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The preservation of tissue fine structure during rapid freezing
... It requires over a 10 4 k/sec to prevent a cryohydrate phenomenon and a warp form to occur when freezing cells where over 60% of components was water. The ultrathin sectioning method by the quick freezing substitution fixation was established a long time ago, but the technique was complicated and there was a big problem in reproducibility (6)(7)(8)(9)(10)(11)(12)(13). This method has an advantage that fine structures are preserved which is suitable for immunoelectron microscopy. ...
It is challenging to demonstrate the capsule of Streptococcus pneumoniae (S. pneumoniae) under immunoelectron microscopy because of the thick mucopeptide cell wall hampering proper fixation. A novel rapid freeze fixation method was established to observe the capsule of S. pneumoniae. A strain of serotype 3 of S. pneumoniae isolate was analyzed after rapid freezing. An ethanol freezing-substitution fixing method was applied and immunohistochemical staining with osmium tetroxide was tested. The capsule was confirmed using the serotype 3 specific polyclonal antibodies labeled with colloidal gold particles. To the best of our knowledge, this is the first report of S. pneumoniae capsule by immunoelectron microscope.
... Freezing has become an increasingly important method of stabilizing biological structure and is an inherent aspect of such techniques as freezesubstitution, ultracryotomy, and freeze-cleaving' (4,5,21,24,25,29) . Although these techniques have been used primarily in studying ultrastructure, they can also be used as a direct probe to examine the ultrastructural alterations that result from freezing. ...
Freeze-cleaving can be used as a direct probe to examine the ultrastructural alterations of biological material due to freezing. We examined the thesis that at least two factors, which are oppositely dependent upon cooling velocity, determine the survival of cells subjected to freezing. According to this thesis, when cells are cooled at rates exceeding a critical velocity, a decrease in viability is caused by the presence of intracellular ice; but cells cooled at rates less than this critical velocity do not contain appreciable amounts of intracellular ice and are killed by prolonged exposure to a solution that is altered by the presence of ice. As a test of this hypothesis, we examined freeze-fractured replicas of the yeast Saccharomyces cerevisiae after suspensions had been cooled at rates ranging from 1.8 to 75,000 degrees C/min. Some of the frozen samples were cleaved and replicated immediately in order to minimize artifacts due to sample handling. Other samples were deeply etched or were rewarmed to -20 degrees C and recooled before replication. Yeast cells cooled at or above the rate necessary to preserve maximal viability ( approximately 7 degrees C/min) contained intracellular ice, whereas cells cooled below this rate showed no evidence of intracellular ice.
... Combination of fixatives [22, 27] and development of new resins [7, 24, 31] as well as working at subzero temperatures have been found to improve considerably the conditions for successful cytochemistry. Cryo-techniques including, cryo- ultramicrotomy [6, 45], cryo-fixation [11, 15, 20, 34], freeze-drying, freeze-substitution [14, 23, 29, 32, 35, 36], and molecular distillation drying [25], have slowly gained credibility since they were found to enhance preservation of biological properties with retention of structural details [16, 23, 42]. This has opened new perspectives in electron microscopy cytocbemistry. ...
Immunogold labeling of amylase obtained over zymogen granules of rat pancreatic acinar cells processed through cryofixation, molecular distillation drying and embedding in resins was found to be of high intensity and displayed a particular pattern. Indeed, it was concentrated in certain areas of the granules leaving others devoid of gold particles. This pattern of labeling reflects a strong compartmentalization of the secretory proteins within each granule. In order to assess this phenomenon, we have compared the intensities and the pattern of distribution of the labelings in tissues processed through: chemical fixation with embedding in various resins, cryo-ultramicrotomy and cryo-fixation followed by molecular distillation drying. Serials sections and double labeling experiments were performed for further evaluation of the results and for assessing artefactual displacement of proteins during tissue preparation. The results obtained indicate that the secretory proteins are indeed segregated within the granule which appears thus as a well organized structure. Cryo-fixation combined with molecular distillation appears thus to be superior in terms of preservation of protein antigenicity and retention of cellular components close to their living state.
Under certain conditions phosphotungstic acid (PTA) can be used as an effective electron stain for demonstrating polysaccharide moieties in tissue sections. There it selectively combines with the glycoproteins of basement membranes, the mucoid layers associated with brush borders, the mucus of goblet cells, the chondroitin sulfate of cartilage matrix, glyco-gen, and “glycocalyces” in general. The initial published investigation indicated that an acid environment was essential for this specificity, but the effect of pH was not systematically explored.
Rib cartilage of newborn rats was chosen for a detailed study of the effects of pH on PTA staining, particularly to provide ancillary information on the formation, secretion and deposition of chondroitin sulfate. Cartilage was prepared without chemical fixation by “freeze-substitution” with 70% glycol, or by “inert dehydration” with glycol at room temperature. Dehydrated material was embedded in prepolymerized hydroxy-propyl methacrylate without an intermediate solvent.
