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PubMed1981

Polyvinyl alcohol coating: an improvement of the freeze-fracture technique.

A modification of the freeze-fracture technique, involving the coating of tissues with polyvinyl alcohol (Vinol) prior to freezing, is described. This results in substantial improvements over the more conventional method in which material is cryoprotected and frozen in buffered glycerol alone. The high vacuum is produced more rapidly and can be better than in the absence of vinol and the speed of replica recuperation is greatly increased.

Erythrocyte Membrane↗
PubMed1981

Freeze-fracture study of water-soluble, standard proteins and of detergent-solubilized forms of sarcoplasmic reticulum Ca2+-ATPase.

Conventional freeze-fracturing electron microscopy was used to study water-soluble proteins and different forms of Ca2+-ATPase-detergent complexes. Freeze-fracture images of solutions containing proteins larger than myoglobin showed the presence of distinct, randomly dispersed particles on smooth fracture surfaces. The distribution of sizes of these particles was closely to Gaussian, with a mean size which was correlated to the Stokes diameter. Monomeric Ca2+-ATPase from sarcoplasmic reticulum, solubilized by deoxycholate or a non-ionic detergent, showed a bimodal distribution of particle sizes. Even more complex distributions were found for dimeric and trimeric preparations of Ca2+-ATPase. The results can be interpreted on the assumption that the Ca2+-ATPase molecule is elongated, with an overall length of about 110 A and a width in its largest part of about 75 A. It is concluded on the basis of the presented results that freeze-fracture electron microscopy can be successfully used for morphological studies of protein molecules in solution.

Animals↗
PubMed1978

A scanning electron microscopic study of normal human oxyntic mucosa using blunt dissection and freeze fracture.

Biopsies from the fundic mucosa of healthy volunteers were examined by scanning electron microscopy following blunt dissection and freeze fracture. The mucosal surface exhibited a cobblestone appearance. With increased magnification, microvilli could be demonstrated on the luminal surface of individual surface cells. Blunt dissection of the gastric mucosa revealed tubular gastric pits descending from the surface until they opened into branched tubular gastric glands. The gastric glands are irregular in outline due to the knoblike projections of individual parietal cells. The internal structure of the component cells also was examined with the scanning electron microscope following freeze fracture. Mucous granules were observed in the apical portion of surface epithelial cells. Lumina of parietal cell canaliculi were found to be continuous with the lumen of the gastric glands. Pepsinogen granules could be seen throughout the cytoplasm of chief cells. The blunt dissection-freeze fracture technique utilizing the scanning electron microscope allows, for the first time, a three-dimensional view of human gastric mucosa, including the gastric pits and glands as well as some of the internal architecture of component cells.

Adult↗
PubMed1986

Microtubule structure studied by quick freezing: cryo-electron microscopy and freeze fracture.

Microtubules have been quickly frozen and examined by electron microscopy using several techniques: (1) freezing of a thin layer of solution by plunging into cryogen, followed by cryoelectron microscopy of the unstained vitrified samples; (2) freezing by the propane-jet method, followed by freeze fracturing and metal replication. The unstained frozen-hydrated microtubules show a structure in agreement with X-ray diffraction data; they differ from negatively stained particles mainly by the better preservation of cylindrical shape. Secondly, they reveal a supertwist of the profilaments that is not detected reliably by other methods. This allows a determination of the number of protofilaments and the polarity. The structural resolution of unstained microtubules is similar to that of stained ones (about 2-3 nm); it is limited by low contrast and lack of crystalline order. Propane-jet or cryo-block freezing followed by freeze fracturing reveals the structures of the inner and outer surfaces of the microtubule wall at a resolution of 4 nm or better. The outside is dominated by the longitudinal protofilaments whereas on the inside one observes tilted cross-striations. Although the freezing temperatures of the two methods are different (liquid nitrogen or helium) they yield similar results for the case of thin layers of protein solution.

Animals↗
PubMed1985

A quick-freeze, freeze-fracture study of mouse spermatozoa.

Rapid cryo-fixation of mouse spermatozoa followed by freeze substitution, thin section and freeze-fracture demonstrates the finer detail possible with quick-freeze as compared to chemical fixation. As seen in thin sections the unit membrane is composed of 2-4 nm size particles and the cytoplasm, mitochondria, and axonemal filament components all appear rich in fine structure detail. The filamentous structure of the post-acrosomal sheath and its connection with the plasmalemma is presented and compared to previous studies on this structure. Freeze-fracture data demonstrates 7-9 nm size, plasmalemmal, PF-face particles most heavily concentrated in the region just ahead of the striated ring. The outer acrosomal EF-face contains linear arrays of 7-9 nm size EF-face particles. The inner acrosomal membrane contains scattered, 7-9 nm size PF-face particles. The inner and outer nuclear membranes also contain scattered, 7-9 nm size particles. The results of this study present data which supports and extends previous studies on mouse spermatozoa. The results are discussed in terms of the advantages of cryo-fixation and freeze-substitution compared to conventional fixation in the preservation of fine structure detail.

