A new freeze-etch unit for freeze-etch rotary shadowing, low temperature freeze-fracturing and conventional freeze-etching.
Explore the source record for details and available documents.
Brevibacterium↗
SEARCH · PubMed Health
Results for “Freezing”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Effects of freeze-preservation on some pollen enzymes. II. Freezing and freeze-drying stresses.
Explore the source record for details and available documents.
Acid Phosphatase↗
A comparison of the ultrastructure of spray-frozen and freeze-etched or freeze-dried bull and boar spermatozoa with that after chemical fixation.
The ultrastructure of bull and boar spermatozoa was investigated following different cryopreparation methods and chemical fixation. Spray-freezing was used for cryofixation in both freeze-etching and freeze-drying studies. Freeze-etching of boar spermatozoa revealed that the arrangement of the postnuclear striations differed from that in the bull. Freeze-drying gave excellent results for structural preservation, which were equal to those of chemical fixation. Some structural details not visible in chemically fixed cells were detected in freeze-dried and vacuum-embedded bull and boar spermatozoa, e.g. the arrangement of the lamellar nuclear contents, known from freeze-fractures, and a fine lamellar structure of the acrosomal contents. Cryofixation by spray-freezing combined with freeze-drying makes any contact of the cells with fixatives, buffer solutions and dehydration media unnecessary, and potentially provides all the advantages of ultrathin sectioning required for histochemical studies.
Acrosome↗
[Freezing damage and freezing protection within the scope of deep-freeze preservation of living materials with special reference to mammalian spermatozoa. Contd from issue 10/77].
Explore the source record for details and available documents.
Animals↗
The circumstances of freezing in the freeze-drying process of haemoderivatives.
It appears from the experiments of the authors that the macroscopic changes taken place during freezing and heating could be explained by the formation of an eutectic crystalline structure in the case of human plasma and albumin solutions. The temperature interval of eutectic spot formation showed good agreement with the temperature range of initial thawing as determined by the DTA method, i.e. with the working zone freeze-drying. It was possible to determine the maximum temperature of complete solidification from the resistance curves of the haemoderivatives. Judging from the shape of the resistance curves, the phenomenon of undercooling did not emerge with the freezing rate applied. Examining the optimal freezing rate, it appeared on the basis of some quality characteristics of the freeze-dried end-product, that the best results were obtained with --30 degrees C freezing in the case of albumin and plasma, and with --45 degrees C freezing in the case of normal and iv immunoglobulins. But the authors deem it necessary to conduct further experiments in this respect.
Blood Preservation↗
The use of low temperature X-ray diffraction to evaluate freezing methods used in freeze-fracture electron microscopy.
Two methods of freezing samples for freeze-fracture electron microscopy have been compared using X-ray diffraction and freeze-fracture results from lipid-water model systems. Perturbations of the molecular organization of hydrocarbon chains and the extent of ice crystal formation have been evaluated for lamellar phases of egg lecithin containing 16% water and egg lecithin-phosphatidylinositol containing 55% water, both freeze quenched in liquid Freon-22 near its melting temperature (113 K). Very thin samples sandwiched between copper sheets separated by an electron microscope grid show much less freezing induced structural rearrangement than small smaples contained on conventional Balzers-type gold planchettes. These results show that the rate of freezing in the very thin preparations is greater than in the conventional ones, which is probably due in part to the improved dissipation of heat from a poorly conductive sample through highly conductive copper sheets.
Freeze Fracturing↗
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↗
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↗
The influence of freezing and freeze-drying of tissue specimens on enzyme activity.
In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques. With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD+), malate dehydrogenase (decarboxylating) (NADP+), isocitrate dehydrogenase (NADP+), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, beta-glucuronidase and non specific aryl esterase. Therefore, the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.
Animals↗
Simplification of the methods for adding and removing glycerol during freeze-preservation of human red blood cells with the high or low glycerol methods: biochemical modification prior to freezing.