Modern microprobe analyzers can, under favorable conditions, detect elements in quantities of > 10−18 g and with a spatial resolution of 20–30 nm (Hall, 1977a). The first instrument was developed and introduced by Castaing (1951), and the technology was quickly and advantageously adopted by metallurgists and materials scientists. However, it has only been within the last decade that microprobes have captured the increasingly widespread attention of biologists studying biochemical and physiological processes in soft tissues (Chandler, 1978). The great attraction of microprobe analysis for the biologist is that it provides a facility for extending his knowledge of tissues, cells, organelles, and even molecules from the structural to a chemical or functional level. It is interesting, therefore, to reflect briefly on the relative slowness of biologists to exploit this undoubtedly powerful and exciting technology.
A practical, working histologist or cytologist in a sense requires under-standing of what he is doing at two different levels. For convenience and general productivity he knows very well he has to “fix” tissues and cells as a first preparative step. However, he is quite aware that this is basically a crime against nature, and no matter how sophisticated the fixation procedure may be, there surely are always lingering doubts as to how closely the final image resembles the living object. One has to define terms. I suppose originally fixation carried no more of a connotation than that biological products were rendered insoluble. Later it would have been appropriate to think of fixatives as being mainly protein coagulants. Now we talk of fixatives, at least good ones, as being cross-linking agents which do their job quite subtly, even at macromolecular levels of resolution. However, even in the last decade, we have radically changed our ideas about what constitutes a good fixative, as we relegated osmium tetroxide to a secondary role and advanced aldehyde fixation to a position of primacy. As objective scientists it at least behooves us to find and explore other ways of preserving tissue fine structure so that at the very least we have independent criteria for the evaluation of fixation procedures.
Microplasmodia of the acellular slime moldPhysarum polycephalum have developed an extensive extracellular slime layer which amounts to 75% of the volume of the total biomass. The slime layer covers the entire plasma membrane including the cell surface invagination system and represents a humid compartment protecting the organism against damaging influence from the surrounding environment.
Histochemical treatment with cationic substances revealed that the slime is mostly composed of acid mucopolysaccharides: intensive staining was obtained by colloidal or dialyzed iron, Ruthenium Red, acriflavine and lanthanum hydroxide.
At the macromolecular level the slime layer is composed of thin filaments showing a parallel course to the plasma membrane. The slime filaments are very sensitive to fixation and dehydration procedures and tend to form thicker aggregates.
From feeding experiments using different fine-particulated markers (Thorotrast, Aerosil) it can be concluded that the slime layer exhibits an important function with regard to the controlled transport and uptake of substances. In particular, the indentations of the cell membrane invagination system running perpendicular from the microplasmodial surface to the cell interior represent preferred pathways for the selective diffusion of macromolecules.
Pretreatment of spinal cord with ethylene glycol permits long-term storage of the tissue at -70°C prior to isolation and biochemical analysis of the cell bodies of spinal motoneurons. The method is useful for storing spinal tissue from laboratory animals, as well as from human post mortem specimens, where aliquots of tissue may then be used for motoneuron isolation over an indefinitely long period. In addition to inhibiting the loss of soluble proteins from the neurons during freezing and thawing, cryoprotection increases the yield and improves the appearance of the isolated cell bodies. The method should aid biochemical studies of many kinds of neuronal subpopulations isolated from small amounts of starting material.
Analysis of pulmonary structure-function relationships by microscopy requites that the lung be fixed under carefully controlled physiological conditions, since lung structure is extremely responsive to the relationship between airway and vascular pressures. Unfortunately, standard post-mortem fixation techniques leave some doubt as to the exact relationship between these pressures during fixation. This problem can be circumvented by stabilizing lung structure by rapid freezing under carefully controlled physiologic conditions. Using ethylene glycol in a freeze substitution technique we have developed procedures which yield a degree of preservation compatible with the high degree of resolution of the electron microscope. These can be used to obtain a more detailed understanding of pulmonary structure-function relationships under well-defined physiological conditions.
A method is described by which frozen hydrated bovine muscle is prepared for the transmission electron microscope. The importance of maintaining specimen temperatures at −110°C (163°K), or below, during the preparation of frozen thin sections and during specimen transfer and microscopical observations is emphasized and documented.
Electron micrographs of a section of muscle were followed through three levels of dehydration. Electron micrographs of frozen hydrated muscle were compared with micrographs of fixed and embedded muscle. In general, the micromorphology of muscle prepared by the two methods was found to be quite similar.
A combined technique of the rapid freezing, freeze substitution–fixation method and the osmium–DMSO-osmium method was devised. By this combined method we clearly observed the architecture of intracellular components in three dimensions. Morphological characteristics were generally similar to those of tissue prepared by the osmium–DMSO-osmium method but different in some respects. Mucigen droplets in intestinal goblet cells, for example, appeared as separated spheres, while in specimens prepared by chemical fixation they were observed as a mass of fused droplets. In the Golgi complex, all cisternae were extremely flat, although they usually dilated on the cis side after chemical fixation. Particles on the mitochondrial tubules of liver cells were well distinguished. They were mushroom shaped, as are those observed by negative staining. The combined method, that is, the rapid freezing, osmium–DMSO-osmium method, is thought to be effective for studying the true structure of intracellular components by scanning electron microscopy.
