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PubMed1984

Liquid propane jet-freezing, freeze-drying and rotary replication of the cytoskeleton and plasma membrane associated structures in human monocytes.

We have studied the cytoskeleton and plasma membrane associated structures of human monocytes using liquid propane jet-freezing, freeze-drying and rotary replication. We find that the cytoskeleton of these cells consists mainly of actin filaments. The actin filaments were identified by their ability to bind myosin sub-fragment S1. A large fraction of these actin filaments radiates from dense cytoskeletal foci. The presence of such actin filament rich foci also in unfrozen cells was demonstrated using immunofluorescence light microscopic techniques. In addition to coated pits a network of thin fibrils (diameter 4-5 nm) were observed on the protoplasmic side of the plasma membrane facing the substrate. These results indicate that liquid propane jet-freezing may represent a simple, inexpensive alternative to impact freezing at liquid helium temperature for obtaining the high cooling rate necessary for successful freeze-drying and rotary replication of cytoskeletal structures.

Actins↗

PubMed1979

Preparative techniques for freezing and freeze-sectioning macrophages for energy dispersive x-ray microanalysis.

In order to study the subcellular distribution of normal intracellular electrolytes and of metal pollutants, rabbit alveolar macrophages and mouse peritoneal macrophages were maintained in standard tissue culture medium with or without various concentrations of cadmium chloride or ammonium vanadate. A variety of preparative techniques were employed to study both monolayers and cell pellets by light microscopy, transmission electron microscopy, scanning electron microscopy and energy-dispersive x-ray microanalysis. Pellets of macrophages centrifuged in narrow bore centrifuge tubes were successfully snap-frozen in liquid-nitrogen-cooled liquid propane and either sectioned on a cryoultramicrotome or freeze-substituted with 1% osmium tetroxide in acetone and embedded in Epon. Spot probes of freeze-dried, frozen thin sections for normal intracellular electrolytes such as potassium, phosphorus and sulfur showed good localization to the cells and differences between organelles. Monolayers were freeze-dried and directly embedded in Epon. When Epon thin sections of these cells and of the freeze-substituted, Epon embedded pellets were obtained with a dry knife, intracellular electrolytes such as potassium, phosphorus and cadmium could still be detected by energy-dispersive x-ray microanalysis. It is concluded that in studies using snap-freezing for element localization, maximum information is obtained with the simultaneous application of a combination of preparatory techniques.

Animals↗

PubMedDate not supplied

Long-term preservation of egg and tissue homogenates for determination of organochlorine compounds: freezing versus freeze-drying.

Storage of wet egg homogenates at temperatures from -18 degrees to -28 degrees C was more suitable for long-term preservation than freeze-drying. Changes in residue levels of heptachlor epoxide, oxychlordane, dieldrin, hexachlorobenzene,p,p'-DDE, mirex, and PCBs were not significant over a 3-year period in fresh herring gull egg homogenates stored at -18 degrees to -28 degrees C. Compounds with gas chromatographic retention times shorter than hexachlorobenzene vaporized during freeze-drying at a rate proportional to their volatility. Evaporative losses of components with vapor pressures less than hexachlorobenzene did not occur in naturally contaminated herring gull eggs after storage at room temperature for up to 1 year. Higher losses of all compounds, up to 25% for p,p'-DDE, occurred in freeze-dried whole-body herring gull homogenates. Easily dehydrochlorinated compounds were rapidly degraded in freeze-dried chicken egg homogenate at room temperature: The half-life of p,p'-DDT and p,p'-DDD was about 20 days, and that of alpha- and gamma-hexachlorocyclohexane was much less than 16 days. About one-third of oxychlordane in herring gull eggs was lost in 1 year under these conditions, but none was lost after freeze-drying when the homogenate was stored at -18 degrees to -28 degrees C.

Eggs↗

PubMed1984

Photosynthetic reaction centers in artificial membranes: estimating protein dimensions by freeze-fracture and freeze-etching.

