Equivalence of the projected structure of thin catalase crystals preserved for electron microscopy by negative stain,glucose or embedding in the presence of tannic acid

Thin crystals of beef liver catalase have been examined by electron microscopy following various preservation procedures. In the first part of this investigation, micrographs of three principal projections were obtained from thin sections of micro-crystals embedded in the presence of tannic acid. Computer reconstructions confirmed the space group assignment of P2₁2₁2₁ and permitted the packing arrangement of the catalase tetramers to be deduced to a resolution of about 20 Å. These results corroborate the packing model for this crystal form proposed by Unwin (1975) on the basis of molecular modeling of one projection. In the second part of this investigation, the projected structures of the thin crystals in various preserving media were compared. The negative contrasting of crystals embedded in the presence of tannic acid was confirmed by direct comparison with nonembedded, negatively stained thin platelet crystals. In addition, good agreement at 20 Å resolution was observed between the structure of negatively stained crystals and the structure of crystal platelets preserved in glucose and examined by lowdose methods, while moderate agreement was established with the published data of Taylor (1978) for crystals embedded in thin ice films. Tannic acid alone was also found to serve as a suitable medium for preserving catalase crystals to a resolution of 3.7 Å as judged by electron diffraction. Overall, we demonstrate that projections obtained from thin sections of catalase crystals embedded in the presence of tannic acid can provide a reliable, negatively contrasted representation of the protein structure to 20 Å resolution. Examination of sectioned crystals could thus provide a useful adjunct to X-ray crystallographic studies of protein crystals and three-dimensional reconstruction of crystal thin sections should ultimately be possible.     

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)     

Ice Morphology: Fundamentals and Technological Applications in Foods

Freezing is the process of ice crystallization from supercooled water. Ice crystal morphology plays an important role in the textural and physical properties of frozen and frozen-thawed foods and in processes such as freeze drying, freeze concentration, and freeze texturization. Size and location of ice crystals are key in the quality of thawed tissue products. In ice cream, smaller ice crystals are preferred because large crystals results in an icy texture. In freeze drying, ice morphology influences the rate of sublimation and several morphological characteristics of the freeze-dried matrix as well as the biological activity of components (e.g., in pharmaceuticals). In freeze concentration, ice morphology influences the efficiency of separation of ice crystals from the concentrated solution. The cooling rate has been the most common variable controlling ice morphology in frozen and partly frozen systems. However, several new approaches show promise in controlling nucleation (consequently, ice morphology), among them are the use of ice nucleation agents, antifreeze proteins, ultrasound, and high pressure. This paper summarizes the fundamentals of freezing, methods of observation and measurement of ice morphology, and the role of ice morphology in technological applications.     

Ice crystals formed in tissue during cryosurgery. II. Electron microscopy

Tissues frozen by means of a cryosurgical probe have been examined by electron microscopy following techniques designed to preserve the ice crystal spaces.Ice crystals appeared similar whether tissues were quenched or not following cryosurgery and the various techniques of dehydration resulted in similar ice crystal architecture.Ice crystal spaces in the area deep to the freezing probe were intracellular both in epithelium and muscle although in the muscle zone some fibers contained large and others small crystal spaces. It is suggested that this might be due to variations in the local blood supply.At the periphery of the frozen area ice crystals were usually extracellular producing gross distortion of the cells which, however, retained intracellular structural integrity. These results are consistent with the belief of many workers that intracellular ice is lethal while extracellular ice is not, but no evidence of penetration of cell membrane by ice crystals was seen.     

DEVELOPMENT AND EVALUATION OF A FREEZE-ETCH,FREEZE-FRACTURE TECHNIQUE APPLIED TO DEVELOPING WHEAT ENDOSPERM

A technique was developed and evaluated whereby differentiating wheat endosperm tissue could be processed for the freeze-etch, freeze-fracture technique without the use of chemical fixatives. Field grown, developing hard red winter wheat (cv. Newton) caryopses were infiltrated with glycerol prior to freezing in liquid nitrogen-cooled Freon-22. Frozen samples were placed in cryogenic vials and stored in liquid nitrogen until needed. Freeze-fracture was carried out under standard conditions. Evaluation of replicas from glycerol-imbibed endosperm was made by comparing them to replicas obtained from freshly frozen untreated endosperm, and endosperm that had been prefixed in glutaraldehyde and paraformaldehyde. Other evaluations were made by comparing replicas of glycerol-treated endosperm to thin sections obtained from wheat that had been imbibed with glycerol, fixed, dehydrated, and infiltrated and embedded in epoxy resin for routine electron microscopy. The results indicate that if the unfixed glycerol-treated wheat endosperm is handled carefully, replicas can be obtained which show few artifacts due to glycerol and freezing. Typical artifacts such as vesiculation of RER, ice crystal damage, production of fusion intermediates, and membrane particle segregation can be nearly eliminated when this technique is applied to developing wheat endosperm.     

