Influence of chemical and freezing fixation methods in the freeze-fracture of stratum corneum

A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum-carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist.     

Ultrastructural and thermocouple evaluation of rapid freezing techniques

The quality of freeze-fixation for electron microscopy is dependent upon the size of intracellular ice crystals. In the absence of cryoprotectants, ice crystal growth is thought to be related to the speed with which the specimen is cooled. The purpose of this study was to investigate the relationship between the cooling rate and ultrastructural preservation in commonly used freezing techniques. The techniques studied included immersion in stirred and unstirred forms of five quenching fluids: liquid nitrogen, isopentane, Freon 12, Freon 22, and propane. Also studied were freezing in a flowing stream of coolant using liquid nitrogen and liquid helium and freezing on a metal surface using cooper and mercury chilled to liquid nitrogen temperature. For each technique a cooling curve was obtained with a 0.360-mm thermocouple which was dropped into the quenching fluids or brought into contact with the metal surfaces. From oscilloscope tracings, the cooling rates were determined in degrees centigrade per second to −100 °C. To evaluate ultrastructural preservation 0.5-mm-thick slices of rat kidney were frozen by each of the techniques and dried in an all glass freeze-drier. The final evaluation was made from electron micrographs of the best morphological preservation yielded by each technique. The results indicate that the copper and mercury surfaces and propane gave the highest cooling rates and the best morphological preservation. The other techniques cooled at decreasing rates and correspondingly showed decreasing abilities to preserve ultrastructure. This work demonstrates that the preservation of cellular ultrastructure by freezing is dependent upon the cooling rate and that as the cooling rate is increased, ultrastructural preservation is enhanced.     

Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization

Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.     

Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Summary Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.Abbreviations HPF high-pressure freezing - MES 2-(N-morpholino)ethanesulfonic acid - PBM peribacteroid membrane - PBS peribacteroid space - PJF propane-jet freezing - RER rough endoplasmic reticulum     

Cell surface arabinogalactan-proteins and their relation to cell proliferation and viability

Summary We have used high-pressure freezing followed by freeze substitution (HPF/FS) to preserve in vivo grown lily pollen tubes isolated from the style. The results indicated that HPF/FS (i) allows excellent preservation of the pollen tubes, (ii) maintains in situ the stylar matrix secreted by the transmitting tract cells, and (iii) preserves the interactions that exist between pollen tubes. Particular attention has been given to the structure of the pollen tube cell wall and the zone of adhesion. The cell wall is composed of an outer fibrillar layer and an inner layer of material similar in texture and nature to the stylar matrix and that is not callose. The stylar matrix labels strongly for arabinogalactan proteins (AGPs) recognized by monoclonal antibody JIM13. The zone of adhesion between pollen tubes contains distinct matrix components that are not recognized by JIM13, and apparent cross-links between the two cell walls. This study indicates that HPF/FS can be used successfully to preserve in vivo grown pollen tubes for ultrastructural investigations as well as characterization of the interactions between pollen tubes and the stylar matrix.Abbreviations AGPs arabinogalactan proteins - FS freeze substitution - HPF high-pressure freezing     

In situ localization of sulphur in the thioautotrophic symbiotic model Lucina pectinata (Gmelin, 1791) by cryo-EFTEM microanalysis

BACKGROUND INFORMATION: Lucina pectinata is a large tropical lucinid known to harbour sulphide-oxidizing bacteria in specialized gill cells. Conventional TEM (transmission electron microscopy) has shown that bacteriocytes also harbour visibly 'empty' vesicles whose chemical content remains, to date, only roughly determined. RESULTS: In the present study, L. pectinata gill tissues were cryo-fixed as fast as possible by performing high-pressure freezing before a freeze-substitution process and finally performing a cryo-embedding in Lowicryl. Ultrathin sections were then used for a cryo-EFTEM (where EFTEM stands for energy-filtered TEM) microanalysis. Results show that bacteriocytes within the gill tissues contain elemental sulphur in small vesicles produced by the host itself. In instances of sporadic depletion of sulphur in the environment, such structures may act as energy sources for bacterial endosymbionts. CONCLUSIONS: The cryo-EFTEM techniques represent (i) the only method used to date to locate and preserve sulphur at the cellular level and (ii) a powerful tool for sulphur metabolism analysis in thioautotrophic symbiont relationships.     