Because estimates of size and shape for membrane proteins are difficult to obtain directly, many workers have incorporated purified proteins into artificial lipid bilayers. Freeze-fracturing has then been used to provide a measure of the approximate size and shape of the membrane protein. We have formed reconstituted membranes containing the photosynthetic reaction center of Rhodopseudomonas viridis, a photosynthetic bacterium. The size and shape of this reaction center is accurately known from studies of negatively stained crystals of the protein to be approximately 4.5 X 6.0 nm. Freeze-fracture images of the reaction center in phosphatidyl choline liposomes show particles formed after reconstitution with an average diameter of 12.3 nm, much larger than the actual size of the protein. Deep-etched images of the surfaces of the liposomes, in which each individual reaction center complex can be seen clearly, show why the diameter of the protein is exaggerated in freeze-fracture. The individual reaction centers tend to cluster into large groups, allowing several individual reaction centers to be visualized as a single (much larger) particle in freeze-fracture. Freeze-fracturing, although a valuable tool in the analysis of membrane structure in natural and artificial membranes, must be used with caution in the estimation of molecular sizes and shapes.

Bacterial Proteins↗

PubMed1981

Investigation of membrane complexes of myelin and gregarine pellicle by freeze-drying and freeze-etching methods.

By the changes introduced in the standard procedures of the freeze-drying method (fixation, transportation of specimens from a vacuum apparatus into glass capillary tubes, and embedding) we have managed to simplify the method and to increase the reliability of the results. Fixation of the tissue was carried out after freeze-drying in molecular beams of OsO4 or in an OsO4 solution in carbon tetrachloride. The samples were embedded in araldite through mixtures of carbon tetrachloride and araldite. Membranes of myelin and gregarine pellicle were investigated. The repeat period of myelin at fixation of nerves after freeze-drying in OsO4 molecular beams is 11.2 +/- 0.5 nm and in an OsO4 solution in carbon tetrachloride, 12 +/- 0.4 nm. A tetragonal reticulum was revealed on the plasma-membrane extracellular surface of gregarine which had been prepared by freeze-drying. This type of structure is never observed after the routine fixation in water, while an analogous structure is revealed after freeze-etching.

Animals↗

PubMed1999

Inoculative freezing by environmental ice nuclei in the freeze-tolerant wood frog, Rana sylvatica.

Efficacy of inoculative freezing by ice nuclei in a simulated winter environment was studied in the wood frog (Rana sylvatica), a freeze-tolerant species that overwinters on the forest floor beneath organic detritus. Adult frogs were confined to plastic canisters and cooled to -2 degrees C over 24 hr with their ventral skin in contact with substrate (humic soil hydrated to 40, 10, or 5%, or soil/peat mixture hydrated to 20 or 10%, w/w), or their dorsal skin in contact with damp leaf mould. Whereas only 20% of control frogs cooled in dry, plastic canisters froze, freezing occurred in nearly all (98%) frogs contacting soil or leaf mould. Inoculation was briefly delayed in frogs exposed to drier substrates. Frogs exposed to an unfreezable substrate (humic soil, 5% moisture) themselves froze, apparently due to the action of constituent nuclei which commonly occur in natural materials. Although the surface over which inoculation can occur is greater in larger frogs, inoculation susceptibility was not correlated with body mass in our frogs (mean +/- SE body mass = 14.0 +/- 0.2 g; range, 9.8-17.8 g). We conclude that the high susceptibility to inoculative freezing in R. sylvatica, which is conferred by its moist, highly permeable integument, promotes freeze tolerance by ensuring that inoculation commences at relatively high temperatures.

Animals↗

PubMed2004

Seasonal effects of dehydration on glucose mobilization in freeze-tolerant chorus frogs (Pseudacris triseriata) and freeze-intolerant toads (Bufo woodhousii and B. cognatus).

It has been hypothesized that freeze-tolerance in anurans evolved from a predisposition for dehydration tolerance. To test this hypothesis, we dehydrated summer/fall-collected and winter acclimated freeze-tolerant chorus frogs and dehydration-tolerant, but freeze-intolerant, Woodhouse's and Great Plains toads to 25% and 50% body water loss (BWL). Following treatments, we measured glucose, glycogen, and glycogen phosphorylase and glycogen synthetase (summer/fall only) activities in liver and leg muscle. Hepatic glucose levels were not significantly altered by dehydration in either summer/fall-collected frogs or toads. Conversely, winter acclimated frogs did show an increment (2.9-fold) in hepatic glucose with dehydration, accompanied by a reduction in hepatic glycogen levels. Winter acclimated toads did not mobilize hepatic glucose in response to dehydration. Further, hepatic glycogen and phosphorylase activities did not vary in any consistent manner with dehydration in winter toads. Mean leg muscle glucose values were elevated at 50% BWL relative to other treatments, significantly so compared to 25% BWL for summer/fall-collected frogs. The pattern of hepatic glucose mobilization with dehydration in winter frogs is consistent with that in other freeze-tolerant frog species, and provides additional support for the hypothesis that freezing tolerance evolved from a capacity for dehydration tolerance. However, the lack of hepatic glucose mobilization in response to dehydration in fall frogs suggests that a seasonal component to dehydration-induced regulation of glucose metabolism exists in chorus frogs. Furthermore, the absence of a dehydration-induced mobilization of hepatic glucose at both seasons in toads suggests that this dehydration response is not universal for terrestrial anurans.

