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PubMed1981

Propane jet-freezing: a valid ultra-rapid freezing method for the preservation of temperature dependent lipid phases.

We have developed a temperature controlled specimen stage on the existing jet-freezing apparatus, in order to quench samples from elevated temperatures up to 330 K. We have shown that the phase transition both from the liquid-crystalline to solid phase for pure phosphatidylcholines and from hexagonal II to lamellar phase for phosphatidylethanolamine/4% egg phosphatidic acid is prevented. Moreover, cooling rates exceeding 10,000 K s-1 were recorded indicative of ultra-rapid freezing capacity. Cryoprotectants can be omitted, since phase separation due to ice crystal growth does not occur.

Crystallization↗

PubMed1987

Evaluation of matrix effects in ion microscopic analysis of freeze-fractured, freeze-dried cultured cells.

SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze-fractured, freeze-dried cultured cells at the approximately 0.5 micron spatial resolution of the Cameca IMS-3f ion microscope. Cell lines studied include normal rat kidney (NRK), 3T3 mouse fibroblast, L6 rat myoblast, chinese hamster ovary (CHO) and rat kangaroo kidney (PtK2) cells. High mass resolution studies indicated that the secondary ion signals of H-, C-, O-, Na+, Mg+, CN-, P-, S-, Cl-, K+ and Ca+ were free from major mass interferences. However, a large mass interference was observed for nitrogen at mass 14. No significant sputtering yield difference between the nuclear and cytoplasmic compartments of the cells studied was observed. The subcellular distributions of the major (H, C, N and O) and minor (P, S, K, Cl, Na, Mg and Ca) matrix elements were found to be largely homogeneous with the exception of Ca, which was observed mainly in the cell cytoplasm. Practical ion yield variations were compared by three different approaches: (i) by the use of cells doped with known electrolyte concentrations, (ii) by quantitative ion implantation, and (iii) by analysis of the same cell with both electron probe and ion microscope. Each approach indicated an absence of significant practical ion yield differences between the nuclear and cytoplasmic regions of these specimens. These observations indicate that secondary ion signals in this type of sample are not significantly affected by local matrix effect variations. Hence, qualitative imaging of such specimens provides a true representation of subcellular elemental distributions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.

Cells, Cultured↗

PubMed1997

Ultrastructural study of glomerular capillary loops at different perfusion pressures as revealed by quick-freezing, freeze-substitution and conventional fixation methods.

Wistar rat kidneys were perfused with some fixatives at different pressures of 100, 150, 200 or 250 cm H2O via the aorta and were studied by the quick-freezing and freeze-substitution (QF-FS) or deep-etching (QF-DE) methods, in addition to the conventional immersion or perfusion fixation method. Different parts of glomerular capillary walls were selected for morphometric analyses. It was demonstrated that the widths of glomerular slit diaphragms and the spaces between foot processes were more widely dilated at higher perfusion pressures (200 and 250 cm H2O) than those seen at both normal perfusion pressure (150 cm H2O) and lower perfusion pressure (100 cm H2O). On the other hand, the glomerular basement membranes were thinner at higher perfusion pressures. By the QF-FS and QF-DE methods, the foot processes showed different shapes from those revealed by the conventional preparation methods, even at the same perfusion pressure. It is concluded that the widths of glomerular slit diaphragms and glomerular basement membranes and the spaces between foot processes may be significantly changed in vivo, depending on the hemodynamics in the glomerular capillary.

Animals↗

PubMed1993

The ultrastructure of the connective tissue matrix of skin and cartilage after high-pressure freezing and freeze-substitution.

Studies designed to investigate the ultrastructure of the connective tissue matrix have historically relied on chemical fixatives to stabilize tissue microarchitecture. However, conventional fixatives are not completely effective in retaining many matrix constituents, including proteoglycans. Fixative recipes have been modified to include agents that retain proteoglycans, but they precipitate the glycosaminoglycan moiety into electron-dense granules and therefore do not preserve native microstructure. To avoid the structural artifacts introduced by aqueous fixatives, we prepared cartilage and skin by a cryostabilization procedure that included high-pressure freezing and freeze-substitution. Although similar approaches have been applied previously for study of connective tissue, our results and interpretation of matrix structure are significantly dissimilar. In optimally preserved areas of cartilage, collagen fibrils are continually surrounded by a densely staining sol. Empty fluid spaces are absent. In less optimally preserved areas, artifacts are noted and described, including a network that mimics the expected structure of proteoglycan. Similarly, the dermal matrix of human skin contains a preponderance of densely staining material that almost fills the voids commonly seen after aqueous fixation. Decorin, immunolocalized to the surface of dermal collagen fibrils, appears to be retained after this procedure.