Animals↗
PubMed1978

Phagocytosis of bacteria by polymorphonuclear leukocytes. A freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure.

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25mum(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.

Animals↗
PubMed1984

Isolated rat brain capillaries possess intact, structurally complex, interendothelial tight junctions; freeze-fracture verification of tight junction integrity.

Populations of isolated brain capillaries have been proposed as useful models for in vitro studies of the blood-brain barrier. Preliminary investigations of barrier properties using such preparations of brain microvessels have suggested that the tight interendothelial junctions (zonulae occludentes) are intact and retain the impermeability to the protein tracer horseradish peroxidase, exhibited by them in vivo. The endothelial junctions of isolated capillaries are therefore assumed to be functionally "tight' in vitro. In order to determine the precise structural organization of these occluding junctions, including an estimate of their tightness (complexity), and to demonstrate a method for simple but precise assessment of junctional integrity, pellets of isolated rat brain capillaries were freeze-fractured and then replicated with platinum and carbon. The freeze-fracture images of interendothelial zonulae occludentes revealed complex arrays of intramembrane ridges and grooves characteristic of tight junctions. Longitudinal fractures of the cellular lining of capillaries exposed vast expanses of interendothelial plasma membrane interfaces and the junctional complexes situated between the cells. From such arrays, the elaborate and complex architecture of the zonulae occludentes could be readily appreciated. Situated on the PF fracture faces are 6-8 parallel ridges which display a high degree of anastomosing between adjacent strands. The EF fracture face contains grooves complementary to the PF face ridges. The zonulae occludentes of these capillary endothelial cells are similar in complexity to those reported in the literature for reptilian brain capillaries and therefore can be presumed "very tight'. This study demonstrates that freeze-fracture of pellets of brain capillaries alleviates sampling problems inherent in whole tissue preparations and, in addition, demonstrates the usefulness of freeze-fracture as a tool to monitor junction structure during in vitro investigation of the blood-brain barrier.

Animals↗
PubMed1987

Fine structure of Blastocrithidia culicis as seen in thin sections and freeze-fracture replicas.

The fine structure of epimastigotes of Blastocrithidia culicis was studied by transmission electron microscopy of thin sections and freeze-fracture replicas. This parasite presents a well developed endoplasmic reticulum and Golgi complex systems. Differences in the density and organization of the intramembranous particles were observed between the membranes which enclose the cell body and the flagellum. Ridge-like elevations, visualized in freeze-fracture replicas, were observed in sites where the mitochondrial branches touched the plasma membrane. A special array of membrane particles was observed on both faces of the flagellar and the cell body membranes at the region where the flagellum adheres to the cell body. It appeared as strands made of two rows of membrane particles. Filipin-treated cells were used for the localization of membrane sterols in freeze-fracture replicas. The number of filipin-sterol complexes varied from cell to cell. In some cells, rows of filipin-sterol complexes were seen. No complexes were observed in the region of the attachment of the flagellum to the cell body.

Animals↗
PubMed1989

Applications of freeze-fracture replication to problems in materials and colloid science.

Understanding the relationship between the molecular structure and the macroscopic properties of polymer solutions and gels, oil-water-surfactant emulsions, lyotropic and thermotropic liquid crystals, colloidal dispersions, detergents, and other such "microstructured fluids" is essential to the optimal use of these commercially important materials. Modern rapid-freezing methods followed by freeze-fracture replication techniques are ideally suited to allow the direct visualization of the three-dimensional structure of the particles or units that make up the dispersion, while simultaneously revealing their orientation and distribution with molecular resolution. This paper reviews the necessary experimental conditions required to successfully exploit the freeze-fracture technique as it applies to microstructured fluid systems. The benefits and limitations of structural studies by freeze-fracture techniques as opposed to the more commonly used light, X-ray, and neutron-scattering methods are discussed. Freeze-fracture replicas can also be imaged by scanning tunneling microscopy to reveal directly three-dimensional fracture contours with improved resolution.