In kidney tissue, prepared without fixation by freeze-substitution or inert dehydration, a system of relatively coarse filaments is found, particularly in the capsular cells of the renal corpuscles and at the base of proximal tubular cells. These filaments are associated with characteristic cytoplasm. This system of filaments and cytoplasm also is seen in the secretory juxtaglomerular cells, and occasionally in cells of the distal convoluted tubules. Small numbers of filaments also are found in agranular “Polkissen” and mesangial cells, and in the visceral epithelial cells of the renal corpuscles, as well as in interstitial cells in tubular areas. In all essential respects these filaments, and the cytoplasm they are embedded in, resemble the system of coarse filaments found in smooth muscle cells under similar circumstances of preservation. It is believed that these are myosin filaments, probably associated with tropomyosin B. Unfortunately, the method of preservation does not disclose thin filaments that might be regarded as actin. Actin filaments are not seen in mammalian smooth muscle either under identical circumstances and are demonstrably largely depolymerized in striated muscle (11). However, the present demonstration of a system presumably containing myosin in epithelial parts of the kidney is interpreted as signifying that these areas have myoid properties, and should be regarded as being myoepithelial in character.
summaryA number of fixation techniques were used on cubes of dry cotyledonary tissue of lettuce seed to develop a satisfactory technique for the anhydrous fixation of dry seed material for electron microscopy. Fixation with formaldehyde or acrolein vapours in combination with Spurr's resin embedding produced satisfactory sections, while protocols involving the use of osmium tetroxide vapours resulted in brittle specimens that were difficult to section. The use of glycerol as a vehicle for anhydrous aldehyde fixation, followed by subsequent aqueous processing, introduced artifacts in membrane infrastructure. When model phospholipid systems, known to exist in the hexagonal phase, were prepared for electron microscopy and compared with anhydrously fixed cotyledonary tissue, the results suggested that dry seed membranes exist in a lamellar phase. It is proposed that leakage from imbibing seeds may be the result of physical/molecular changes in the membranes rather than of a phase change from the hexagonal to the lamellar state.
The muscular system of the ventral diaphragm of Locusta consists of parallel muscle fibers, which are connected by structures like intercalated discs within transverse bridges. Each muscle fiber is enveloped by a thick sheath of connective tissue. The fibers are attached to the cuticule by means of hypodermic cells with tonofibrils.
Uncontracted sarcomeres have a length of 5 μm and more. The H-band is slightly indicated, a M-line is not visible. Actin and myosin filaments (diameter 72 respectively 160 AE) are out of register. Moreover there is a third and very thin type of filaments. The Z-band has an undulating shape and collects the actin filaments into bundles. Mitochondria lie on either side of the Z-band. The T-system invaginates as sarcolemmal clefts and continues its course inwards as tubuli. The sarcolemmal clefts are connected with the Z-band. The T-system and the sarcoplasmic reticulum are joined by diads of irregular distribution.
Limitated deprivation of Ca++ causes waves of contraction with the length of several sarcomeres.
Contrary to standard methods the freeze-substitution causes some modifications such as shrinking of the sarcoplasm, thickening of the myosin filaments, vacuolization of mitochondria and vesicular system. Within the waves of contraction the A-band shortenes with increasing sarcomere contraction. The diameter of the myosin filaments measures 172 AE, the 305 AE-period of the cross-bridges remains constant within the middle of the filaments.
According to chemical data, methanol raises the shrinkage temperature of collagen significantly more than ethanol (86° C versus 70° C). Since increase of shrinkage temperature appears desirable in tissues to be embedded in paraffin, methanol was substituted for ethanol in Carnoy's fluid. This methanol-Carnoy mixture is referred to as methacarn solution. The fixation-embedding procedure was similar to that described in the study of Carnoy fixation. Methacarn-fixed sections showed little or no shrinkage and compared well with material fixed in Carnoy's or Zenker's fluid. Myofibrils, especially in endothelial and epithelial cells, were more prominent in methacarn- than in Carnoy-fixed tissues.
A review of the chemical literature showed that methanol, ethanol and chloroform stabilize or even enhance helical conformations of proteins, presumably by strengthening of hydrogen bonds. Interference with hydrophobic bonds causes unfolding and/or structural rearrangements in globular proteins. The twin-helical structure of DNA collapses in alcoholic solutions. Hence, methacarn fixation can be expected to preserve the helical proteins in myofibrils and collagen, but the conformations of globular proteins and DNA will be significantly altered. Literature on conformational effects produced by fixatives used in electron microscopy was also reviewed. Glutaraldehyde and OsO4 cause considerable loss of helix (22–29% and 39–66% respectively). KMnO4 and glutaraldehyde followed by OsO4 produce extensive transitions from helical to random-coil conformations similar to those seen in powerful denaturants such as 8 M urea. Evidently these fixatives are unsuitable for studies of helical proteins. In contrast ethylene glycol preserves helical conformations.
Die Gefriertrocknung von Kryostatschnitten kann, im Gegensatz zur Trockung von Gewebsblcken, manometrisch kontrolliert werden. Das in die herkmmlichen Gefriertrockungsanlagen eingebaute Pirani-Element gengt fr diesen Zweck vollauf. Es ist damit mglich, das Verfahren ohne jedes Risiko durch Regelung der Khltemperatur abzukrzen. Die Druckkurve zeigt eindeutig, wann die Schnitte eine fr histologische und histochemische Reaktionen optimale Restfeuchtigkeit erreicht haben.In contrast to freeze-drying of tissue blocks the freeze-drying of cryostat sections can be controlled manometrically. The Pirani-element has been found to be fully sufficient for this task. It is therefore possible to shorten the procedure without any risk by regulating the cooling temperature. The pressure curve shows clearly, when the sections have reached optimum humidity for histological and histochemical staining.