Acclimatization↗

PubMed1992

Characterization of inner ear sensory hair cells after rapid-freezing and freeze-substitution.

In order to determine the ultrastructural organization of normal cells and to understand better the anatomical substrates for outer hair cell motility, cryofixation was performed on the sensory epithelium of the inner ear of guinea pigs prior to substitution of frozen water with organic solvents containing chemical fixatives. In this way cells would not be altered by the direct application of the chemicals commonly used for preservation, which are also known to cause fixation-induced shape changes in outer hair cells. Following rapid freezing and freeze-substitution, preservation of cells within the isolated sensory epithelium containing the organ of Corti was similar to that seen in conventionally fixed cells. However, in rapidly frozen and freeze-substituted outer hair cells the cytoplasm and the cellular membranes differed from those seen in conventionally fixed preparations. The cytoplasmic matrix was densely packed with filaments and stained homogeneously, suggesting better preservation of the cytoskeleton and less extraction of the soluble ground substance. Cell membranes were smooth, indicating that fixation-induced shape changes and shrinkage had been avoided. The subsurface cisternal system of intracellular membranes lining the lateral wall of the outer hair cells was composed of continuous, tightly packed, parallel rows of membranous lamellae. Thus rapid-freezing and freeze-substitution are important techniques by which structural alterations correlated with outer hair cell motility can be separated from fixation-induced cell shape changes.

Animals↗

PubMed2006

Preparation of 3-D regenerated fibroin scaffolds with freeze drying method and freeze drying/foaming technique.

Although three-dimensional fibroin scaffolds have been prepared with freeze drying method, the porosity and pore sizes still can not satisfy the requirement of tissue engineering. In this article, fibroin porous scaffold with high porosity and > 100 microm diameter interconnected pores was firstly prepared with freeze drying method through adjusting fibroin concentration. The morphology of different scaffolds lyophilized from different fibroin concentration was observed by SEM. A novel freeze drying improved method, freeze drying/foaming technique, was also devised to prepare fibroin scaffolds at different fibroin concentrations. Using the said method, the porosity and pore size of fibroin scaffolds prepared from 12% concentration were 85.8 +/- 4% and 109 +/- 20 microm respectively with yield strength up to 450 +/- 6 KPa while the porosity and pore size of fibroin scaffolds prepared from 8% concentration were 96.9 +/- 3.6% and 120 +/- 30 microm respectively with yield strength up to 30 +/- 1 KPa. The freeze drying/foaming technique produced scaffolds with a useful combination of high yield strength, interconnected pores, and pore sizes greater than 100 microm in diameter. Through adjusting fibroin concentration and thawing time, the porosity, pore sizes and mechanical properties could be controlled to satisfy the different requirements of tissue engineering. The results suggested that fibroin scaffolds prepared with the above methods could be formed for utility in biomaterial application.

Animals↗

PubMed1976

Correlated x-ray diffraction and freeze-fracture studies on membrane model systems. Perturbations induced by freeze-fracture preparative procedures.

Lipid-water and protein-lipid-water phases have been examined by X-ray methods before and after freezing. Frozen samples have been subsequently fractured and replicated, thus permitting an evaluation of the nature of structural perturbations in samples examined by freeze-fracture electron microscopy. Important results are summarized: (1) Freezing low water content (approx. less than 25%) phases causes perturbations in the packing of hydrocarbon chains. The results suggest that freezing liquied paraffin chains produces a condensed "glass-like" packing. (2) Additional perturbations occur in high water content samples. After freezing, much smaller lamellar repeat distances, intense ice reflections, and extensive perturbation of fracture faces are consistant with the expulsion of water from between lamellae. Presence of glycerol generally relieves these perturbations but in some cases introduces additional lattice disorder. (3) Surprisingly, cooling by a stream of cold N2 gas (-140 degrees C) produces qualitatively the same results as rapid cooling in liquid Freon-22 (-160 degrees C). (4) Complex perturbations occur in phases containing integral membrane proteins. Interesting results have been obtained with cytochrome b5-lecithin lamellar associations which display both smooth and rough fracture faces without clearly defined particles.