Simple methods have been developed for adding and removing glycerol during freeze-preservation with 20 per cent W/V glycerol at minus 150 C, or with 40 per cent W/V glycerol at minus 80 C. A one-step method with a 35 per cent W/V glycerol solution is used to prepare 20 per cent W/V glycerolized red blood cells, and a two-step method with a 57 per cent W/V glycerol solution is used to prepare 40 per cent W/V glycerolized red blood cells. The systems for washing glycerolized red blood cells have been simplified. This method consists of dilution of the thawed glycerolized red blood cells prior to recovery, followed by on-line dilution of these red blood cells with wash solutions during continuous flow centrifugation. This can be done in any of three commercially available washing systems, and they all use the same sodium chloride solutions. For the 40 per cent W/V glycerolized red blood cells, this process takes about 30 minutes and uses 2.2 to 3.2 liters of the sodium chloride solutions, whereas the 20 per cent W/V glycerolized red blood cells can be processed in about 20 minutes using 1.5 to 2.5 liters. After storage in CPD for three days at 4 C, red blood cells can be freeze-preserved with 40 per cent W/V glycerol at minus 80 C or with 20 per cent W/V glycerol at minus 150 C. When the thawed red blood cells are washed in the Fenwal Elutramatic, the IBM Blood Processor, or the Haemonetics Blood Processor and stored at 4 C in sodium chloride-glucose-phosphate for at least 24 hours before transfusion, they have excellent posttransfusion survival values and normal or slightly decreased oxygen transport function. Alternatively, these red blood cells can be rejuvenated before freeze-preservation so that their 2,3-DPG levels are increased and their affinity for oxygen is reduced. Red blood cells that are stored in CPD at 4 C for as long as 28 days can be rejuvenated with a solution containing pyruvate, inosine, glucose, phosphate, and adenine (PIGPA, Solution A) before freeze-preservation with 40 per cent W/V glycerol at minus 80 C. Any one of the above systems can be used to wash these red blood cells and they can be stored at 4 C in a sodium.
Adenine↗
Understanding the relationship between lateral spread of freeze and depth of freeze.
The predictable relationship between lateral spread of freeze and depth of freeze is a keystone concept in the clinical evaluation of "depth dose" of cryosurgical procedures. This relationship can be readily determined and understood by carrying out simple experiments using potato models and checked directly by measurements on patients. When using specialized treatment methods such as the "cone-spray" technique with liquid nitrogen, it is necessary to limit the technique to properly selected cases and to spray the treated surfaces evenly if the predictable ratio of depth of freeze to lateral spread of freeze is to be used.
Animals↗
Freezing of rat lymphocytes. V. The effect of suppressor cells in phytohaemagglutinin-stimulated spleen cell cultures before and after freeze-thawing.
Spleen lymphocyte proliferation, as measured by 3H-thymidine incorporation induced by phytohaemagglutinin (PHA), was increased after freeze-thawing with 10% dimethyl sulphoxide. Depletion or intoxication of macrophages in fresh spleen cell preparations also increased lymphocyte proliferation in response to PHA. On the other hand, freezing of macrophage-depleted spleen cell suspensions lowered 3H-thymidine uptake of stimulated cultures. At concntrations above 3%, macrophages added to cultures of fresh purified lymphocytes showed a dose-dependent inhibitory effect on the PHA response, and fresh macrophages were more inhibitory than frozen-thawed macrophages. Purified lymphocytes mixed with 10% macrophages showed a higher response after freeze-thawing. It is concluded that macrophages suppress the lymphoproliferative response to PHA in rat spleen cell cultures, and that these macrophages are more sensitive than lymphocytes to the present freeze-thaw process.
Animals↗
[Rapid freezing of biologic tissue. Measurement of temperature and rate of freezing by thin-layer thermocouple].
An apparatus for rapid freezing of biological tissues by contact with a copper block cooled by liquid helium has been devised to reduce the contamination of copper block surface. It prevents the precooling of the specimen while going through the layers of cold helium gas surrounding the copper block and reduces the quantity of helium necessary for freezing. It also enables one to obtain easily reproducible results from freezing by immersion in liquid coolants. Freezing rates are measured directly, related to the specimen thickness, by a thin film thermocouple; its low thermal inertia gives speed measurements of the order of 100,000 degrees C/s.
Animals↗
Membrane fusion during secretion: cortical granule exocytosis in sex urchin eggs as studied by quick-freezing and freeze-fracture.