The interpretation of freeze-etching pictures of biological membranes is difficult but very important for the resulting informations. The results of our studies of various biological membranes led to the following conclusions:
1.
The membranes are splitted during freeze fracturing and the visible membrane faces are views at one half of the membrane (from the membrane interior).
2.
The two membrane faces which arise from the splitting of a membrane are different. In all cases there are more particles found on the face which is in close connection with the plasma [the (+)(−) distinction of the membrane faces].
3.
The number of particles at the membrane faces shows differences which may be in relation to the function of every type of membrane. A membrane model is proposed.
The seminiferous tubule of the hamster is surrounded by a layer of specialized cells. The fine structure of these cells is similar to that of smooth muscle. Their cytoplasm contains numerous fine filaments and dense areas; in addition, vesicles can be seen forming on the plasmalemma and are also free in the cytoplasm. It is possible that their contractions aid in the transport of spermatozoa through the seminiferous tubules.
Freshly dissected bone from embryonic chicks and young postnatal mice has been prepared for electron microscopy by ultracryomicrotomy. Nuclei, ribosomes, endoplasmic reticulum, and a large number of dense mitochondrial granules within osteogenic cells are observed in unfixed, undecalcified, and unstained sections, obtained with dry knives in the temperature range of −60 to −80°C. The presence of granules in mitochondria from tissues not exposed to solvents strongly suggests that the granules contain a solid phase of calcium phosphate in vivo. Small clusters of lathe-shaped and dense needle-like particles are observed in the extracellular tissue space closest to the osteoblasts. Tilting of unstained frozen sections indicates that the needle-like particles are platelets viewed obliquely or on edge. The rapid and massive changes in the amount of mineral deposited arise principally by an increase in the number of mineral particles, rather than their growth in size. Sections prepared by alternative methods combined with ultracryomicrotomy present similar features.
Selected area electron diffraction and high spatial resolution, nondispersive electron probe Xray microanalysis have been used to examine the nature of the solid phase mineral deposits of specific ultrastructural components in freshly dissected, undecalcified bone tissue from embryonic chicks, prepared anhydrously with either 100% ethylene glycol or dry ultracryomicrotomy. No electron diffraction patterns of a specific calcium phosphate solid phase are generated from the dense mitochondrial granules of osteoblasts, shown to contain calcium and phosphorus by electron probe microanalysis, and from the early mineral deposits from certain regions of newly synthesized bone. Similar results are obtained from a synthetic preparation of an amorphous calcium phosphate. Absence of an electron diffraction pattern is not caused by an insufficient mass of the calcium phosphate solid phase. The electron diffraction patterns of the more heavily mineralized older regions of the bone show the reflections and characteristics of poorly crystalline hydroxyapatite. There is a progressive change in the electron diffraction pattern approaching that of crystalline hydroxyapatite with increasing distance from the periosteal surface. Electron probe microanalysis of the same tissue components and regions shows that the changes in the electron diffraction characteristics are accompanied by an increase in the Ca/P ratios of the solid mineral phase.
Ultrastructure of freeze-substituted hyphal tip cells of Fusarium acuminatum was compared to that after conventional chemical fixation. As a result of improved preservation by a modified freeze-substitution procedure, we document here: (i) an extensive system of smooth membrane cisternae with electron-dense contents, including tubules and single, fenestrated cisternae; (ii) two types of apical vesicles with a homogeneously electron-dense interior; (iii) an extensive system of microtubules which extend well into the apical dome; (iv) four distinct layers in the apical “primary” cell wall, with basipetal thickening due almost exclusively to enlargement of the outer cell wall layer; (v) hexagonal microvesicles; and (vi) a smooth profile of all cellular membranes and most organelles.
Within the stated limits of cell fine structure preservation and within the observed anatomical limits specified in the Results and Discussion sections, a freeze-substitution method using an ethylene glycol-Hank's solution eutectic mixture with a glutaraldehyde additive can be used to effectively prepare undecalcified human dentin for electron microscopy. The ultrastructual appearance of the odontoblast cell body and the odontoblastic process subjected to freeze-substitution differs from that seen with conventional chemical fixation. Artifacts produced by freeze-substitution differ in appearance and frequency of occurrence from those produced by glutaraldehyde-osmium tetroxide sequential double fixation. The cellular component of dentin shows greater structural preservation of protein when it is subjected to freeze-substitution that when it is prepared by conventional chemical fixation. The absence of ice crystal defects in the odontoblastic process in calcified dentin and the presence of ice crystal defects in the odontoblast cell body suggest that intracellular water in the odontoblastic process in the calcified dentin may exist in a more highly structured state than intracellular water in the odontoblast cell body. If intracellular water exists in a more highly structured state in the odontoblastic process of the calcified dentin than in the cell body, the ratio of protein molecules to cytoplasmic volume may be greater in the odontoblastic process than in the cell body. After glutaraldehyde-osmium tetroxide sequential double fixation, the use of graded alcohol dehydration obtained cell fine structure preservation and artifact control superior to that obtained by use of ethylene glycol cryodehydration. Further refinements of the freeze-substitution technique, as it applies to the preparation of undecalcified human dentin, are necessary to increase the amount of cellular preservation, to decrease the number of ice crystal artifacts, and to improve the overall quality of cell fine structure preservation.