Animals↗

PubMed1989

Ultrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching.

Intima from aortas of normal Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed (10 days - 3 months) rabbits were examined by ultra-rapid freezing without chemical fixation followed by rotary shadow freeze-etching. The extracellular matrix in areas devoid of cells was seen in extraordinary detail and consisted of a reticulum of thick filaments, finer branching filaments, collagen fibrils, and granules of varying sizes. No lipid deposits were seen in normal intima. However, the subendothelial region of WHHL intima was filled with collagen fibrils surrounding and entwined between clusters of discrete lipid vesicles that ranged in size from 23 to 169 nm. Approximately 80% of the lipid vesicles in the WHHL rabbit intima measured between 70 and 169 nm. The lipid particles in the WHHL intima always appeared in clusters, many of which appeared to be fusing into larger size vesicles. These aggregates were clearly linked to the matrix filaments. A similar deposition of lipid particles was seen in the extracellular matrix of cholesterol-fed rabbits but in contrast to the particle size distribution of the WHHL intima, more than 75% of the particles in the cholesterol-fed intima had a diameter between 23 and 68 nm and 51% were between 23 and 45 nm. We conclude that in cell-free areas of WHHL and after only 10 days of cholesterol feeding, lipoprotein-derived lipid is present in the intima as clusters of vesicles enmeshed in the complex extracellular matrix.

Animals↗

PubMed1988

The bulk specimen X-ray microanalysis of freeze-fractured, freeze-dried tissues in gerontological research.

The rationale for choosing the freeze-fracture freeze-drying (FFFD) method of biological bulk specimen preparation as well as the theoretical and practical problems of this method are treated. FFFD specimens are suitable for quantitative X-ray microanalysis of biologically relevant elements. Although the spatial resolution of this analytical technique is low, the application of properly selected bulk standard crystals as well as the measurement of the intracellular water and dry mass content by means of another method developed in the same laboratory, allow us to obtain useful information about the age-dependent changes of ionic composition in the main intracellular compartments. The paper summarizes the problems with regard to specimen preparation, beam penetration and the quantitative analysis of FFFD specimens. The method has been applied so far mainly for the analysis of intranuclear and intracytoplasmic concentrations of Na, C1 and K in various types of cells and has resulted in a significant contribution to our understanding of the cellular mechanisms of aging.

Aging↗

PubMed1981

Investigation about the surface structure of some viruses by the freeze-etching and freeze-drying methods.

An electronmicroscopic study of a suspension of Venezuelan equine encephalomyelitis (VEE) virus by freeze-drying and freeze-etching methods showed that glycoprotein peplomers are located on the surface of the lipoprotein shell. These peplomers are trimeric in shape and form a regular icosahedral surface lattice corresponding to T = 4. The modes of glycoproteins clustering for the two clones of VEE are different. Venezuelan equine encephalomyelitis (VEE) virus belongs to the alpha-viruses of the toga-virus family. Virions of VEE virus have the icosahedral nucleocapsid surrounded by a lipoprotein envelope (peplos) and contain single-stranded RNA. During budding, alpha-virus nucleocapsid acquires a host cell lipid membrane in which virus specific glycoproteins are built in (Bonsdorff et al. 1975, Harrison et al. 1974). Earlier the surface peplomer clustering was investigated only for the sindbis virus (Bonsdorff et al. 1975, Bonsdorff et al. 1978).

Enterovirus↗

PubMed1998

High-pressure freezing and freeze-substitution of native rat brain: suitability for preservation and immunoelectron microscopic localization of myelin glycolipids.