Exocytosis of cortical granules was observed in sea urchin eggs, either quick-frozen or chemically fixed after exposure to sperm. Fertilization produced a wave of exocytosis that began within 20 s and swept across the egg surface in the following 30 s. The front of this wave was marked by fusion of single granules at well-separated sites. Toward the rear of the wave, granule fusion became so abundant that the egg surface left with confluent patches of granule membrane. The resulting redundancy of the egg surface was accommodated by elaboration of characteristic branching microvilli, and by an intense burst of coated vesicle formation at approximately 2 min after insemination. Freeze-fracture replicas of eggs fixed with glutaraldehyde and soaked in glycerol before freezing displayed forms of granule membrane interaction with the plasma membrane which looked like what other investigators have considered to be intermediates in exocytosis. These were small disks of membrane contact or membrane fusion, which often occurred in multiple sites on one granule and also between adjacent granules. However, such membrane interactions were never found in eggs that were quick-frozen fixation, or in eggs fixed and frozen without exposure to glycerol. Glycerination of fixed material appeared to be the important variable; more concentrated glycerol produced a greater abundance of such "intermediates." Thus, these structures may be artifacts produced by dehydrating chemically fixed membranes, and may not be directly relevant to the mechanism by which membranes naturally fuse.
Animals↗
A copper block method for freezing non-cryoprotected tissue to produce ice-crystal-free regions for electron microscopy. I. Evaluation using freeze-substitution.
A wide variety of plant and animal tissues were prepared for electron microscopy by freeze-substitution, after rapid freezing on a liquid nitrogen cooled copper block by the van Harreveld method. Measurements were made of ice crystal size versus depth in tissues that had not been treated with any cryoprotectants. Ice crystal size increased exponentially with depth. It was confirmed that a narrow surface band increased exponentially with depth. It was confirmed that a narrow surface band (approximately 12 mum) was frozen sufficiently rapidly to prevent the formation of electron microscopically-visible ice crystals.
Animals↗
Freezing of rat lymphocytes. III. Freezing of plaque-forming cells and restoration by frozen-thawed normal cells of antibody production in irradiated rats.
The present investigation is an extension of earlier work with dimethyl sulphoxide (DMSO) protected frozen-thawed rat lymphocytes. In the present work it is shown that some 85-90% of the haemolytic plaque-forming cells (PFC) survived the freeze-thaw process. Irradiated rats were restored with fresh and frozen-thawed cells and immunized against sheep red blood cells (SRBC). Evidence is presented that a restrictive control of the PFC response by suppressor cells present in the spleen cell suspension is lost during the freeze-thaw process, giving a higher number of PFC/spleen in recipients of frozen-thawed mixed spleen and lymph node cells than in rats receiving the corresponding fresh preparations.
Animals↗
Freeze-fracturing and freeze-etching of cardiac myosin filaments.
Myofilament structure was studied in freeze-etch replicas of unfixed, glycerinated beef cardiac muscle. The information which is revealed depends upon the direction of metal shadowing in relation to the filament axis. Shadows oblique to this axis reveal that the outer surface of a longitudinal half of a thick filament comprises three, sometimes four, rows of myosin molecules. These molecules are generally assembled in a braided manner with both left and right-handed helical components. Occasionally a more parallel to the myofilament axis reveal cross-bridges linking thick and thin filaments. These bridges are readily detectable by optical diffraction techniques, giving an axial bridge spacing of approximately 40 nm. In unetched preparations cross bridges appear as vertical rows of beads. In all replicas the effects of plastic deformation of proteins must be considered.
Animals↗
Short fixation-shock freezing and freeze-drying versus chemical fixation and dehydration: computer assisted image analysis of morphological variables and immunogold labeling density on pituitary secretory granules.
We have performed a computer assisted image analysis evaluation of the effects of two preparation protocols on morphological variables and immunolabeling density of secretory granules using rat adenohypophysis as a model. Glutaraldehyde (GA) fixation for 2 hr and chemical dehydration was compared with short GA fixation (15 min) followed by cryofixation and freeze-drying (CF-FD). The 2 hr GA fixed specimens showed spherical nuclei and secretory granules regardless of their size, contrasting with the more irregularly shaped nuclei and secretory granules after short GA-CF-FD. In the latter group of specimens a correlation could be found between smaller nuclear areas and more irregular shapes. The differences in morphological variables between the two preparation protocols might be due to a better protein stabilization and a reduced collapse of macromolecules after the 2 hr GA fixation, strengthened by the chemical dehydration. The specific immunolabeling with antiserum to growth hormone was much greater but more varied after short time GA-CF-FD than after long GA fixation. Epon surface topography over the granule area also differed: smooth after short time GA-CF-FD and furrowed after long GA fixation. Our results, demonstrating important differences in morphological parameters and immunolabeling density between two common preparation protocols, seem critical for more reliable interpretation in quantitative immunoelectron microscopy. We also emphasize the need for computer assisted image analysis and measurements in immunoelectron microscopy to ensure objective evaluations.
Animals↗