Artifacts associated with quick-freezing and freeze-drying

We have studied the structures produced when nonbiological samples were subjected to quick-freezing and freeze-drying with a liquid helium cooled freeze-slamming device. Samples examined in this way included sodium chloride, sucrose, and Tris buffer. A variety of filamentlike and trabeculumlike structures were formed in these preparations. These structures may represent eutectic mixtures formed during the growth of small ice crystals during the freezing process, and exposed during the rapid sublimation of pure ice during the etching process. Samples of biological membranes (isolated chloroplast membranes) were prepared in various buffers by means of this technique. In distilled water, excellent replicas of membrane surfaces were obtained. In salt solutions, however, the membranes appeared to be embedded in a network of thin filaments appearing very much like a cytoskeletal lattice. It is concluded that extreme caution must be used when employing this preparation technique for studies of cell architecture, and that extensive washing of cell components in distilled water may be necessary to obtain faithful representations of cell structure.     

Studying the Morphology of Lyophilized Protein Solids Using X-ray Micro-CT: Effect of Post-freeze Annealing and Controlled Nucleation

The objective of this study was to determine how different techniques used during the freezing step of lyophilization affect morphology of the dried protein solids. Aqueous solutions containing recombinant human albumin, trehalose, and sodium phosphate buffer were dried after their freezing by shelf-ramp cooling, immersion in liquid nitrogen, or controlled ice nucleation. Some shelf-frozen solutions were heat treated (annealed) before the vacuum drying. We used three-dimensional (3D) X-ray micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) to study the morphology of solids. The X-ray micro-CT images of the lyophilized microporous solids showed traces of varied size and structure ice crystals that were comparable to corresponding SEM images. A post-freeze heat treatment and a controlled nucleation both induced larger ice crystal ghosts in the solids. The variations in the structure of walls surrounding ice crystals, formed by the different freezing procedures, should affect the water vapor transition during the primary and secondary drying. Some solids also showed higher-density layer in the upper surface. Overall, the simple sample preparation procedures and the ample morphological information make the X-ray micro-CT appropriate for analyzing lyophilized pharmaceuticals.     

A Rapid Plastic Embedding Technique for Preparation of Three-Micron Thick Sections of Decalcified Hard Tissue

A 24 hour start-to-finish method is described for the preparation of three-micronthick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerizd by four hours heating at 42 C. Three-micron-thick sections are then easily prepared using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.     

A rapid plastic embedding technique for preparation of three-micron thick sections of decalcified hard tissue.

A 24 hour start-to-finish method is described for the preparation of three-micron-thick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerized by four hours heating at 42 C. Three-micron-thick sections are then easily prepared by using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.     

The formation and distribution of ice within dormant and deacclimated peach flower buds

Abstract A freeze-fixation technique was used to examine the distribution of ice crystals and the pattern of freezing in peach flower buds. In dormant buds, ice crystals formed at localized sites within the bud axis and scales. Ice crystal formation disrupted tissues and mechanical injury from repetitive freezethaw cycles was apparent. There was evidence of ice formation in the floral organs of dormant buds exposed to ?25°C but none observed in buds exposed to either ?5 or ?10°C. The distribution of ice crystals was different in deacclimated buds. In addition to large ice crystals within the subtending bud axis and scales, evidence of large crystals within the developing floral organs was noted. These crystals were most prominent in the lower portions of the developing flower and peduncle, and caused a separation of the epidermal layer from adjacent cells. The distribution of ice crystals within both dormant and deacclimated peach flower buds corroborated the results of previous thermal analysis experiments.     

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.     

The basis for hyperactivity of antifreeze proteins

Antifreeze proteins (AFPs) bind to the surface of ice crystals and lower the non-equilibrium freezing temperature of the icy solution below its melting point. We have recently reported the discovery of three novel hyperactive AFPs from a bacterium, a primitive insect and a fish, which, like two hyperactive AFPs previously recognized in beetles and moths, are considerably better at depressing the freezing point than most fish AFPs. When cooled below the non-equilibrium freezing temperature, ice crystals formed in the presence of any of five distinct, moderately active fish AFPs grow suddenly along the c-axis. Ice crystals formed in the presence of any of the five evolutionarily and structurally distinct hyperactive AFPs remain stable to lower temperatures, and then grow explosively in a direction normal to the c-axis when cooled below the freezing temperature. We argue that this one consistent distinction in the behaviour of these two classes of AFPs is the key to hyperactivity. Whereas both AFP classes bind irreversibly to ice, the hyperactive AFPs are better at preventing ice growth out of the basal planes.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

Influence of lipids on ice formation in cryoprotective media

Cryomicroscopic analysis demonstrated that two lipid preparations from marine vertebrates (<0.1%) and liposomes prepared from rainbow trout sperm lipids (<0.5%) efficiently hindered the growth of ice crystals during freezing of multicomponent cryoprotective media used for trout sperm cryopreservation. At higher lipid concentrations, crystals either did not form at all or had altered shape and blurred boundaries. Addition of egg yolk (10%) together with these lipids increased the size of crystal structures and markedly changed their shape.     