A quick, simple protocol is described for the preparation of tissue for electron immunocytochemistry without the use of fixatives or deleterious solvents. Fresh, normal human colon was rapidly dehydrated in ethanediol (ethylene glycol) then embedded directly in low-acid glycol methacrylate. Using both mono- and polyclonal antibodies, in conjunction with colloidal gold probes, a range of intra- and extracellular epitopes were localized; these epitopes included lysozyme, chromogranin, desmin and collagen IV. Overall, the tissue compared well with material fixed in glutaraldehyde, partially dehydrated and embedded in LR White acrylic resin. Ultrastructural detail was good and was further enhanced, without affecting probe density and epitope localization, by the addition of 1% tannic acid or 1% uranyl acetate to the dehydrant. The technique is applicable to a wide range of tissues, allowing excellent antigen retention which might prove useful for the immunolocalization of sensitive epitopes.
The author presents a rather detailed review of the varied morphological techniques available for the study of ribosomal structure. It is pointed out that preparation and technique has not developed to the point of fulfilling the theoretical ability of the electron microscope to resolve structure, but that negative staining and dark field electron microscopy offer new structural information. The author further points out the preparative techniques and source of material studied, and the structural differences found in membrane related ribosomes and poly ribosomes. (McNary - Boston, Mass.)
Promitochondria of anaerobically grown Saccharomyces cerevisiae were selectively labeled in vivo by incubating the cells with [(3)H]leucine and cycloheximide. When the labeled cells were washed free of cycloheximide and adapted to oxygen in the presence of unlabeled leucine, the respiring mitochondria formed during adaptation proved to be radioactive. In contrast, only insignificant label was found in iso-1-cytochrome c which is synthesized de novo during adaptation. Respiratory adaptation of anaerobically grown yeast thus involves differentiation of promitochondrial organelles.
Eggs of the marine clam Spisula solidissima were examined by the technique of freeze-substitution. Morphology of the cytoplasm was investigated as a function of cellular water content using osmotic means to control water concentration. In eggs frozen in less than double strength sea water, ice crystallization usually occurred throughout the cell. In eggs frozen in double to triple strength sea water, 10–75% of the eggs were devoid of ice crystals in the cytoplasm and in triple strength sea water, also in the nucleus. From these results and from a detailed analysis of cells in which both ice crystals and ice crystal-free regions occurred it is concluded that cells or parts of cells may be frozen free of ice crystals if their free water content is reduced. Reduction in free water content may occur in several ways: deliberate dehydration by osmotic means; accidental dehydration by evaporation of water from cells prior to freezing; osmotic dehydration of one region by an adjacent one in which ice crystallization is in progress. In ice crystal-free cells, all common cell inclusions could be recognized.
Studies of ice crystal-free eggs of the clam Spisula solidissima using freeze-substitution techniques are described. Formation of ice in cells may be avoided by removing sufficient intracellular water prior to freezing. After such treatment, most subcellular inclusions can be recognized and have dimensions similar to those seen after chemical fixation. Golgi regions, ribosomes, endoplasmic reticulum, nuclear envelope, annulate lamellae, yolk and the vitelline membrane resemble their counterparts in chemically fixed eggs. Mitochondria, however, lack the space normally found between cristae walls, and the resulting structure resembles two unit membranes “back to back.” Several inclusions and structures not seen in chemically fixed eggs are described: light bodies are found in groups of two to eight near the Golgi regions; cores occur in the microvilli of the vitelline membrane, microtubules are found in the cytoplasm of the unfertilized egg and 220 to 250 Å particles, which are not ribosomes, occur regularly arranged along microtubules.
The size and distribution of ice crystals formed during cryosurgical procedures in intact animals are not clear. In the present experiment oral mucosa was frozen in situ by means of a surface applied cold probe and was excised and freeze substituted while in the frozen state. It was shown that the form of the frozen tissue was preserved during this procedure and the area frozen was divisible into a zone representing the central part of the lesion and a peripheral zone separating this from normal tissue. Ice crystals within the body of the lesion were intracellular in location but varied somewhat in size. Ice crystals in the boundary zone appeared to be intracellular in the epithelium and both intra- and extracellular in the muscle fibres.It is suggested that the intracellular crystals in the body of the frozen area result in cell death while the extracellular ice in the boundary zone results in a less predictable response.
There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from −20 ° to −50 °C and then rapidly cooled to −196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above −40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as −45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as −20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.
The effect of different freeze-thaw regimes on the ultrastructure of tomato fruit parenchyma tissue has been presented. Ice patterns formed as a result of the freezing phase were also determined, using freeze-substitution and freeze-etching techniques.Fine structure of these highly vacuolated, high moisture cells typically comprising most plant tissues was extensively altered by all freeze-thaw combinations tried. Membranous components of the protoplasm were especially affected. Nevertheless, apparent damage was minimal following intermediate rates of freezing and thawing. A possible explanation based on the size of ice crystals formed during freezing was discussed. The general results were discussed in relation to freeze-thaw-induced ultrastructural changes that have been reported for animal cells and tissues.
