High Freezing Temperature Research Articles

3D-microstructure analysis of hydrated bentonite with cryo-stabilized pore water

In saturated bentonite, free water is hosted in the meso- and macropores. Microscopic characterization of free water and the associated pore structure is very difficult because of the swelling- and shrinking-behaviour of montmorillonite. In this article, we present state of the art cryo-preparation techniques including high pressure freezing and low temperature freeze substitution, which enable the stabilization of bentonite microstructures. Microscopic analyses of cryo-stabilized bentonite samples are then performed with conventional SEM, with cryo-SEM and with FIB-nanotomography. From the resulting 2D- and 3D-images, so-called “continuous pore size distributions” are calculated and the 3D-connectivities of the mesopores are documented. Furthermore, from the comparison with pore size analyses that are based on conventional preparation techniques (oven drying and freeze drying), it is shown that high pressure freezing leads to more reliable results. Overall, it is demonstrated that reliable quantitative 3D-characterization can be achieved from the bentonite pore structure when high resolution 3D-imaging by FIB-nanotomography is combined with modern cryo-preparation techniques (i.e. high pressure freezing).

Hydrogen Clusters that Remain Fluid at Low Temperature

Superfluidity of hydrogen, predicted three decades ago, continues to elude experimental observation, due to the high freezing temperature of H2 at T=13.8 K. Here, large para-H2 clusters are obtained in a cryogenic pulsed free jet expansion and are studied via nonlinear Raman spectroscopy. Clusters formed from neat pH_{2} are solid as evidenced by the characteristic splitting of the rotational S0(0) line. However, clusters formed of highly diluted pH_{2} (< or = 1%) in He have a single S0(0) line and remain liquid at the estimated superfluid transition temperature of T=1-2 K.

The substitution effect of boron on reentrant behavior of rapidly solidified Fe–Mn–Zr alloys

We have studied the magnetic properties of boron-substituted amorphous reentrant Fe–Mn–Zr alloys. The magnetic characterization of a-Fe 82Mn 8− x B x Zr 10 (numbers indicate at.%) alloys has been done through the studies of thermal variation of magnetization, ac susceptibility and ferromagnetic resonance. Low-temperature spin freezing was observed at 54 K with large coercivity and low magnetic moment for a-Fe 82Mn 8Zr 10. The large coercivity at low temperatures and high freezing temperature for this sample is attributed to magnetic hardening. With an increase of the concentration of B, it is observed that the Curie temperature increases and the spin-glass-like transition observed at low temperature moves to lower temperatures and finally vanishes at x = 8 at.%. A single resonance peak in FMR spectrum was observed below T C and at least two distinct line shapes were observed well beyond the T C for two of the compositions, namely, x = 4 and 6. These observations could be qualitatively understood in terms of a finite spin clusters plus an infinite ferromagnetic matrix model. It can also be concluded that the magnetic disorder is reduced as boron concentration is increased.

A random-field model for polarization reversal in Pb(Yb1/2Nb1/2)O3–PbTiO3 single crystals

Polarization switching and dielectric temperature behavior have been investigated in 0.55Pb(Yb1/2Nb1/2)O3–0.45PbTiO3 (PYN–PT) single crystals. These compositions are of interest owing to their unusually high Curie temperature and high coercive fields. The dielectric permittivity was determined as function of temperature and the polarization was measured as a function of applied field for different drive frequencies. Analysis of the experimental results with a random-field model was found to show similar trends to recent reports for the Pb(Mg1/3Nb2/3)O3 relaxor materials. The frequency dispersion of the maximum in dielectric spectra, Tmax, was also analyzed with the Vogel–Fulcher relation yielding a high freezing temperature of 577 K. Polarization behavior was found to follow a stretched exponential time dependence and a fractal dimension of ∼2.2 was obtained. It is proposed that the polar cluster state in PYN-PT single crystals do not freeze in random orientations but rather arrange locally in preferred configurations. The fraction of the clusters decreases under increasing external field drive, resulting in the formation of the percolated network at higher fields.