An Ice-Solvent Method of Drying Frozen Tissue for Plant Cytology

A freeze-dry method where cold absolute ethanol is used as a dehydrating agent in place of vacuum dehydration has been applied to various plant materials with good cytological results. The method involves: (a) freezing rapidly small pieces of tissue 1 cubic mm or less in partly frozen isopentane cooled with liquid nitrogen, (b) transferring quickly to vials of cold absolute ethanol at -41° to -45°C, and (c) holding within this temperature range for 3 days to dissolve the ice. A simply constructed cryostat is used to maintain the vials of absolute alcohol and tissue at the cold temperature. This consists of a semi-frozen constant temperature bath of either 65% ethanol or pure diethyl oxalate in a tightly covered beaker which fits within a large dewar flask half filled with dry ice. The bath is arranged so that it will be on top of and in contact with the dry ice but properly insulated to prevent freezing completely.

The resulting dried tissue is very unstable in either water or hot absolute ethanol; therefore, to prevent loss of cytological detail during further processing, the tissue must be treated to render the proteins insoluble. Either (a) replace the cold absolute ethanol in the tissue vials with cold (approx. -40°C) 75% ethanol, warm slowly to 60°C, and hold for 1 hour, or (b) replace with cold acidulated 95% ethanol (100 ml. of 95% ethanol + 0.30 ml. of glacial acetic acid), warm to room temperature, and hold for 30 minutes. Following either treatment the tissues are dehydrated to absolute alcohol and embedded in paraffin by the usual technics. Sections are attached to slides by flattening over warm water and drying.

When epidermis from onion bulbs was used as a basis of comparison of fixed and living material with the phase-contrast microscope, the mitochondria, plastids, and other fine structures in fixed preparations appear to be nearly identical with the living. Fat droplets disappear. With larger tissues such as onion root tips, thin freehand sections must be prepared before freezing to obtain good cytological results. The application of the method to cytochemical studies is discussed and in many ways it seems to be as useful as the freeze vacuum-dry method.     

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.     

Freezing of nonwoody plant tissues: IV. Nucleation sites for freezing and refreezing of onion bulb epidermal cells

A freezing microscope and videotape recorder were used to study ice formation in onion bulb epidermal cells during repeated freezing. Behavior during refreezing after freezing and thawing indicated that the barrier to ice crystal propagation previously reported in living cells was not damaged by freezing. Refreezing in many cells began at a site other than that responsible for the initial freezing.     

Theoretical analysis of the ice crystal size distribution in frozen aqueous specimens.

To estimate theoretically how suited different freezing techniques are for freezing of freeze-etch specimens, it is necessary to know the relationship between specimen cooling rate and the resulting average ice crystal size. Using a somewhat simplified theoretical analysis, we have derived the approximate ice crystal size distribution of nonvitrified frozen aqueous specimens frozen at different cooling rates. The derived size distribution was used to calculate the relationship between relative change in average ice crystal size, (delta l/l), and relative change in specimen cooling rate delta (dT/dt)/(dT/dt). We found this relationship to be (delta l/l) = -k X delta (dT/dt)/(dT/dt) where k = 1.0 when specimen solidification takes place at about -6 degrees C, and k congruent to 1.3 when it takes place at about -40 degrees C.     

ULTRASTRUCTURE AND CALCIUM TRANSPORT IN CRUSTACEAN MUSCLE MICROSOMES

Fragmented sarcoplasmic reticulum (FSR) from crustacean muscle was examined following preparation by a variety of electron microscopic techniques. The 30–40 A particles which appeared on the outer surface of FSR vesicles following negative staining were not observed following preparation by freeze-drying, freeze-etching, thin sectioning, or critical-point drying. Crustacean FSR exhibited high values of calcium uptake and extensive nodular formation in the presence of oxalate. 80–90 A diameter membrane particles were seen in freeze-etch preparations of both intact lobster muscle and FSR vesicles. Thin sections of FSR vesicles revealed a membrane thickness of 60–70 A. The membrane appeared to be triple layered, each layer having a thickness of 20–25 A.     

Potassium binding sites in muscle: Electron microscopic visualization of K,Rb, and Cs in freeze-dired preparations and autoradiography at liquid nitrogen temperature using86Rb and134Cs

Summary Normal frog sartorius muscles and muscles in which a major portion of the intracellular K⁺ was reversibly replaced by Rb⁺ or Cs⁺ were frozen, freeze-dried and embedded without chemical fixation or staining. Dry-cut sections of these preparations reveal striation patterns with higher contrast than those of wet-cut sections of the same preparation. The results suggest that in the living state the alkali metal ions are mainly localized in the A bands and Z lines of myofibrils. This idea is confirmed by a new autoradiographic technique by means of which the distribution of Rb⁺ and Cs⁺ in frozen-hydrated single muscle fibers has been investigated. The findings support the association-induction hypothesis according to which most cell K⁺ and other alkali-metal ions are not free in cell water but are adsorbed to beta- and gamma-carboxyl groups of cell proteins.