Colloids↗
PubMed1986

Tight and gap junctions in the intestinal tract of tunicates (Urochordata): a freeze-fracture study.

The intestinal tracts from seven different species of tunicates, some solitary, some colonial, were studied fine-structurally by freeze-fracture. These urochordates occupy an intermediate position phylogenetically between the vertebrates and the invertebrates. The various regions of their gut were isolated for examination and the junctional characteristics of each part investigated. All the species examined exhibited unequivocal vertebrate-like belts of tight-junctional networks at the luminal border of their intestinal cells. No septate junctions were observed. The tight junctions varied in the number of their component strands and the depth to which they extended basally, some becoming loose and fragmented towards that border. The junctions consisted of ridges or rows of intramembranous particles (IMPs) on the P face, with complementary, but offset, E face grooves into which IMPs sometimes fractured. Tracer studies show that punctate appositions, the thin-section correlate of these ridge/groove systems, are sites beyond which exogenous molecules do not penetrate. These junctions are therefore likely to represent permeability barriers as in the gut tract of higher chordates. Associated with these occluding zonular junctions are intermediate junctions, which exhibit no identifiable freeze-fracture profile, and macular gap junctions, characterized by a reduced intercellular cleft in thin section and by clustered arrays of P face particles in freeze-fractured replicas; these display complementary aggregates of E face pits. The diameters of these maculae are rarely very large, but in certain species (for example, Ciona), they are unusually small. In some tissues, notably those of Diplosoma and Botryllus, they are all of rather similar size, but very numerous. In yet others, such as Molgula, they are polygonal with angular outlines, as might be indicative of the uncoupled state. In many attributes, these various junctions are more similar to those found in the tissues of vertebrates, than to those in the invertebrates, which the adult zooid forms of these lowly chordates resemble anatomically.

Animals↗
PubMed1975

A freeze-fracture replication apparatus for biological specimens.

A freeze-fracture apparatus of original design has been constructed which can be fitted onto a standard vacuum evaporator unit. In it, cell suspensions and organized tissue may be processed by inserting a sample into a cylindrical holder. By leaving a small part of the tissue protruding from the holder, pre-selected and aligned portions of the specimen can subsequently be revealed by fracture under vacuum. After rapid freezing, the specimen remains firmly attached to the inner wall of the sample holder, preventing its possible loss during fracturing. A mechanism, in the form of a double-sided converging wedge, which is operated from outside the vacuum chamber, is used to produce a fracture in the specimen. The device gently induces a fracture in the desired part of the tissue and lifts the protruding part of the specimen out of the way. In this way, reasonably flat fracture faces are produced for subsequent replication. As the fracturing mechanism comes into contact only with the outer edges of the specimen, damage and contamination liable to occur when the entire specimen is traversed by a blade, is avoided. In addition the specimen stage is surrounded by a cold metal shroud which acts as an efficient trap for contaminants. In this way, favourable vacuum conditions are produced in the vicinity of the specimen. Such effective enclosing of the specimen also facilitates controlled sublimation of the sample.

Freeze Fracturing↗
PubMed1983

P-face particle density of freeze-fractured vertebrate cardiac plasma membrane.

P-face particle density of freeze-fractured cardiac plasmalemma provides an estimate of the total population of membrane channels, carriers, and receptors that insert into or traverse unit area of lipid bilayer. We have determined the size of this population of integral membrane protein assemblies by counting the number of P-face particles per square micron of plasmalemma in freeze-fractured hearts, using stereoimaged replicas tilted with a goniometer stage in the electron microscope. Particle numbers per square micron were 4,525 +/- 231, 4,799 +/- 235, 4,122 +/- 153, 4,281 +/- 218, and 5,848 +/- 300 for ventricular myocyte plasmalemmas of rat, rabbit, 8-day chick, adult chicken, and frog, respectively. These values are at least two times greater than published values, in which stereo imaging was not used. Published ligand-binding studies indicate that only the surface density of the Na+-K+ pump sites can account for a significant fraction of P-face particles; the rest so far lack functional correlates. Particle density of frog heart plasmalemma significantly exceeded particle densities of chicken and mammalian plasma membranes.

Aging↗
PubMed1978

A freeze fracture study of the developing tegumental outer membrane of Schistosoma mansoni.

