL within 15 days. The phase-transformation process of amorphous CEL along the temperature scale was examined visually, as well as spectrally. This propensity for devitrification of amorphous CEL seemed to depend on the strength of differential molecular interactions between the amorphous and crystalline form.     

High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.