Electron microscopic examination of Chinese hamster tissue-culture cells showed that freezing and thawing result in structural alterations, the type and magnitude of which depend on the cooling and warming velocities used. Cells suspended in Hanks balanced salt solution and cooled to −196 °C at rates exceeding the survival optimum exhibit different patterns and extents of ultrastructural alterations than do cells cooled or warmed at rates lower than optimum. Even though the addition of 0.4 M dimethyl sulfoxide confers some protection, in terms of survival, it does not prevent structural alterations. The types of alterations, however, differ from those found in cells frozen in Hanks alone. Freeze-thaw treatments producing similar percentages of cell survival do not necessarily cause similar structural alterations, nor is there a simple correlation between structural alterations and survival.
When Chinese hamster tissue-culture cells are frozen in a variety of suspending media, the percentage of cells surviving is maximal at optimum cooling rates, rates that are 2–4 orders of magnitude lower than those used to freeze cells for subsequent processing by the electron microscopy techniques of freeze-cleaving and freeze-substitution. The existence of such optima suggests that at least two factors dependent on cooling rate interact to determine the ultimate survival of a frozen-thawed cell. Other data are consistent with the view that the causes of injury in rapidly and slowly frozen cells are different. First, cells frozen rapidly in 0.4 M solutions of sucrose, glycerol, and dimethyl sulfoxide, or in 0.004 M polyvinylpyrrolidone, are inactivated to a much greater extent by slow warming than are cells frozen slowly in those solutions; that is, cells frozen at rates greater than the optimum are considerably more sensitive to slow warming. Second, the inactivation rate of cells frozen rapidly in glycerol is greater at −40 °C than that of cells frozen slowly. Third, the temperatures at which cells are killed as they are slowly frozen are very different from those observed during the slow warming of rapidly frozen cells. The precise nature of the two factors remains uncertain, but indirect evidence suggests that cells cooled slower than optimum are killed by alterations in the properties of the extracellular and intracellular solution induced by ice formation (e.g., high solute concentrations), and that cells cooled faster than optimum are killed by the formation of intracellular ice and its subsequent recrystallization during warming. Such intracellular recrystallization may be a potentially serious source of artifacts in frozen material processed for electron microscopy at temperatures above −60 °C, and perhaps even above −100 °C.
Freeze-thaw-induced disruption and disorganization of tomato fruit ultrastructure were reduced by immersing the tissue in hypertonic sucrose solution before freezing. Other “protective” agents similarly used appeared less effective. The results with sucrose, while not indicative of viability in this very frost-sensitive tissue, are discussed from the standpoint of plasmolytic and other possible protective mechanisms.
In the past decade some success has been achieved in the cryofixation of biological materials for use in ultrastructural research. The general change in attitude toward the need for advanced cryofixation methods, which allow the processing of biomaterials in the native state (i.e., without any chemical pretreatments), must also be viewed as a success. Previously, attempts along these lines were not always adequately appreciated. Four factors mainly have caused some rethinking: First, evidence has accumulated that any chemical pretreatment introduces the hazard of ultrastructural artifacts. Second, the new techniques for element analysis (from advanced cryosectioning to X-ray microprobe analysis) cannot be fully exploited with chemically altered materials. Third, rapid freezing offers a new possibility to catch fast dynamic processes. Fourth, it allows the investigation of dissolved hydrated proteins and molecular assemblies by electron microscopy. With this in mind, we believe that cryofixation will be of increasing importance for cellular and molecular biology.
In order to study the structure of membranes of tubular myelin and lamellar bodies (pulmonary surfactant), tissue from adult rat lung was prepared for freeze fracture by the rapid freezing method of Heuser et al. which requires no prior fixation or cryoprotection. Other tissues were freeze substituted and thin sectioned. A layer of tissue 50-100 mu thick was well preserved, but alveoli were partially collapsed. Freeze-fractured lamellar bodies were composed of tightly packed stacks of smooth lamellae about 100 A thick. Cross-fractures through tubular myelin exposed membranes organized into the square lattice described earlier. Fractures parallel to the longitudinal axis exposed particles arranged in rows which coincided with the corners. Membrane faces between the rows were smooth. These observations suggest (1) that in vivo the content of lamellar bodies is most likely arranged in layers, (2) that tubular myelin is present in tissues unexposed to fixatives or lipid solvents, (3) that smooth-surfaced lamellar body membranes become particulate when they form tubular myelin, and (4) that chemical fixation does not alter the general appearance of tubular myelin but it may affect lattice dimensions.