Galactocerebroside (GalC) and sulfatide are major constituent lipids in vertebrate myelin. Their precise immunolocalization in electron microscopy so far has been hampered by the fact that lipids are not immobilized by chemical fixation and thus get extracted during dehydration with organic solvents. Here, we examined the suitability of cryotechniques for the preservation and immunohistochemical localization of myelin glycolipids in rat brain at the ultrastructural level. Native cerebral cortex tissue, obtained by fine-needle biopsy, was cryoimmobilized by high-pressure freezing and dehydrated by freeze-substitution before embedding in Epon. This procedure resulted in an excellent preservation of brain ultrastructure. Concomitantly, immunogold labeling of ultrathin sections with the well-defined monoclonal antibodies (mAbs) O1, O4, and R-mAb, which were shown to react with GalC and/or sulfatide and some structurally related glycolipids, revealed a good conservation of relevant epitopes. These data suggest that in adult rat cerebral cortex, the most relevant antigens recognized by R-mAb, O1, and O4, namely GalC and sulfatide, are exclusively expressed in myelin structures. Because these mAbs are common markers for the identification of developing oligodendrocytes, this "postembedding glycolipid-labeling technique" holds great potential for studying oligodendroglial differentiation in normal and pathological conditions at the ultrastructural level.

Animals↗

PubMed2006

High-pressure freezing and freeze substitution of rat myocardium for immunogold labeling of connexin 43.

The value of high-pressure freezing (HPF) and freeze substitution (FS) for immunoelectron microscopy (immuno-EM) of the heart was investigated in bioptic specimens taken from isolated hearts of 0-, 5-, and 14-day-old rats at baseline and at 15, 30, 45, and 60 min after induction of ischemia. The target antigen chosen here was the gap junction protein connexin 43 (Cx43). After HPF and FS, immunogold labeling was applied for detection of Cx43. Gold particles associated with gap junction areas, free plasma membrane, and annular gap junctions (AGJs) were counted and distributions compared by contingency table analysis. HPF and FS resulted in excellent preservation of antigenicity for Cx43. The mostly good preservation of the ultrastructure was limited by mechanical damage at the border and by ice crystal formation in the center of the tissue blocks. In normal myocardium of newborns, gold particles associated with free plasma membrane were frequently observed, with AGJs only seldom. In older rats, the opposite relation was found. During ischemia, no distribution changes occurred in newborn or 14-day-old rats. In 5-day-old rats, however, ischemia induced a shift of Cx43 from gap junction plaques to AGJs. In conclusion, HPF and FS are an ideal alternative to chemical fixation for immuno-EM as the excellent preservation of antigenicity is combined with a well-preserved ultrastructure. The results indicate that the process of degradation of gap junctions via AGJs gradually increases during postnatal rat heart development, a process that may be accelerated by ischemia in an early developmental state.

Animals↗

PubMed1983

Inhibitory and excitatory synapses in crayfish stretch receptor organs studied with direct rapid-freezing and freeze-substitution.

Crayfish abdominal stretch receptor organs are innervated by inhibitory (GABA) and excitatory (glutamate) synapses. Previous studies with aldehyde fixation showed that synaptic vesicles in the inhibitory synapse are flat and small, whereas those in the excitatory synapse are rounder and larger. We have reexamined these inhibitory and excitatory synapses by using direct rapid-freezing and freeze-substitution in order to preserve synaptic structure closer to its living state. Fine details of synaptic structure appear to be better preserved by this method. Synaptic vesicles in inhibitory as well as excitatory synapses are round, so the conventional flattened shape of vesicles in the inhibitory synapse must depend on some aspect of aldehyde processing. However, the average size of vesicles in the inhibitory synapse is significantly smaller than that of vesicles in the excitatory synapse, so synaptic vesicle size is regarded as having functional significance.

Animals↗

PubMed1993

Freeze substitution after fast-freeze fixation in preparation for immunocytochemistry.

As compared to classical chemical fixation, the physical immobilization of ultrastructures by fast-freeze fixation (FFF) and the subsequent exchange of water in its solid state by freeze substitution (FS) improve the preparation procedure for immunogold labeling (IGL). FFF-FS results in a morphological preservation of unchallenged quality, as well as in a better preservation of antigenic reactivity, thus allowing remarkable precision of labeling on sections. However, FFF, particularly over a cooled metal plate, requires a heavy and expensive machine. It is not suitable for all biological specimens and in the best conditions, which remain difficult to standardize, the thickness of the well-preserved portion of the specimen does not exceed a few microns for compact tissues, and exceptionally 30-40 microns for isolated cells. The FS procedure is long and must be adjusted empirically for every new specimen and antigenic detection. The preservation of a given antigen's reactivity in the presence of fixative agents and embedding resins remains unpredictable. The action of fixative agents is different and milder in FS than when they are used classically in chemical fixation. By chance, one of the best FS procedures for the preservation of both ultrastructure and antigenicity appears to be by using acetone alone, together with a molecular sieve to improve the water exchange process. A large choice of embedding resins usually allows us to find a compromise between ultrastructural and antigenic preservation.

