Freeze-fracture Replication Technique Research Articles

Formation and characteristics of aqueous two-phase systems formed by a cationic surfactant and a series of ionic liquids

Aqueous two-phase systems (ATPS) were obtained in the aqueous mixtures of a cationic surfactant and a series of ionic liquids (ILs). The effects of IL structure, temperature and additives on the phase separation were systematically investigated. The microstructures of some ATPS were observed by freeze-fracture replication technique. Lyotropic liquid crystal was found in the bottom phase besides micelles under different conditions. Remarkably, both IL structure and additives profoundly affected the formation and properties of the ATPSs. The phase separation can be attributed to the existence of different aggregates and the cation-π interactions of the cationic surfactant with the ILs, which has a significant role in the formation of ATPS. The extraction capacity of the studied ATPS was also evaluated through their application in the extraction of two biosubstances. The results indicate that the ILs with BF4(-) as anion show much better extraction efficiencies than the corresponding ILs with Br(-) as anion do under the same conditions. l-Tryptophan was mainly distributed into the NPTAB-rich phase, while methylene blue and capsochrome were mainly in the IL-rich phase.

Temperature-induced micelle transition of gemini surfactant in aqueous solution

The viscosity and morphology of gemini surfactant in aqueous solution at various temperatures are investigated by rheometer and transmission electron microscopy with a freeze fracture replication technique. The viscosity of 18-3-18 or 16-3-16 reaches its maximum when the solution temperature is near the Krafft temperature of the surfactant. The morphology of a gemini surfactant in aqueous solution is strongly influenced by the temperature: vesicles or sphere-like aggregates are formed in the dilute solution of 18-3-18 under 25 °C environment; when the solution temperature goes up to 50 °C, such aggregates will transit to worm-like micelles with the length of each from 100 nm to 400 nm; meanwhile, the increase of concentration may lead to the formation of network; however, the worm-like micelles or network will transit to vesicles or sphere-like aggregates again when the temperature reaches 80 °C.

High-Resolution Scanning Tunneling Microscopy of Fully Hydrated Ripple-Phase Bilayers

A modified freeze-fracture replication technique for use with the scanning tunneling microscope (STM) has provided a quantitative, high-resolution description of the waveform and amplitude of rippled bilayers in the P beta' phase of dimyristoylphosphatidylcholine (DMPC) in excess water. The ripples are uniaxial and asymmetrical, with a temperature-dependent amplitude of 2.4 nm near the chain melting temperature that decreases to zero at the chain crystallization temperature. The wavelength of 11 nm does not change with temperature. The observed ripple shape and the temperature-induced structural changes are not predicted by any current theory. Calibration and reproducibility of the STM/replica technique were tested with replicas of well-characterized bilayers of cadmium arachidate on mica that provide regular 5.5-nm steps. STM images were analyzed using a cross-correlation averaging program to eliminate the effects of noise and the finite size and shapes of the metal grains that make up the replica. The correlation averaging allowed us to develop a composite ripple profile averaged over hundreds of individual ripples measured on different samples with different STM tips. The STM/replica technique avoids many of the previous artifacts of biological STM imaging and can be used to examine a variety of periodic hydrated lipid and protein samples at a lateral resolution of about 1 nm and a vertical resolution of about 0.3 nm. This resolution is superior to conventional and tapping mode AFM to soft biological materials; the technique is substrate-free, and the conductive and chemically uniform replicas make image interpretation simple and direct.

New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicateMetandrocarpa uedai

Cellulose synthesizing enzyme complexes (terminal complexes, TCs) have been found in the plasma membrane of epidermal cells in the tunicateMetandrocarpa uedai by using freeze-fracture replication techniques for electron microscopy. Assembly of cellulose microfibrils by TCs is a universal phenomenon in the biological kingdoms. The TCs are locally distributed in the plasma membrane of the epidermal cells facing the tunic, and no TCs are observed on the lateral membranes bordered by tight junctions. The TCs consist of two types of membrane subunits: large particles (14.5 nm in diameter) on the periphery and small subunit particles (7.2 nm) filling the center; the latter are hypothesized to be involved in cellulose synthesis. The TCs are the linear type (ca. 195 nm in length and 78 nm in width). Direct connections of TCs with the termini of microfibrils were observed. Amorphous regions, which were hypothesized the nascent microfibrils, were associated with the depressions of the TCs. The distortion of microfibrils on their terminus indicates that the crystallization may occur at the margin of TCs from which the microfibrils are discharged. This report provides evidence that: (1) The outer cell membrane of epidermis is the site for the assembly of cellulose microfibrils in the tunic; (2) a new type of TC is involved in the biosynthesis of cellulose microfibrils in the tunicates; (3) disorganized glucan chains may be synthesized in the depression of TCs and crystallized outside the E-surface of the epidermal cell membrane.