Thaw effects on cold-hardiness parameters in yellow birch

One-year-old, cold-hardened, container-grown yellow birch (Betula alleghaniensis Britt.) seedlings were exposed to cold treatments after being pretreated with a simulated winter thaw. Freezing injury to roots and shoots was assessed by relative electrolyte leakage and triphenyltetrazolium chloride reduction. Growth characteristics were also determined after 60 days under greenhouse conditions. Relative electrolyte leakage and triphenyltetrazolium chloride reduction measurements showed that roots became increasingly damaged with decreasing cold-treatment temperatures. However, plants pretreated with thaws showed significantly lower stem increment, shoot length, and leaf area in response to the cold temperatures than did the unthawed plants. Variation in these growth parameters was also significantly correlated with both root and shoot freezing injury parameters. Cold hardiness under different thaw pretreatments was assessed using the highest freezing temperature that caused significant injury, referred to as the critical temperature. For seedlings without the thaw pretreatment, shoot and root critical temperatures were estimated as –52.5 and 23.8°C, respectively. Following 12 days of thaw, these temperatures increased to –24.08°C for shoots and –13°C for roots. Twelve days of thaw, or growing degree-day (&gt;4°C) accumulations greater than 66 during a thaw, could sufficiently deharden roots and shoots such that they would be susceptible to freezing damage at ambient temperatures commonly encountered in the Canadian Maritimes. We also observed that root pressure declined significantly with increasing root freezing injury. Sufficient root pressure is required for springtime refilling of xylem embolisms caused by winter cavitation of the vessels in this species. Weak root pressure caused by freezing injury would represent a risk of shoot dieback and tree decline due to the remaining embolisms reducing water flow to the developing foliage. The rapid reduction of shoot cold hardiness may also indicate the threat of late-spring frosts to this species. These induced changes are especially important under climate change scenarios that suggest increases in winter temperatures and changes in seasonality in eastern Canada.Key words: climate change, cold hardiness, electrolyte leakage, growth, root pressure, TTC reduction.

Ultrastructure of hypertrophic cartilage: Histochemical procedures compared with high pressure freezing and freeze substitution

The effect of cationic dyes on the ultrastructure of hypertrophic cartilage was compared with results obtained with modern cryotechniques in studies on rat epiphyseal growth plate. Addition of alcian blue, acridine orange, cupromeronic blue, ruthenium hexamine trichloride, ruthenium red, or safranin O to conventional glutaraldehyde/osmium tetroxide fixatives to a large extent resulted in prevention of chondrocyte shrinkage except for alcian blue which showed poor tissue penetration. The fine structure of the matrix in pericellular and territorial compartments appeared very coarse with areas of high contrast in tissue exposed to fixatives containing cationic dyes. This indicates structural collapse and precipitation of electron-dense material, a pattern clearly differing from that observed in specimens prepared by the cryotechniques. The dyes giving a pattern most similar to that seen after high pressure freezing, freeze substitution, and low temperature embedding were acridine orange and safranin O. It is concluded that studies of matrix ultrastructure down to the molecular level necessitate the application of cryotechniques.

Identification of glycoproteins specific to biotrophic intracellular hyphae formed in the Colletotrichum lindemuthianum-bean interaction.

Monoclonal antibodies (MAbs) specific for intracellular for hyphae (IH, i.e. infection vesicles and primary hyphae). Appressoria/germ tubes and conidia of Colletotrichum lindemuthianum (Sace, & Magn.) Briosi & Cav. isolated from infected leaves of Phaseolus vulgoria L. were obtained using a co-immunization procedure. One of the MAhs; UB25, bound specifically to IH in immunofluorescence immunogold and Western blot assays: it showed no affinity for conidia, conidial germ tubes, appressoria or appressorial germ tubes growing in vitro, of for any plant components. Immunogold labeling of infected tissue prepared by high pressure freezing, freeze-substitution and low temperature embedding showed that the UB25 antigen was present in the interfacial matri surrounding IH and in the fungal wall. The antigen was confined to infection vesicles and primary hyphae in contact with host protoplast and could not be detected in primary hyphae growing in intercellular spaces. UB25 recognizes a protein epitope present in a set of N-linked glycoproteins. These glycoproteins are expressed at an early stage of intracellular development, suggesting a possible role in biotrophy or recognition.

Spin-Glass Behavior in Co-Ferrite Amorphous Films

Amorphous Co-ferrite thin films not containing vitrifying elements were prepared by ion beam sputter deposition. Their magnetic properties were investigated by means of low-field dc magnetization and ac susceptibility measurements, which gave evidence of spin glass behavior. The temperature dependence of magnetization in a 50 Oe external field exhibited an irreversibility just below room temperature, which decreased with increasing field strength. A cusp in the ac susceptibility (80 Hz, 1 Oe) corresponding to the spin freezing temperature was observed at 284 K. Below this temperature, relaxation of the thermoremanent magnetization was observed. Besides this transition at 284 K, a sharp peak in the ac susceptibility at 86 K, suggestive of another transition, was observed. It is thought that spin glass ordering with a high freezing temperature is caused mainly by transition to an amorphous structure for strong superexchange interactions.

Collagen-based wound dressings: control of the pore structure and morphology.