Animals↗

PubMed1994

Improved ultrastructural preservation of rat ciliary body after high pressure freezing and freeze substitution: a perspective view based upon comparison with tissue processed according to a conventional protocol or by osmium tetroxide/microwave fixation.

Conventional fixation of the delicate, highly folded rat ciliary body and its iridial extension, as well as of vitreal structures, is associated with the induction of a number of artifacts, thus limiting the reliability of morphological interpretations. Improved ultrastructural preservation may be achieved by microwave heating in combination with osmium tetroxide fixation. This protocol, although simple and cheap, yields results, particularly with respect to the extracellular matrix compartment between inner and outer ciliary epithelial cells, which are not greatly inferior to those obtained by implementing the sophisticated high pressure freezing and freeze substitution technique. The latter affords good to very good ultrastructural preservation of epithelium and stromal components, such as blood vessels, neural elements, smooth muscle cells, fibrocytes, and free cells, up to a depth of 50-100 microns from the tissue surface. Its superiority over osmium tetroxide/microwave fixation is revealed in the cytoplasmic, intraorganellar, and vitreal matrix compartments, which incur no obvious losses.

Animals↗

PubMed2005

Chloroplast ultrastructure in leaves of Urtica dioica L. analyzed after high-pressure freezing and freeze-substitution and compared with conventional fixation followed by room temperature dehydration.

In this article, we report on the adaptation of high-pressure freezing and freeze-substitution (HPF-FS) for ultrastructural analysis of leaf tissue with special emphasis on chloroplasts. To replace the gas in the intercellular spaces, a mixture of water and methanol (MeOH) was employed. We compared three different supplements for FS--osmiumtetroxide, uranyl acetate, and safranin--with regard to the preservation of the ultrastructure of chloroplasts and other cellular compartments. The results show that (i) replacement of air within intercellular spaces by 8% (v/v) MeOH has no influence on the ultrastructure of the chloroplasts, (ii) undulation of membranes frequently observed after conventional preparation of specimens does not occur during chemical fixation but during room temperature dehydration, and (iii) uranyl acetate or osmium tetroxide employed during FS are not superior over safranin.

Atmospheric Pressure↗

PubMed2000

Purification and characterization of protein kinase A from liver of the freeze-tolerant wood frog: role in glycogenolysis during freezing.

Freeze tolerance by various amphibians includes cryoprotectant production in the form of glucose. Activation of the catalytic subunit of liver cAMP-dependent protein kinase (PKAc) facilitates activation of glycogenolysis, a critical biochemical process necessary for production of glucose. Here, we purified PKAc from Rana sylvatica liver to determine the extent to which cold temperature, which stimulates cryoprotectant production, affected PKAc activity and function. PKAc was purified to greater than 95% homogeneity, with a final specific activity of 71 nmol phosphate transferred/min/mg protein. The molecular weight of frog liver PKAc was 47.6 +/- 1.1 kDa and K(m) values for the phosphate acceptor kemptide and Mg-ATP were 9.0 +/- 0.1 and 51.8 +/- 1.0 microM at 22 degrees C, respectively. K(m) values for both substrates dropped significantly at 5 degrees C. The enzyme was sensitive to specific inhibitors of mammalian PKAc (PKA(i), H89) but was only moderately inhibited by high salt concentrations. Furthermore, salt inhibition was reduced at low temperature. The effect of temperature on enzyme activity indicated a conformational change in PKAc at 10 +/- 2 degrees C, with calculated activation energies of 51 +/- 4 kJ/mol at temperatures above 10 degrees C and 110 +/- 9 kJ/mol below 10 degrees C. PKAc in wood frog liver plays a crucial role in mediating the freeze-induced glycogenolysis that is responsible for the production of 200-300 mM levels of glucose as a cryoprotectant. Differential effects of low temperature on enzyme function, increased substrate affinity and reduced ion inhibition, appear to be central to this role.