Colloidal structures of three Chinese petroleum vacuum residues

Transmission electron micrographs obtained with the freeze-fracture replication technique on three typical Chinese vacuum residues and their fractions demonstrate that petroleum vacuum residues are colloidal systems, in which asphaltenes (the n-heptane-insoluble, toluene-soluble fraction) and heavier resins constitute the dispersed phases and the remaining lighter resins, aromatics and saturates the dispersing media.

A freeze-fracture study of the perineurium in galactose neuropathy: morphological changes associated with endoneurial oedema.

Feeding rats with galactose as 40% of their diet results in peripheral nerve oedema related to the intrafascicular accumulation of galactitol and sodium. In this study, associated changes in the perineurium were examined by the freeze-fracture replication technique. Perineurial cells are linked by tight junctions (zonulae occludentes). In normal animals these are made up of anastomosing strands organized in a belt-like arrangement along the margins of continuous cells. The majority of the tight junctions in the galactose-fed animals displayed structural abnormalities. These ranged from slight separation of the strands to fragmentation and dispersal, with looping of isolated strands. Some of the tight junctions contained large dilated compartments within the junctional network. Short lengths of intramembranous particles, probably representing assembly or disassembly of tight junctional strands, were also observed. The membranes of perineurial cells normally possess numerous openings of caveolae. A quantitative assessment showed that the mean density of these caveolae openings was increased in the galactose-fed rats as compared with controls. The alterations in the tight junctions resemble those that have been produced experimentally in epithelia by subjecting them to abnormal osmotic gradients. They also resemble those seen in human diabetic neuropathy in which osmotic disturbances involving the perineurium have been considered to occur. If the alterations involve the inner layers of the perineurium, they are likely to impair its barrier function. The increased number of caveolae openings in galactose neuropathy may represent a reaction to the endoneurial oedema and indicate that the pinocytotic-like vesicles have a transport function.

A freeze‐fracture study of the perineurium in normal and protein‐deprived rats

Observations have been made using the freeze-fracture replication technique on the perineurium of normal and protein-deprived rats in which its permeability barrier function is known to be deficient. The perineurial cells of young normal rats possessed belt-like tight junctions (zonulae occludentes) at the borders and maculae occludentes at sites remote from their borders. In older rats, the zonulae occludentes were more prominent and the maculae occludentes relatively less frequent. No abnormalities were detected in the tight junctions of young rats with early induction of protein deficiency but this may have been related to sampling problems. In older severely protein-deficient animals, although many of the tight junctions were normal, some were abnormal and contained focal regions of dispersed strands. The density of caveolae in the surface membrane of the perineurial cells of older rats with severe protein deficiency was significantly greater than in the control animals. This provides support for the view that the pinocytotic-like vesicles of perineurial cells are involved in transport of substances across the cells. The increased numbers of caveolae in the protein deficient rats may reflect increased transcellular traffic. There were considerable differences in the density of P-face IMPs between the different perineurial lamellae, but the results did not allow a decision to be made as to whether there was a polarization of the cells between their endoneurial and epineurial aspects. No differences were detected in the density of P-face IMPs between the young control and protein-deprived rats. In the perineurium of the older rats with protein deficiency, IMP density was significantly greater in the E face than in the controls but not different in the P face. The delay in the development of enzymatic activity in the perineurium of protein-deficient rats that has been demonstrated histochemically is therefore not paralleled by a reduction in IMPs.

An Optimized Freeze-Fracture Replication Procedure for Human Skin

This in vitro study aimed at substantial modification of the freeze-fracture replication technique (FFRT) which should result in an optimal visualization of the ultrastructure of human skin. The technique was modified in two ways: firstly, the conventional sample holders such as gold cups and copper plates were replaced by silver cylinders (83.5% silver, 16.5% copper) resulting in almost perpendicular cross fractures through the skin. Secondly, the replica cleaning procedure was optimized through the following sequence of treatments. Firstly, a mild tissue destruction was obtained by simultaneous lipid solvation and water extraction with absolute methanol (20 h), followed by protein denaturation with dimethyl sulfoxide (DMSO, 24 h). Subsequently, a final treatment was given using an alkaline sodium hypochlorite solution (20% KOH/13% NaClO; 1:3 v/v, 4 d). After rinsing the replicas for 45 min in aqua bidest, they were mounted on copper grids and examined in the transmission electron microscope (TEM). The combination of the unorthodox fracturing method and the optimized cleaning procedure yielded large, practically undamaged and very clean replicas of near perpendicular cross fractures through human skin. Common handicaps related to current freeze-fracture procedures when applied to skin, such as incomplete cleaning and fragmentation of replicas and oblique or irregular fracturing planes, can largely be avoided in this way. In this paper a complete description of the method is given, and a number of advantages are illustrated with the aid of TEM micrographs.