The ultrastructure of nerve and glial cells in the cerebral and cerebellar cortices of mice was studied after rapid freezing followed by substitution fixation. The cerebral and cerebellar cortices were frozen by bringing them into contact with a polished pure copper block cooled at a temperature of about -196 degrees C. The tissues were fixed and substituted in acetone containing 2-4% OsO4 at -78 degrees C for 2-3 days and then prepared for electron microscopy. Tissue fixed by this method displayed the following characteristics. (1) The contour of cells, processes and intracellular membrane systems was smooth. (2) The extracellular spaces were of variable widths. (3) Microtubules were well preserved and were often observed to extend into nerve terminals and to run close to presynaptic membranes. (4) The matrix of cytoplasm and mitochondria was electron dense. Dense granules, possibly binding sites of divalent cations, were often found in the mitochondrial matrix. (5) The plasma membrane of neuronal processes was thicker than that of glial processes. (6) The plasma membranes of nerve fibres and glial processes appeared asymmetrical, the inner leaflet being slightly thicker than the outer leaflet, whereas membranes of cell organelles such as smooth endoplasmic reticulum. Golgi bodies, lysosomes, multivesicular bodies, mitochondria and synaptic vesicles, were symmetrical.
New data on the ultrastructural features of the elasmoid scales of Carassius auratus have been obtained by use of rapid freezing with subsequent freeze-substitution in anhydrous solvents. These are compared with the results obtained using conventional aqueous fixatives. The external layer of the scales is composed of randomly oriented collagen fibres. In the first stages of mineralization, mineral deposits are located in the interfibrillary substance where dense granules appear to be active sites of mineralization. Spheritic mineralization occurs in this layer. The fibrillary plate is composed of two kinds of collagen fibres. Most of them are organized in lamellae forming the "plywood-like structure". They are thicker than the so-called "TC fibres", which are oriented from the basal part towards the superficial layer. These TC fibres are involved in the first stages of mineral deposition in the fibrillary plate where inotropic mineralization occurs. The mineral phase is almost always located in the interfibrillary matrix in both layers of the elasmoid scale. In this respect, teleost scales differ from those described so far in other lower vertebrates.
The rapid-freezing and freeze-substitution method fixes a specimen as if it were prepared before excision. We used this method to study the stria vascularis of guinea pigs using electron microscopy. Findings were essentially the same as those obtained with conventional chemical fixation, although freeze substitution made it possible to observe the membrane structures in a smoother and more linear manner. This method seems to be the procedure of choice for studying the instantaneous movement and behavior of cells.
Small pieces of mouse pancreas were rapidly frozen in helium II, substituted in methanol at -75°C., and embedded in methacrylate
by ultraviolet polymerization in the cold. The unstained cells show a structure similar to that after OsO4 fixation, except that the RNP particles have little or no contrast and the mitochondria and Golgi zones appear as grey areas
without internal structure. After staining the sections by floating them on solutions of lead acetate or osmium tetroxide,
there is an increase in contrast of RNP particles, ergastoplasmic membranes, and zymogen granules. Mitochondrial and Golgi
membranes, zymogen granule membranes, and a membrane along the outside of the ergastoplasmic cisterna appear in negative contrast.
The structure of the ergastoplasm, the existence of RNP particles, and the production of negative contrast are discussed.
A modification of Gomori's method for acid phosphatase produces a lead deposit around the periphery of the zymogen granules.
Possibly this deposit does not represent the true site of the enzyme, but the results show the feasibility of histochemistry
at the level of resolution of the electron microscope.
It was attempted to preserve the water distribution in central nervous tissue by rapid freezing followed by substitution fixation at low temperature. The vermis of the cerebellum of white mice was frozen by bringing it into contact with a polished silver mirror maintained at a temperature of about -207 degrees C. The tissue was subjected to substitution fixation in acetone containing 2 per cent OsO(4) at -85 degrees C for 2 days, and then prepared for electron microscopy by embedding in Maraglas, sectioning, and staining with lead citrate or uranyl acetate and lead. Cerebellum frozen within 30 seconds of circulatory arrest was compared with cerebellum frozen after 8 minutes' asphyxiation. From impedance measurements under these conditions, it could be expected that in the former tissue the electrolyte and water distribution is similar to that in the normal, oxygenated cerebellum, whereas in the asphyxiated tissue a transport of water and electrolytes into the intracellular compartment has taken place. Electron micrographs of tissue frozen shortly after circulatory arrest revealed the presence of an appreciable extracellular space between the axons of granular layer cells. Between glia, dendrites, and presynaptic endings the usual narrow clefts and even tight junctions were found. Also the synaptic cleft was of the usual width (250 to 300 A). In asphyxiated tissue, the extracellular space between the axons is either completely obliterated (tight junctions) or reduced to narrow clefts between apposing cell surfaces.
The hydroxylation of trimethylethylene and cyclohexene in t-butyl alcohol with hydrogen peroxide in the presence of osmium tetroxide as catalyst has been studied spectroscopically. It has been found that the maximum absorption band due to osmium tetroxide-hydrogen peroxide in t-butyl alcohol shifted during hydroxylation from 244 to 286 mμ with trimethylethylene and 288 mμ with cyclohexene. These maxima were also observed to occur when osmium tetroxide was allowed to react in t-butyl alcohol with vicinal glycols in the absence of hydrogen peroxide. In the presence of excess hydrogen peroxide these maxima shifted back to the maximum of osmium tetroxide-hydrogen peroxide mixture. It has been concluded that the maxima at 286-288 mμ are due to complexes between osmium tetroxide and the glycols. These complexes were also studied by means of paper chromatography and in the case of ethylene glycol and pinacol they were actually detected and separated.