A comparison of cryo- versus chemical fixation in the soil green algae Jaagiella

Chemical fixation protocols provided unsatisfactory preserved material for ultrastructural studies on Jaagiella alpicola Vischer (Chlorophyta). Instead, several methods of rapid freeze fixation followed by freeze substitution were applied. For fast freeze fixation, two methods were tried: plunge immersion freezing in liquid propane using a home-made device, and projection against a copper block cooled by either liquid nitrogen or liquid helium. Each method furnished well fixed material. The quality of the fixed samples was quite similar whether propane or the cryoblock cooled with liquid nitrogen was used. Liquid helium, however, provided superior results. After fixation the samples were cryosubstituted, using acetone or methanol as organic solvent with a chemical fixative added. Acetone gave better results than methanol as a substitution solvent when high temperature embedding was performed. The best cryosubstitution for ultrastructural studies was that in which osmium tetroxide or a mixture of osmium tetroxide and urany acetate was used.     

Protein recovery and identification from the gulf killifish, Fundulus grandis: comparing snap-frozen and RNAlater® preserved tissues

Reliable proteomic analysis of biological tissues requires sampling approaches that preserve proteins as close to their in vivo state as possible. In the current study, the patterns of protein abundance in one‐dimensional (1‐D) gels were assessed for five tissues of the gulf killifish, Fundulus grandis, following snap‐freezing tissues in liquid nitrogen or immersion of fresh tissues in RNAlater^®. In liver and heart, the protein profiles in 1‐D gels were better preserved by snap‐freezing, while in gill, the 1‐D protein profile was better preserved by immersion in RNAlater^®. In skeletal muscle and brain, the two approaches yielded similar patterns of protein abundance. LC‐MS/MS analyses and database searching resulted in the identification of 17 proteins in liver and 12 proteins in gill. Identified proteins include enzymes of energy metabolism, structural proteins, and proteins serving other biological functions. These protein identifications for a species without a sequenced genome demonstrate the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.     

Relationships between ice crystal size, water content and proton NMR relaxation times in cells

Biological specimens were frozen under controlled conditions. We questioned how the size of ice crystals, as measured in cryosectioned and cryoadsorbed sections of these biological specimens, relates to the water content and to the proton NMR relaxation times (T1 and T2) of the unfrozen specimens. The results permit the following conclusions: After rapid freezing in liquid propane cooled in a liquid nitrogen bath, the average size of ice crystals at distances of 150 microns or more from the surface of a particular tissue was always the same. Thus, the average size of the ice crystals was found to be characteristic of the type of biological tissue studied. Linear regression analysis showed average ice crystal size to have a significant correlation coefficient to T1 relaxation time and to water content. Specifically ice crystal size increased with T1 relaxation time and with water content. Multiple regression and path analysis demonstrated a positive correlation between the T1 relaxation time and the ice crystal size variation. Path analysis showed that both water content and T2 relaxation time were less directly correlated with ice crystal size. The findings from the path analysis and other observations show that the average size of ice crystals in subcellular compartments is best predicted by the proton T1 relaxation time. A working model is put forth to explain differences in ice crystal size observed between specimens enriched in globular or in parallel filamentous proteins.     

A method for preserving ultrastructural properties of mitotic cells for subsequent immunogold labeling using low-temperature embedding in LR White resin

The best available approach of biological sample preparation for transmission electron microscopy currently includes cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method has been well established for interphase cells; however, a reliable and easy procedure is still missing for mitotic cells especially because of their fragility and sensitivity to treatments. Here, we present a fast and effective method for HPF/automated FS and LR White embedding of mitotic cells which allows for their controlled and reproducible quality processing. It should be useful in various ultrastructural studies on mitotic cells especially in combination with immunocytochemistry.     

The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 3, Pages 387-397 (1977)

Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was demonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.     