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.

Brain Edema in Delayed Radiation Necrosis: Study of Capillary Ultrastructure

A 42-year-old male was found to have cerebral radiation necrosis 2 years after a split course of radiation therapy following subtotal excision of a left parasagittal meningioma. The surgical specimens were studied by means of conventional ultrathin sectioning and freeze-fracture replication techniques. The electron density of the capillary endothelium was decreased, as was the degree of surface infolding. The width was irregular, and there was an unusually large number of pinocytotic vesicles (37/micron 2). The endothelium was also discontinuous, and the basal laminae were irregular in thickness. The pericapillary space was markedly enlarged, and accumulation of a fibrin-like substance was noted in the extravascular space. The pericapillary zone contained inflammatory cells. However, the tight junctions were preserved and the intercellular junctions were composed of five strands that appeared to consist of continuous particles. The authors conclude that activated pinocytotic vesicles play an important role in increasing vessel permeability in delayed cerebral radiation necrosis and that leakage may occur through interruptions in the endothelium.

51] Electron microscopy: Assays involving freeze-fracture and freeze-etching

Publisher Summary This chapter discusses the assays involving freeze-fracture and freeze-etching. The freeze-fracture replication technique, developed by Moor and Muhlethaler has two unique features. First, it exposes structures residing within the lipid bilayer matrix for electron microscopic examination, and second, it permits the study of biological structures “fixed” only by freezing, thus circumventing a number of artifacts linked to other preparation methods for electron microscopy. Though the latter advantage is the main impetus for development of the technique, it is rarely fully exploited because of the major difficulties encountered in freezing native biological fluids without introducing ice-crystal artifacts. The standard geometry of shadowing is unidirectional at an angle of 45° relative to the average fracture plane. The inclination of a particular small area of the fracture face is not known, however, and it can be quite different from the average plane. Therefore, calculations of heights based on the lengths of shadows are very approximate.

Freeze-fracture study of the mechanoreceptive digital corpuscles of mice.

The freeze-fracture replication technique was used to study the mechanoreceptive digital corpuscles in toe pads of mice. The axon terminal plasmalemma had intramembranous particles (IMPs) at a density of 2367 +/- 517 microns-2 (mean +/- S.E.M.) in the P-face and 84 +/- 4 microns-2 in the E-face. Particles were 10 +/- 1.8 nm in diameter in the P-face and 10 +/- 1.5 nm (mean +/- S.D.) in the E-face. Particle-rich and particle-free areas were noted in the P-face. The lamellar cell plasmalemma had IMPs at a density of 3359 +/- 224 microns-2 in the P-face and 265 +/- 95 microns-2 in the E-face. Particles were 10 +/- 1.4 nm in diameter in the P-face and 10 +/- 1.6 nm in the E-face. Non-terminal unmyelinated fibres in the connective tissue compartment of toe pads were also examined: the P-faces of the axolemma and Schwann cell plasmalemma had IMPs at a density of 1356 +/- 283 microns-2 and 1514 +/- 514 microns-2, respectively, while the E-face of these membranes had only a few particles. Particles were 9 +/- 1.2 nm and 10 +/- 1.6 nm in diameter in the P-faces of axon and Schwann cell plasmalemmata, respectively. The results show that the IMPs in terminal axolemma and in lamellar cell plasmalemma have a much higher density than those of non-terminal axons or Schwann cells in myelinated and unmyelinated fibres. In addition, IMPs in the terminal axolemma are larger than those in non-terminal axolemma except for the nodal axolemma. It can be said that plasmalemmata of both the axon terminals and lamellar cells of digital corpuscles are specialized in terms of IMPs, suggesting that they have specific physiological properties in mechanoreceptive functions including mechano-electric transduction.

Intercellular junctions in the cells of the human enamel organ as revealed by freeze-fracture

Examined by thin sections and freeze-fracture replication techniques, secretory ameloblasts possessed two sets of the junctional complexes at both proximal and distal ends of the cell bodies, which consisted of tight junctions and occasional gap junctions and desmosomes. The proximal tight junction was fascia occludens, whereas the distal tight junction was zonula occludens. Between adjacent ameloblasts, mature gap junctions were frequent. The stratum-intermedium cells were connected to each other and to the stellate-reticulum cells and ameloblasts by well-developed desmosomes, gap junctions and fascia or macula-type tight junctions. Stellate-reticulum cells were inter-connected by many extensive cytoplasmic processes, in which well-developed desmosomes, small gap junctions and occasional macula-type tight junctions appeared. Thus fascia or macula-type tight junctions as well as many desmosomes seem to serve in mechanical, cell-to-cell adhesion during tooth formation. Frequent and large gap junctions between adjacent stratum-intermedium cells and between the stratum intermedium and the base of the ameloblast suggest that, in relation to enamel formation, these two cell layers form a functional unit.