Binding Sites↗

PubMed1989

Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution.

Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Intracellular freezing and survival in the freeze tolerant alpine cockroach Celatoblatta quinquemaculata.

The alpine cockroach Celatoblatta quinquemaculata is common at altitudes of around 1500 m on the Rock and Pillar range of Central Otago, New Zealand where it experiences freezing conditions in the winter. The cockroach is freeze tolerant, but only to c. -9 degrees C. The cause of death at temperatures below this is unknown but likely to be due to osmotic damage to cells (shrinkage). This study compared the effect of different ice nucleation temperatures (-2 and -4 degrees C) on the viability of three types of cockroach tissue (midgut, Malpighian tubules and fat body cells) and cooling to three different temperatures (-5, -8, -12 degrees C). Two types of observations were made (i) cryomicroscope observations of ice formation and cell shrinkage (ii) cell integrity (viability) using vital stains. Cell viability decreased with lower treatment temperatures but ice nucleation temperature had no significant effect. Cryomicroscope observations showed that ice spread through tissue faster at -4 than -2 degrees C and that intracellular freezing only occurred when nucleated at -4 degrees C. From temperature records during cooling, it was observed that when freezing occurred, latent heat immediately increased the insect's body temperature close to its melting point (c. -0.3 degrees C). This "rebound" temperature was independent of nucleation temperature. Some tissues were more vulnerable to damage than others. As the gut is thought to be the site of freezing, it is significant that this tissue was the most robust. The ecological importance of the effect of nucleation temperature on survival of whole animals under field conditions is discussed.

Acclimatization↗

PubMed2000

Review article fine structure of the exocrine cells of rat sublingual gland revealed by rapid freezing and freeze substitution method.

The ultrastructure of mucous cells of rat sublingual gland processed by rapid freezing, followed by freeze substitution, was compared with that obtained by the standard chemical fixation technique. The rapid freezing method gave a very good preservation of membrane structure with round and discrete mucous droplets (granules) not showing any sign of coalescence. The cisterns of the Golgi apparatus and the trans Golgi network also were well preserved. Upon secretory stimulation by pilocarpine, mucous droplets were discharged by the usual mechanism of exocytosis. From all these findings it emerged that mucous cells had the same structural characteristics as serous cells. In the endpieces of rat sublingual gland prepared by the rapid freezing method, serous cells aligned with mucous cells around the central lumen, and no cap-like arrangement of serous cells (demilunes) was observed. Furthermore, computer reconstruction of stereo images from serial section light micrographs prepared by the rapid freezing method showed that, within a given endpiece, all serous cells had direct access to the lumen and that they were disseminated throughout it and not only in its fundus. From our observations it seems very likely that, at least in rat sublingual gland, serous demilunes are an artificial product caused by the compression exerted on serous cells by the mucous cells distended during the conventional fixation procedure.

Animals↗

PubMed2005

Slow controlled-rate freezing of sequentially cultured human blastocysts: an evaluation of two freezing strategies.

BACKGROUND: To optimize blastocyst cryopreservation, the prerequisite is to develop a better understanding of factors that influence their survival and implantation potential. Therefore, the aim of the present work was to evaluate, retrospectively, the outcome of blastocyst cryopreservation in a day 2/3 fresh embryo transfer programme. METHODS: Two different freezing strategies were compared: a first strategy (strategy A: 3007 blastocysts frozen) consisted of freezing those blastocysts that had at least a cavity; a second strategy (strategy B: 3831 blastocysts frozen) consisted of freezing only more advanced stage blastocysts with a good quality inner cell mass and trophectoderm. The outcome of cryopreservation, as related to the two different freezing strategies, was analysed. In addition, after freezing and thawing, we evaluated the influence of blastocyst developmental characteristics on immediate morphological survival and further development in vitro. RESULTS: The immediate morphological survival after thawing was higher for early blastocysts as compared to advanced and hatching blastocysts. The further developmental potential in vitro of thawed blastocysts was higher for advanced and hatching blastocysts as compared to early blastocysts. As a result, the percentage of deliveries, calculated as a percentage of started thawing cycle, and the percentage of children born, calculated as a percentage of embryos transferred, was not different for strategies A and B. CONCLUSION: The results clearly indicate that culture conditions and cryopreservation procedures of blastocysts need to be further improved.