Acclimatization↗

PubMed1993

Further observations on the morphological alterations of the glomerular capillary wall of the rat kidney caused by chemical and physical agents: standard procedures versus quick-freezing and freeze-substitution.

The purpose of the present study was to pin-point which of the various preparatory steps required by different histological procedures (conventional, cryosubstitution, freezing followed by cryosubstitution or replication) induces alterations in the fine structure of the endothelial and epithelial surface coat (SC) and of the interposed glomerular basement membrane (GBM). Samples of rat kidney cortex prefixed by vascular perfusion, and isolated glomeruli fixed by immersion, were used. The results demonstrate a continuous, homogeneous and amorphous SC and a GBM devoid of laminae rarae when freezing is used and followed either by cryosubstitution and embedding or by fracture, deep etching and replication. Postfixation in OsO4, and especially dehydration in organic solvents at room temperature, generate a filamentous SC and a GBM with laminae rarae. The different morphology may be due to an extraction of glycoconjugates from SC and GBM during postosmication and/or especially to precipitation of these components during dehydration by organic solvents at room temperature. Studies on isolated glomeruli show comparable results and, in addition, demonstrate that the distance of the capillary loop from the surface of the block influences the morphology of SC and GBM. Rinsing the vascular bed before isolation does not induce a loss of SC or GBM components.

Acetone↗

PubMed1994

Ultrastructure of hypertrophic cartilage: histochemical procedures compared with high pressure freezing and freeze substitution.

The effect of cationic dyes on the ultrastructure of hypertrophic cartilage was compared with results obtained with modern cryotechniques in studies on rat epiphyseal growth plate. Addition of alcian blue, acridine orange, cupromeronic blue, ruthenium hexamine trichloride, ruthenium red, or safranin O to conventional glutaraldehyde/osmium tetroxide fixatives to a large extent resulted in prevention of chondrocyte shrinkage except for alcian blue which showed poor tissue penetration. The fine structure of the matrix in pericellular and territorial compartments appeared very coarse with areas of high contrast in tissue exposed to fixatives containing cationic dyes. This indicates structural collapse and precipitation of electron-dense material, a pattern clearly differing from that observed in specimens prepared by the cryotechniques. The dyes giving a pattern most similar to that seen after high pressure freezing, freeze substitution, and low temperature embedding were acridine orange and safranin O. It is concluded that studies of matrix ultrastructure down to the molecular level necessitate the application of cryotechniques.

Animals↗

PubMed1982

Ultrastructure of scales in a teleost (Carassius auratus L.) after use of rapid freeze-fixation and freeze-substitution.

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.

Animals↗

PubMed1988

Ultrastructure of matrix vesicles in chick growth plate as revealed by quick freezing and freeze substitution.

The ultrastructure of extracellular membrane-bound matrix vesicles (MVs), their biogenesis, and the surrounding matrix in chick tibial growth plate were studied after quick freezing and freeze substitution (FS) in an organic solvent. There were several notable differences in the ultrastructural preservation of cartilage when FS was used as compared with conventional fixation. The ultrastructural appearance of MVs after FS was extremely variable. Within the MVs, intravesicular filaments, amorphous material, and membrane-associated undercoat structures were observed. Intravesicular filaments, similar in diameter to microfilaments seen in the cytoplasm, were attached to the inside of MV membranes. This observation indicates the similarity of MV membranes and the plasma membrane. In some MVs in the proliferative zone an electron-dense material was present along the inner side of the MV membrane. In the prehypertrophic zone, crystalline material often appeared within the electron-dense material, which may be a precursor form of hydroxyapatite. The earliest crystals observed were in MVs but not in the extracellular matrix. Regarding MV formation, in addition to budding from cell surfaces and to cellular disintegration, this study also indicates that a sequential process of extrusion of preformed cytoplasmic structures may occur. Also, small MVs measuring 25-40 nm seem to arise from the disruption of large MVs. This is a previously unreported observation on MV biogenesis. FS preserves proteoglycans in the cartilage matrix as a fine, filamentous network. Initial extracellular calcification was not associated with this network.

Animals↗