Collagen-based sponges have been used as both temporary and permanent coverings for dermal defects in animals and humans. Cellular ingrowth within such a sponge has been shown to depend on the porosity and the presence of fibrous structure. Collagen sponges were made by freezing and freeze-drying dispersions under acidic conditions. These studies involved the effects of dispersion pH and viscosity as well as freezing temperature on the surface and bulk morphology of collagen-based sponges. Using scanning electron and light microscopy, the results of these studies indicated that large surface pores that form connections (channels) with the interior of the sponge were formed using low-viscosity collagen dispersions. At high dispersion pH (3.2) and at a moderate freezing temperature (-30 degrees C), fibrous structure and a large number of channels were present. When a lower dispersion pH (2.0) and freezing temperature (-80 degrees C) were used, pores sizes were smaller with channels and fibrous structure, whereas a higher freezing temperature (-20 degrees C) resulted in a sheet-like structure and increased pore sizes. Differences in pore size and surface morphology were explained on the basis of ice crystal growth. In the case of abundant free water (high pH) and high freezing temperature, the pore size was greatest because of enhanced ice crystal growth.

Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure - preservation of proteoglycans in their native state.

The extracellular matrix of epiphyseal cartilage tissue was preserved in a state believed to resemble closely that of native tissue following processing by high pressure freezing, freeze substitution, and low temperature embedding (HPF/FS). Proteoglycans (PG) were preserved in an extended state and were apparent as a reticulum of fine filamentous threads throughout the matrix. Within this network, two morphologically discrete components were discernible and identified with the carbohydrate and protein components of PG molecules. Numerous points of contact were clearly visible between components of the PG network and cross-sectioned collagen fibrils and also between PG components and chondrocytic plasmalemmata. These observations provide direct morphological indication that such relationships may exist in native epiphyseal cartilage tissue.

Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure--implications for the theories of mineralization and vascular invasion.

Electron microscopic examination of epiphyseal cartilage tissue processed by high pressure freezing, freeze substitution, and low temperature embedding revealed a substantial improvement in the preservation quality of intracellular organelles by comparison with the results obtained under conventional chemical fixation conditions. Furthermore, all cells throughout the epiphyseal plate, including the terminal chondrocyte adjacent to the region of vascular invasion, were found to be structurally integral. A zone of degenerating cells consistently observed in cartilage tissue processed under conventional chemical fixation conditions was not apparent. Hence, it would appear that cell destruction in this region occurs during chemical processing and is not a feature of cartilage tissue in the native state. Since these cells are situated in a region where tissue calcification is taking place, the implication is that the onset and progression of cartilage calcification are, at least partially, controlled by the chondrocytes themselves. The observation that the terminal cell adjacent to the zone of vascular invasion is viable has important implications in relation to the theory of vascular invasion. This may now require reconceptualization to accommodate the possibility that active cell destruction may be a precondition for vascular invasion.

Fluid inclusion studies in the northern Arkansas zinc district

Many zinc-lead deposits of the Mississippi Valley type occur in northern Arkansas along the southern flank of the Ozark uplift. Geologically the deposits are similar to the much larger deposits in the Tri-State region 200 kilometers to the northwest. Studies of freezing and homogenization temperatures of fluid inclusions in minerals from the Arkansas deposits have been made to see if the ore fluids were also similar. Primary inclusions in sphalerite, quartz, and dolomite homogenized in the range 80 degrees to 132 degrees C. Pressure corrections to be added probably do not exceed 10 degrees C. Most of the primary inclusions in calcite were single phase, suggesting temperature of formation of less than approximately 50 degrees C. The majority of the freezing temperatures for sphalerite and quartz were in the range --20 degrees to --23.6 degrees C, corresponding to salinities greater than 22 weight percent salts; the highest freezing temperature for sphalerite was --10.8 degrees C, corresponding to approximately 15 weight percent salts. The freezing data for calcite show that calcite was deposited from solutions having salinities from less than 4 weight percent salts to nearly fresh water.The close similarity of the mineralogy, geology, and fluid inclusion data for the two districts suggests that they represent the same period of mineralization. The origin of the ore deposits may be explained by the movement of heated, saline, formational waters out of the Arkoma or Anadarko basins.

Types of cell freezing and the post-thawing survival of mammalian ascites sarcoma cells

Summary By the use of mammalian tumor cells of MTK sarcoma III, an ascites sarcoma of rat, functional survival of frozen-thawed tumor cells was examined. Freezing of ascites drops containing the tumor cells was observed on the stage of a special refrigerated microscope. The types of cell freezing, intra- and extracellular, in the tumor cells were determined at various freezing temperatures above −30°C. Despite their small size, the tumor cells tended to freeze internally even at a high freezing temperature of −10°C. If an ascites drop was frozen at nearzero temperatures and then slowly cooled to −20°C, however, almost all tumor cells in the drop froze extracellularly. The tumor cells thawed after intracellular freezing appeared to be destroyed, while those thawed from extracellular freezing appeared intact. The rats transplanted with intracellularly frozen-thawed tumor cells were found to bear no tumor cells and were entirely healthy. On the other hand, the rats into which extracellularly frozen-thawed tumor cells had been transplanted died on the 23rd and 24th day after transplantation as a result of an abundant growth of tumor cells in their ascites.