Experimental hepatic cryosurgery

In 28 dogs, a major portion of a lobe of the liver was frozen, either by contact with a cryoprobe or by spraying liquid nitrogen (?196 °C) over the exposed liver or by pouring liquid nitrogen into a plastic barrel placed on the lobe of the liver, which was wrapped in a Dacron velour cloth. With the probe technique, the amount of tissue that could he frozen was not as great as with direct application of liquid nitrogen to the liver. The spray technique allowed freezing of a larger area but with cracking of the liver substance with considerable mortality from bleeding. With the pour technique, even a larger area of liver could be frozen and bleeding was much less because of the protection afforded by the Dacron cloth. There were no toxic effects from the devitalized liver which was left to be resorbed. These techniques are applicable for treating localized lesions in the liver in humans, but the experiments demonstrate the difficulty of achieving great depth of freezing with any currently available technique of freezing tissue in situ. Clearly, advances in cryosurgical equipment are needed.     

Effect of ice nucleation by droplet of immobilized silver iodide on freezing of rabbit and bovine embryos

Silver iodide was immobilized by applying the insoluble reaction between sodium alginate and calcium chloride. The immobilized silver iodide was immersed into a freezing solution in order to trigger ice nucleation. Temperature change during cooling and postthaw in vitro development of embryos were examined in order to evaluate the effectiveness of the immobilized silver iodide (AgI alginate-gel droplet) on embryo development. Samples containing the AgI alginate-gel droplets released the latent heat of fusion at a higher subzero temperature than samples without the AgI alginate-gel droplets. When the AgI alginate-gel droplet was added to the freezing solution of rabbit and bovine embryos, they were successfully preserved in liquid nitrogen.     

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.     

The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels.

Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was deomonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.     

Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation/dehydration

Summary A cryopreservation process using encapsulation/dehydration was set up for apices sampled on in vitro plantlets of sugarcane. After dissection, apices were cultured for one day on standard medium and then encapsulated in medium with 3% alginate. Optimal conditions comprised preculture for 2 days in liquid medium with 250 g.l^–1 sucrose, desiccation for 6 hours under the laminar flow or for 10–11 hours with silicagel followed by rapid freezing and slow thawing. Survival after freezing in liquid nitrogen ranged between 38 and 91% for the 5 varieties experimented. Cryopreservation did not modify the electrophoretic profiles for aminoleucine peptidases and amylases with plants of the variety Co 6415.Abbreviations BAP 6-benzylaminopurine - KIN Kinetin - EDTA ethylenediamine tetracetic acid - AMP aminoleucine peptidases - AMY amylases - RFLP restriction fragment length polymorphism     

Effect of fixation time on measurement of 2''-deoxyribonucleoside 5''-triphosphate pools in the rat embyro.

Values for the concentrations of deoxyribonucleoside triphosphates in rat embryos on day 12 of gestation, determined by high-pressure liquid chromatography, were artifactually two to three times as high in embryos fixed by cooling in ice/water followed by freezing on solid CO2, in 20s, as in those more rapidly/fixed in liquid N2, in 1 s.     

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.     

High-pressure freezing of isolated gastric glands provides new insight into the fine structure and subcellular localization of H+/K+-ATPase in gastric parietal cells.

High-pressure freezing (HPF) is currently the most reliable method to obtain an adequately frozen sample for high-resolution morphological evaluation. Here we applied the HPF technique to isolated rabbit gastric glands to reveal structural evidence that may be correlated with functional activity of gastric parietal cells. This approach provided well-preserved fine structure and excellent antigenicity of several parietal cell proteins. Microtubules were abundant in the cytoplasm and frequently appeared to be associating with tubulovesicles. Interestingly, many electron-dense coated vesicles were apparent around the intracellular canaliculi (IC) of resting parietal cells, consistent with active membrane retrieval from the apical membranes. Immunolabeling of H+/K+-ATPase was evident on the endocytic components (e.g., multivesicular bodies) and tubulovesicles. After histamine stimulation, the parietal cells characteristically showed expanded IC membranes with varied features of their apical microvilli. The labeling density of H+/K+-ATPase was four-fold higher on the IC membrane of stimulated parietal cells than on that of resting parietal cells. Immunolabeling of ezrin was clearly identified on the IC and basolateral membranes of parietal cells, corresponding to their F-actin-rich sites. The present findings provide a new insight into the correlation of cell structure and function in gastric parietal cells.     

On the preparation of cryosections for immunocytochemistry

The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.