The preceding discussion indicates the value of the erythrocyte as a simple biological system in studying biophysical and biochemical aspects of freezing injury and protection. A beginning has been made toward an understanding of the mechanisms of action of cryoprotective compounds. Observations and concepts have been described which may lead toward a more generalized interpretation of freezing injury. Cryobiologists should strive to find similarities among their different systems and freezing phenomena rather than to compartmentalize their observations. Only then can basic cryobiology become more than a descriptive science and applied cryobiology more than design by empiricism.
Remarkable cytoplasmic detail can be preserved without chemical fixation if living tissues are properly dehydrated with increasing concentrations of inert, water-soluble, permeable, organic substances. Success is thought to depend upon adequate stabilization and consequent immobilization of the macromolecular systems before physiologically damaging the cytomembranes, particularly the plasma membranes. Thus, the most critical period is midway in dehydration when the rate and kind of exchange appears to be of great significance. Once immobilization is achieved, the final stages of dehydration can be completed in a variety of ways without importantly altering the end result. The simplest successful procedure is to use ethylene glycol as the dehydrating agent and to transfer the tissue directly from this into prepolymerized hydroxypropyl methacrylate. Glycerol and glucose syrup can be substituted for the first stage of dehydration, and then tissue is transferred to ethylene glycol to complete dehydration and allow subsequent infiltration with the hydroxypropyl methacrylate embedding mixture. If other embedments are desired, Cellosolve can serve as an intermediate solvent. Dimethyl sulfoxide and urea have not so far worked well as primary dehydrating agents. Methanol has been useless. Postdehydration fixation with anhydrous crotonaldehyde adds little or nothing to the final image. Treatment with osmium tetroxide dissolved in anhydrous Cellosolve in general degrades the final pattern. Fixation with glutaraldehyde and/or osmium tetroxide after partial dehydration is possible.The described method of tissue preparation, which may be termed “inert dehydration,” is basically a physical method, presumably leaving proteinaceous systems preserved essentially in their native states. Hopefully, the technique will find histochemical, immunological, and autoradiographic applications. The general method of dehydration is easily performed, and quantities of tissue can be processed reliably. Thus tissue can be stored for long periods in glycol or glycerol, and subsequently be further processed for biochemical or morphological study.
Immersion of frozen-dried tissue into methacrylate under high vacuum at a temperature below the boiling point of methacrylate at that pressure makes it possible to obtain a perfect penetration of the methacrylate. The polymerization according to Müller (3) by u.v. light in the cold gives satisfactory results. These techniques for immersion and polymerization prevent an extensive vacuolization of the tissue due to improper embedding. The vacuolization due to ice crystal formation does not interfere in a critical way with the structural organization of the pancreas cells studied so far and represents an easily recognizable artifact.Preliminary observations on frozen-dried pancreas tissue confirm the geometrical structural pattern observed in mitochondria in osmium-fixed material. The basic membrane of the α-cytomembranes also has its counterpart in the frozen-dried material. However, the contrast conditions are such that the picture appears as the negative of that of osmium-fixed material.The opaque 150 Å particles (“RNA particles”), or any component that would correspond to them, have not been observed.
Microsomal preparations from rat brain were centrifuged in various density gradients obtained with different concentrations of sucrose. The different zones of the gradients were analyzed chemically as to their protein and RNA content and examined with the electron microscope as to their content of particles and membrane structures. A satisfactory separation of the two components of the microsomal preparations was obtained. The membranes were recovered in the upper zones with low density, whereas the particles were obtained in high amount, with only a few membranes left, in the lower zones, with high density. The concentration of particles was fairly well correlated with the concentration of RNA in the different zones, thus giving further evidence of the association of RNA with the particle component of the microsome preparations. The centrifugation in density gradient represents a quick and simple method for the isolation of RNA-rich particles.
An equipment and technique for fixation of tissues by freeze-drying is described. Satisfactory preservation of the structures is obtained both for light microscopy and electron microscopy.In many of the specimens, however, damage to the ultrastructure resolvable by the electron microscope is impossible to avoid. An analysis has been made of the cause of these artifacts and, probably, the critical stage in the treatment is the impregnation of the dry specimens with the fluid plastic. The frequency of occurrence of the damage may be decreased by treating the dry specimens with osmium-tetroxide vapor. It is also important to maintain the specimens below − 70°C until they are dry to avoid ice crystal formation. The artifacts are characteristic and easy to recognize and thus the method gives reliable results.Exocrine pancreas was used as test specimen when checking the preservation of the ultrastructure. The observations upon conventionally osmium-fixed material could be confirmed to a great extent. However, there is one important exception from this. There are no 150 Å particles (RNA particles) in the cytoplasm in the frozen-dried cells.
A method for rapid freezing is described in which use is made of the good heat conducting properties of silver. The freezing was accomplished by bringing the tissue in contact with a polished silver surface at the temperature of liquid nitrogen either at atmospheric or reduced pressure. Helium gas flowing over this surface prevented the condensation of water or air on the silver. After freezing the tissue was placed in a substituting solvent. The best results were obtained with 2% osmium tetroxide in acetone at −85°C. The ultrastructure of the tissue was well preserved in a narrow surface layer only.
Biological Effects of Freezing and Supercooling
- Smith
- Saunders