The freeze fracture technique has been used to quantify changes in the integral components of the double outer membrane of Schistosoma mansoni during the 6-week period of development within the mouse. The intramembraneous particle (IMP) density on the P1 face begins to rise within 6 h of host penetration, reaches a maximum at day 4 and then falls rapidly after day 9, so that it is at a low level between 3 and 6 weeks. The E1 face IMP density follows the same course as that of the P1 face except that maximum particle density is recorded on day 1 and the counts begin to fall on day 5. The IMP density on the P2 face remains at a consistently low level throughout development. The E2 face IMP density rises gradually to a peak at day 4, when the parasites have migrated to the lungs, and remains thereafter at a similar level, so that by 6 weeks the E2 face has a higher IMP density than the other three fracture faces. The E2 face IMP show a marked increase in size on day 4. Morphological studies indicate that a different type of inclusion body makes a transient appearance in the tegument of the lung worms, and immunocytochemical techniques show the lung worms to be nonimmunogenic. It is suggested, therefore, that the E2 face IMP may represent complexes of parasite antigens and acquired host antigens. The tegumental membranes of cultured specimens have also been examined by freeze fracturing and the IMP densities compared with those obtained from in vivo parasites; the cultured schistosomula have a lower E2 face particle density than the in vivo specimens.

Animals↗
PubMed1984

Freeze-fracture analysis of isolated liver mitochondria in different metabolic states.

Isolated liver mitochondria were crosslinked with glutaraldehyde during different respiratory states and then analyzed either by freeze-fracturing, or in thin sections prepared according to a low denaturation embedding technique. In freeze-fractured material, large intracristal spaces were found in 86% of the mitochondria freshly isolated in a sucrose medium. These results were in good agreement with mitochondria analyzed in thin sections, in which case 88% of the mitochondria contained intracristal spaces. In freeze-fractured mitochondria, only 24% of the mitochondria crosslinked after 3 min in respiratory state 4 contained intracristal spaces, while 36% of the mitochondria contained intracristal spaces during state 3. These values compared favorably with those obtained in embedded material in which case 21 and 41% of the mitochondria contained intracristal spaces during states 3 and 4, respectively. The agreement between the observations made with the two methods demonstrated that the disappearance of intracristal spaces is independent of the embedding technique, and is not a special effect caused by dehydration in ethylene glycol.

Animals↗
PubMed1989

Advantages of the use of intact plant tissues in freeze-fracture electron microscopy.

The use of whole, intact plant tissue for freeze-fracture electron microscopy provides important information that cannot be obtained from the use of isolated biological membranes or of artificial (phospholipid) membrane preparations. This is not to imply that these examinations of such preparations are not useful, since it would be difficult to interpret our observations of intact cells and tissues without the analysis of these model systems. Analysis of intact tissue and cells reveals the relative densities of membrane proteins of the different membranes within a cell; the three-dimensional organization of various organelles, especially the endoplasmic reticulum; changes in intramembranous particle (IMP) distribution due to stress or injury; and, in conjunction with the use of filipin, membrane sterol content and relative distribution. It is our intention that this survey of freeze-fracture images of intact plant tissues will illustrate the uniqueness of the information gained from an analysis of whole plant tissues compared to isolated membrane fractions.

Filipin↗
PubMed1980

Chromatin freeze fracture electron microscopy: a comparative study of core particles, chromatin, metaphase chromosomes, and nuclei.

Chromatin gels, metaphase chromosomes, and intact nuclei were studied by freeze fracturing followed by electron microscopy. The results complement and extend those obtained by classical electron microscopy techniques as they are obtained without fixation or dehydration. The freeze fracturing technique permits a determination of the hydrated diameters of nucleosomes in chromatin and in nuclei to be 13 nm by comparing to simultaneously studied test objects. Nucleosomes in chromatin fibers are closely spaced but are discrete particles in all conditions studied. In the presence of divalent ions, most chromatin in solution, chromosomes, and nuclei is organized into fibers whose thickness is larger than 40 nm. The images are not at all compatible with a super bead organization of the nucleofilament. Freeze fractures of intact nuclei provides information on the distribution of chromatin in a hydrated unfixed state. The images suggest that most of the chromatin is localized in large domains in contact with the inner nuclear membrane.

Animals↗
PubMed1985

Rotary-shadowed freeze-fracture replicas: a simple method for the analysis of fracture faces.

In rotary-shadowed freeze-fracture replicas, intramembrane particles on the periphery of a membrane fracture face are not uniformly shadowed from all sides. Those eccentrically positioned intramembrane particles with a net centripetally directed shadowing are on a convex fracture face. In contrast, those eccentrically positioned intramembrane particles with a net centrifugally directed shadowing are on a concave fracture face. Centrally positioned intramembrane particles on convex faces are uniformly shadowed from all sides; however, central depressions of concave faces are often unshadowed.

Animals↗