Blastocyst↗

PubMed1982

Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21 degrees C).

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethylsulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configuration at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0.3 K min-1 or 2 K min-1 were related to the distribution of the ice phase within the tissue.

Animals↗

PubMed2005

Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at -80 degrees C without postthaw processing.

BACKGROUND: Platelets (PLTs) can be frozen with 6 percent dimethyl sulfoxide (DMSO) at -80 degrees C for up to 2 years. This method has been modified by concentrating the PLTs and removing the supernatant before freezing. STUDY DESIGN AND METHODS: High-yield leukoreduced PLTs stored at 22 degrees C for up to 5 days were divided into three equal volumes: one was frozen with 6 percent DMSO at -80 degrees C, thawed, washed, and resuspended in plasma (old method with DMSO); the second was treated with 6 percent DMSO, concentrated to remove the supernatant DMSO, frozen at -80 degrees C, thawed, and diluted with 0.9 percent NaCl (new method with DMSO); and the third was treated with 0.9 percent NaCl without DMSO, concentrated to remove the supernatant solution, frozen at -80 degrees C, thawed, and diluted with 0.9 percent NaCl (new method without DMSO). RESULTS: Freeze-thaw-wash recovery of PLTs frozen by the old method with DMSO was 74 +/- 2 percent with 5 percent PLT microparticles. Freeze-thaw recovery was 94 +/- 2 percent with 7 percent PLT microparticles (new method with DMSO) and 69 +/- 9 percent with 15 percent PLT microparticles (new method without DMSO). Total DMSO in washed PLTs was 400 and 600 mg in PLTs concentrated before freezing. In vivo recovery of PLTs frozen by the new method with DMSO and transfused into normal volunteers was 30 percent and the life span was 7 days. CONCLUSION: Concentrating PLTs before freezing simplified the procedure by eliminating postthaw washing. PLTs frozen by this method had more PLTs with reduced GPIb and increased annexin V binding than those frozen by the old method.

Blood Banking↗

PubMed1987

Rapid-freezing and malachite green-acrolein-osmium tetroxide freeze-substitution fixation improve visualization of extracellular lipids in rat incisor pre-dentin and dentin.

Rat incisor tissue sections were fixed by a modified version of the malachite green-aldehyde method (MGA) composed of rapid-freezing, malachite green-acrolein staining, and osmium tetroxide freeze-substitution (Fr.MGAO). In the pre-dentin, a thick, dense network of branched fibrous structures was observed. Cryotechniques allowed visualization of complexes about twice as thick and dense as the aggregates visualized on MGA-treated sections. Pretreatment of rapid-frozen samples with methanol before freeze-substitution fixation and staining prevented staining of the complexes otherwise revealed by the Fr.MGAO method. Electron-dense material stained by this procedure resisted de-mineralization with EDTA, while intramitochondrial granules and dentin crystallites were dissolved. EDTA treatment demonstrated unequal distribution of Fr.MGAO staining in dentin in the form of tiny dots underlining the collagen fibers. These results support the concept that rapid-freezing, followed by staining and freeze-substitution fixation, improves preservation of the phospholipids visualized as extracellular matrix components in pre-dentin and dentin of rat incisors.

Acrolein↗

PubMed1985

A quick-freeze, freeze-fracture study of mouse spermatozoa.

Rapid cryo-fixation of mouse spermatozoa followed by freeze substitution, thin section and freeze-fracture demonstrates the finer detail possible with quick-freeze as compared to chemical fixation. As seen in thin sections the unit membrane is composed of 2-4 nm size particles and the cytoplasm, mitochondria, and axonemal filament components all appear rich in fine structure detail. The filamentous structure of the post-acrosomal sheath and its connection with the plasmalemma is presented and compared to previous studies on this structure. Freeze-fracture data demonstrates 7-9 nm size, plasmalemmal, PF-face particles most heavily concentrated in the region just ahead of the striated ring. The outer acrosomal EF-face contains linear arrays of 7-9 nm size EF-face particles. The inner acrosomal membrane contains scattered, 7-9 nm size PF-face particles. The inner and outer nuclear membranes also contain scattered, 7-9 nm size particles. The results of this study present data which supports and extends previous studies on mouse spermatozoa. The results are discussed in terms of the advantages of cryo-fixation and freeze-substitution compared to conventional fixation in the preservation of fine structure detail.

Animals↗