Connection of lamellar body to intracellular membrane system in type II alveolar cells of human lung.

We examined the type II alveolar cells of the human lung with an electron microscope, using the freeze-fracture replication technique, and found connections of some lamellar bodies to barlike structures, which seemed to be a kind of endoplasmic reticulum system. This morphologic finding suggested that the human lung has an additional pathway for surfactant synthesis in type II alveolar cells.

509—Ordered protomembranes

Lipid-like structures synthesized according to the Cold Theory of the origins of life self-assemble in multitudes of stable microspheres. The walls of the microspheres present membrane-like properties. Optical (conventional and polarized light images) and electron microscope (conventional and freeze-fracture replication techniques) clearly show order and bilayer structure in the wall-membrane of the vesicles.

Ordered protomembranes

Lipid-like structures synthesized according to the Cold Theory of the origins of life self-assemble in multitudes of stable microspheres. The walls of the microspheres present membrane-like properties. Optical (conventional and polarized light images) and electron microscopy (conventional and freeze-fracture replication techniques)clearly show order a

Freeze-fracture replication technique of human skin.

The freeze-fracture replication technique represents one of the most advanced methods for studying the ultrastructure of biological tissues and has given a new dimension to the ultrastructure research. This technique makes it possible to obtain a cast of a fracture surface of frozen preparations. Apart from allowing samples to be examined without being dehydrated, i.e. under conditions very close to those of living state, it has proved especially useful for the analysis of many tissue functions concerned with membrane activity and exchanges between cells and their disturbance in specific disease conditions. Even through good quality replicas had already been obtained back in 1961 (Moor et al. 1961), only after 1970, mainly thanks to the contribution made by Breathnach and his collaborators (Breathnach et al. 1972, Breathnach 1973, Breathnach et al. 1973) was the freeze-fracture replication technique successfully used in the study of the skin, since this tissue had proved especially difficult to process. In this review paper, technical principles will be summarized and the most important findings so far obtained in the study of normal and pathologic skin will be illustrated.

The permeability barrier in the epidermis of the grass snake during the resting stage of the sloughing cycle.

Tracer and freeze-fracture replication techniques show that there are two morphologically and topographically distinct permeability barriers in the epidermis of the grass snake. Tight junctions interconnect the apico-lateral plasma membranes of the uppermost living cells, establishing an ionic or osmotic gradient between the stratum germinativum and alpha layer. The second barrier is formed by intercellular lipid sheets in the overlying mesos layer, which are very similar to the barrier found in the stratum corneum of mammals.

Freeze-fracture study of alveolar lining layer in adult rat lungs

The freeze-fracture replication technique was utilized to study the structure of the alveolar lining layer in the rat lung. The surface film appeared to be a finely granular membranous film devoid of membrane-associated particles. The density of its granularity varied from place to place, particularly near zones of emergence of linear grooves on the film. Linear grooves overlay and constituted the upper wall of underlying tubular myelin. Occasionally there were small saccular depressions in the surface film. The hypophase generally appeared thin but was abundant over niches in the alveolar walls, especially at the junctions of alveolar septa. It was a structureless layer for the most part, but occasionally contained spherical bodies and tubular myelin, which tended to gather in the niches. These observations suggest that the surface film is a phospholipid monolayer containing no bound protein. Tubular myelin, saccules, and spherical bodies may be forms of surfactant molecules in reserve, prior to incorporation into the surface film.

The freeze‐fracture study of alveolar type II cells and alveolar content in fetal rabbit lung

Freeze-fracture replication technique was utilized to study the morphology of type II alveolar epithelial cells and alveolar contents in the late gestation of rabbit fetuses. It was shown that the lamellar inclusion bodies of type II cells were enveloped by the usual type of unit membrane with membrane-associated particles of 15 nm diameter. The interior of the inclusion bodies was composed of multiple stacks and/or whorls of membranes which were devoid of membrane-associated particles. Small vesicles within the inclusion were found more frequently with this technique than in chemically fixed thin-sectioned preparations. The intra-alveolar contents were comprised of two components; sperical bodies, which were identical to the internal contents of the lamellar bodies of type II cells, and tubular elements. These tubules most often appeared rectangular on cross-fractured faces. Triangular hexagonal fracture faces were also noted. The tubules were seen to rest on the surfaces of the spherical components. Our observations suggest that the tubular element of alveolar contents is formed through the interaction between the discharged lamellar body content and the alveolar fluid, and further suggest that at least the major constituent of type II cell lamellar bodies is lipid not bound to protein. Three new observations were made in this study; the absence of membrane-associated particles on the interior of the lamellae of the inclusions, the cross-fractured profiles of tubular elements of the alveolar contents, and the occasional multicompartmental nature of type II cell inclusions.